Lymphoid Lineages Research Articles

Advanced-Stage Chronic Myeloid Leukemia: Options for Difficult Treatment Situations.

A small number of patients with chronic myeloid leukemia (CML) either present with or progress to the accelerated phase (AP) or blast phase (BP). This occurs in approximately 4-7% of patients with CML. Most patients who progress to BP-CML are of myeloid lineage, while approximately 30% are of lymphoid lineage. Due to the rarity of this condition, there are no large or randomized trials that can inform clinical decisions. Most data are from retrospective chart reviews or data from old studies when tyrosine kinase inhibitors (TKIs) were initially approved. In addition, the definition of these categories has been in continuous flux over the last 20 years, making applicability of data even more confusing. In some classifications, the cutoff is 30% blasts for the definition of BP-CML, while in others a cutoff of 20% is used. In addition, more recently the World Health Organization (WHO) classification omitted the accelerated phase and recognized only a two-phase disease, while the International Consensus Classification retained a three-phase definition and retained the accelerated phase. Therapy for patients with AP/BP-CML depends on several factors, including prior therapy, BCR::ABL1 mutation, co-morbidities, cell lineage, and eligibility for allogeneic stem cell transplantation (alloHCT). Patients with AP-CML at presentation have a relatively favorable prognosis and may not need alloHCT if they respond appropriately to therapy. For patients with AP-CML who progressed while on TKI therapy or those with BP-CML, alloHCT is considered the only curative therapy. Our goal is to review the available data on the therapy of patients with AP-CML and BP-CML.

Single cell multiomics reveal divergent effects of DNMT3A and TET2 mutant clonal hematopoiesis in inflammatory response.

DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematological neoplasms. While individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome-ARDS) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant CH on COVID-19-related ARDS and mortality. Using single cell (sc-) proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of IL32, a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19 related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19.

Cell-cell interactions mediating primary and metastatic breast cancer dormancy.

Breast cancer remains one of the leading causes of death in women around the world. A majority of deaths from breast cancer occur due to cancer cells colonizing distant organ sites. When colonizing these distant organ sites, breast cancer cells have been known to enter into a state of dormancy for extended periods of time. However, the mechanisms that promote dormancy as well as dormant-to-proliferative switch are not fully understood. The tumor microenvironment plays a key role in mediating cancer cell phenotype including regulation of the dormant state. In this review, we highlight cell-cell interactions in the tumor microenvironment mediating breast cancer dormancy at the primary and metastatic sites. Specifically, we describe how immune cells from the lymphoid lineage, tumor-associated myeloid lineage cells, and stromal cells of non-hematopoietic origin as well as tissue resident stromal cells impact dormancy vs. proliferation in breast cancer cells as well as the associated mechanisms. In addition, we highlight the importance of developing model systems and the associated considerations that will be critical in unraveling the mechanisms that promote primary and metastatic breast cancer dormancy mediated via cell-cell interactions.

Navigating diagnostic dilemmas: a rare presentation of extramedullary T-lymphoblastic leukemia/lymphoma with chronic myeloid leukemia.

Chronic myeloid leukemia (CML) typically presents in the chronic phase. The blast crisis phase in CML predominantly comprises the myeloid phenotype, while B-cell lymphoblastic crisis is common among the lymphoid lineages. Presentation as a T-lymphoblastic crisis is exceptionally rare. Little is known about its characteristics, treatment, and prognosis. This case study reports a patient who presented as an extramedullary blast crisis with T-lymphoblastic immunophenotype without a known prior diagnosis of CML. We performed hematoxylin and eosin staining, immunohistochemistry on the inguinal lymph node, and bone marrow biopsy. Ancillary studies including flow cytometry and cytogenetic testing were conducted as needed. BCR::ABL1 is quantitative real-time polymerase chain reaction monitored disease progression. Our patient is a 40-year-old male with no previous medical history who presented with neck stiffness and pain of one week in duration. Clinical evaluation revealed diffuse lymphadenopathy and splenomegaly. A biopsy from the inguinal lymph node revealed T-lymphoblastic lymphoma (T-LBL) (90%) and a population of myeloblasts (10%). Subsequent bone marrow biopsy showed myelocyte expansion, dwarf megakaryocytes, scattered myeloblasts (9%), and T-lymphoblasts (6%). Flow cytometry of the bone marrow aspirate revealed myeloblasts (5.4%) and T-lymphoblasts (6.3%). Genetic and molecular studies identified the BCR-ABL1 fusion. This case contributes to the medical literature by documenting a rare occurrence of extramedullary T-LBL with concurrent CML. The absence of a CML history makes the diagnosis particularly challenging and underscores the need for comprehensive and personalized treatment strategies.

TMIC-68. DEEP LEARNING OF THE SPATIAL IMMUNE ENGAGEMENT IN GLIOBLASTOMA UNDER TEMOZOLOMIDE TREATMENT

Abstract Effective immune synapses are a prerequisite for desirable immune surveillance and control of tumour growth. Cell-to-cell interactions form the basis of immune synapses, resulting in either immunoprotected or immunosuppressed manifestations in the tumour brain that could explain glioblastoma (GBM) recurrence. Using state-of-the-art multiplex imaging, spatial statistics, and spatially aware deep learning tools in 14 patient-matching tissues from primary and recurrent GBM, we detected spatial immune networks (SINets). SINets across 105 regions of interest comprising 2.3 million cells show stronger spatial engagement between myeloid and lymphoid lineages at recurrence. SINets’ architecture in primary tumours is associated with relapse timing. Patients, where the SINets are more modular, recur sooner than patients with denser SiNets. In addition, this association presents sexual dimorphism in our cohort (N=3 females, and N=4 males). Phenotype-to-phenotype spatial coexistence also contributes to enhancing phenotype prediction. A spatial-aware deep learning model trained with single-cell data from primary tumours reaches an average balanced accuracy of 0.76 in primary tumour cellular communities unseen during training, compared to a spatial-agnostic model that reached an average balanced accuracy of 0.69. In summary, we detected a distinct spatial engagement in glioblastoma undergoing chemo-radiation treatment. The spatial patterns of coexistence represent ecological niches that contribute to defining cell occurrence and may underpin effective immune synapses. Overall, the spatiotemporal elements provide a newer approach for dissecting glioblastoma’s complexity and discovering opportunities for stratifying patients by harnessing spatial information encoded in multicellular niches.

Molecular characterization of human HSPCs with different cell fates in vivo using single-cell transcriptome analysis and lentiviral barcoding technology.

Hematopoietic stem and progenitor cells (HSPCs) possess the potential to produce all types of blood cells throughout their lives. It is well recognized that HSPCs are heterogeneous, which is of great significance for their clinical applications and the treatment of diseases associated with HSPCs. This study presents a novel technology called Single-Cell transcriptome Analysis and Lentiviral Barcoding (SCALeBa) to investigate the molecular mechanisms underlying the heterogeneity of human HSPCs in vivo. The SCALeBa incorporates a transcribed barcoding library and algorithm to analyze the individual cell fates and their gene expression profiles simultaneously. Our findings using SCALeBa reveal that HSPCs subset with stronger stemness highly expressed MYL6B, ATP2A2, MYO19, MDN1, ING3, and so on. The high expression of COA3, RIF1, RAB14, and GOLGA4 may contribute to the pluripotent-lineage differentiation of HSPCs. Moreover, the roles of the representative genes revealed in this study regarding the stemness of HPSCs were confirmed with biological experiments. HSPCs expressing MRPL23 and RBM4 genes may contribute to differentiation bias into myeloid and lymphoid lineage, respectively. In addition, transcription factor (TF) characteristics of lymphoid and myeloid differentiation bias HSPCs subsets were identified and linked to previously identified genes. Furthermore, the stemness, pluripotency, and differentiation-bias genes identified with SCALeBa were verified in another independent HSPCs dataset. Finally, this study proposes using the SCALeBa-generated tracking trajectory to improve the accuracy of pseudo-time analysis results. In summary, our study provides valuable insights for understanding the heterogeneity of human HSPCs in vivo and introduces a novel technology, SCALeBa, which holds promise for broader applications. KEY POINTS: SCALeBa and its algorithm are developed to study the molecular mechanism underlying human HSPCs identity and function. The human HSPCs expressing MYL6B, MYO19, ATP2A2, MDN1, ING3, and PHF20 may have the capability for high stemness. The human HSPCs expressing COA3, RIF1, RAB14, and GOLGA4 may have the capability for pluripotent-lineage differentiation. The human HSPCs expressing MRPL23 and RBM4 genes may have the capability to differentiate into myeloid and lymphoid lineage respectively in vivo. The legitimacy of the identified genes with SCALeBa was validated using biological experiments and a public human HSPCs dataset. SCALeBa improves the accuracy of differentiation trajectories in monocle2-based pseudo-time analysis.

Age-associated imbalance in immune cell regeneration varies across individuals and arises from a distinct subset of stem cells

The age-associated decline in immunity manifests as imbalanced adaptive and innate immune cells, which originate from the aging of the stem cells that sustain their regeneration. Aging variation across individuals is well recognized, but its mechanism remains unclear. Here, we used high-throughput single-cell technologies to compare mice of the same chronological age that exhibited early or delayed immune aging phenotypes. We found that some hematopoietic stem cells (HSCs) in early aging mice upregulated genes related to aging, myeloid differentiation, and stem cell proliferation. Delayed aging was instead associated with genes involved in stem cell regulation and the response to external signals. These molecular changes align with shifts in HSC function. We found that the lineage biases of 30% to 40% of the HSC clones shifted with age. Moreover, their lineage biases shifted in opposite directions in mice exhibiting an early or delayed aging phenotype. In early aging mice, the HSC lineage bias shifted toward the myeloid lineage, driving the aging phenotype. In delayed aging mice, HSC lineage bias shifted toward the lymphoid lineage, effectively counteracting aging progression. Furthermore, the anti-aging HSC clones did not increase lymphoid production but instead decreased myeloid production. Additionally, we systematically quantified the frequency of various changes in HSC differentiation and their roles in driving the immune aging phenotype. Taken together, our findings suggest that temporal variation in the aging of immune cell regeneration among individuals primarily arises from differences in the myelopoiesis of a distinct subset of HSCs. Therefore, interventions to delay aging may be possible by targeting a subset of stem cells.

Characterization and ontogeny of a novel lymphoid follicle inducer cell during development of the bursa of Fabricius.

The avian bursa of Fabricius (BF) is a primary lymphoid organ, where B-cell development occurs within bursal follicles of epithelial origin. During embryogenesis the epithelial anlage of the BF emerges as a diverticulum of the cloaca surrounded by undifferentiated tail bud mesenchyme. While it is believed that the epithelial-mesenchymal BF primordium provides a selective microenvironment for developing B cells, the initial events inducing lymphoid follicle formation are not fully elucidated. Using wild type and CSF1R-eGFP transgenic chick embryos, we find that separate B cell, macrophage and dendritic cell precursors enter the BF mesenchyme, migrate to the surfaceepithelium, and colonize the lymphoid follicle buds. Detailed immunocytochemical characterization revealed a novel EIV-E12+ blood-borne cell type, colonizing the surface epithelium of the BF rudiment before the entry of myeloid and lymphoid lineages and the appearance of this cell type coincides with the onset of follicle bud formation. Chick-duck chimeras and chick-quail tissue recombination experiments suggest that EIV-E12+ cells represent a transient lymphoid inducer cell population. They are not dendritic or B cells precursors, and they are capable of follicle bud induction in both dendritic cell- and B cell-depleted bursae.

TRIP13 is Critical for the Survival of B-cell Lymphomas and Myeloid Leukemias In Vitro

Hematologic malignancies represent a diverse group of blood tumors accounting for approximately 10% of all cancer cases in the US. Based on the site of manifestation, presumed cell of origin, genetic abnormalities, and clinical features, we recognize more than 100 clinically important subtypes of hematologic malignancies broadly categorized into three main groups: leukemia, lymphoma, and multiple myeloma. While the prognosis for patients with specific subtypes of hematologic malignancies, such as Hodgkin lymphomas or chronic lymphocytic leukemia has significantly improved over the last several years, other subtypes such as acute myeloid leukemia or non-Hodgkin lymphomas still present with significant treatment challenges. A better understanding of molecular drivers of tumor maintenance is needed to improve existing therapies. Thyroid hormone receptor interactor 13 (TRIP13) is an AAA + ATPase that plays an important yet poorly understood role in the regulation of the mammalian cell cycle. The TRIP13 gene is highly expressed in various human tumors including colorectal carcinoma and glioblastoma, and it seems to promote tumorigenesis. Recently, we identified TRIP13 as a gene overexpressed in Peripheral T-cell lymphomas where it plays a critical role in tumor maintenance by regulating the G2-M phase of the cell cycle. However, the biological activities of TRIP13 in non-T cell hematologic malignancies have not been investigated to date. Here we used shRNA-mediated knockdown and a small molecule inhibitor to downregulate TRIP13 in B-cell lymphoid and myeloid lineages. We found genetic downregulation inhibited the proliferation of both B-cell lymphomas and acute myeloid leukemia cell lines in vitro. The pharmacologic inhibition of TRIP13 effectively induced the G0/G1 cell cycle arrest and apoptosis of B-cell lymphomas. Altogether these data demonstrate that TRIP13 is critical for the maintenance of hematologic malignancies of B-cell and myeloid malignancies in vitro. Thus, TRIP13 might be a good target in the treatment of various types of hematologic malignancies.

Spatial Profiling of Ovarian Clear Cell Carcinoma Reveals Immune-Hot Features

Ovarian clear cell carcinoma (OCCC) has a high incidence in Asia, with a frequent occurrence at an early stage, but without sufficient data on molecular stratification for high-risk patients. Recently, immune-hot features have been proposed as indicators of poor prognosis in early stage OCCC. Specific patterns of intratumoral heterogeneity associated with immune-hot features must be defined. NanoString Digital Spatial Profiling technology (Cold spring biotech corp.) was used to decipher the spatial distribution of the 18-plex protein panel. Regions of interest (ROIs) were collected based on the reference hematoxylin and eosin–stained morphology. Areas of illumination (AOIs) were defined according to the ROI segmentation using the fluorescence signals of the visualization markers pan-cytokeratin (PanCK), CD45, or DNA. Unsupervised hierarchical clustering of 595 AOIs from 407 ROIs showed that the PanCK segments expressed different combinations of immune markers, suggestive of immune mimicry. The following 3 immune-hot clusters were identified: granzyme B–high, immune signal-high , and immune-like cells; the following 2 immune-cold clusters were identified: fibronectin-high and immune checkpoint–high cells. In tumor samples at the International Federation of Gynecology and Obstetrics stage IC1/2 experiencing recurrence, there was an increased occurrence of PanCK+ AOIs with immune signal-high and immune-like cell groups in the papillary morphology surrounded by macrophage lineage tumor-infiltrating immune cells (TIIs). In contrast, for tumor samples at the International Federation of Gynecology and Obstetrics stage IC3/II with recurrence, PanCK+ AOIs were prevalent in the fibronectin-high group, particularly in those with a tubulocystic morphology surrounded by lymphoid lineage non-TIIs. Our study on the spatial profiling of early stage OCCC tumors revealed that the immune mimicry of tumor cells, presence of TIIs, and morphologic patterns were associated with recurrence, which switched during tumor progression.

Systemic deficits in lipid homeostasis promote aging-associated impairments in B cell progenitor development.

Organismal aging has been associated with diverse metabolic and functional changes across tissues. Within the immune system, key features of physiological hematopoietic cell aging include increased fat deposition in the bone marrow, impaired hematopoietic stem and progenitor cell (HSPC) function, and a propensity towards myeloid differentiation. This shift in lineage bias can lead to pre-malignant bone marrow conditions such as clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenias of undetermined significance (CCUS), frequently setting the stage for subsequent development of age-related cancers in myeloid or lymphoid lineages. At the systemic as well as sub-cellular level, human aging has also been associated with diverse lipid alterations, such as decreased phospholipid membrane fluidity that arises as a result of increased saturated fatty acid (FA) accumulation and a decay in n-3 polyunsaturated fatty acid (PUFA) species by the age of 80 years, however the extent to which impaired FA metabolism contributes to hematopoietic aging is less clear. Here, we performed comprehensive multi-omics analyses and uncovered a role for a key PUFA biosynthesis gene, ELOVL2 , in mouse and human immune cell aging. Whole transcriptome RNA-sequencing studies of bone marrow from aged Elovl2 mutant (enzyme-deficient) mice compared with age-matched controls revealed global down-regulation in lymphoid cell markers and expression of genes involved specifically in B cell development. Flow cytometric analyses of immune cell markers confirmed an aging-associated loss of B cell markers that was exacerbated in the bone marrow of Elovl2 mutant mice and unveiled CD79B, a vital molecular regulator of lymphoid progenitor development from the pro-B to pre-B cell stage, as a putative surface biomarker of accelerated immune aging. Complementary lipidomic studies extended these findings to reveal select alterations in lipid species in aged and Elovl2 mutant mouse bone marrow samples, suggesting significant changes in the biophysical properties of cellular membranes. Furthermore, single cell RNA-seq analysis of human HSPCs across the spectrum of human development and aging uncovered a rare subpopulation (<7%) of CD34 + HSPCs that expresses ELOVL2 in healthy adult bone marrow. This HSPC subset, along with CD79B -expressing lymphoid-committed cells, were almost completely absent in CD34 + cells isolated from elderly (>60 years old) bone marrow samples. Together, these findings uncover new roles for lipid metabolism enzymes in the molecular regulation of cellular aging and immune cell function in mouse and human hematopoiesis. In addition, because systemic loss of ELOVL2 enzymatic activity resulted in down-regulation of B cell genes that are also associated with lymphoproliferative neoplasms, this study sheds light on an intriguing metabolic pathway that could be leveraged in future studies as a novel therapeutic modality to target blood cancers or other age-related conditions involving the B cell lineage.

42-parameter mass cytometry panel to assess cellular and functional phenotypes of leukocytes in bronchoalveolar lavage of Rhesus macaque.

This Optimized Multiparameter Immunofluorescence Panel (OMIP) reports on the development of a mass cytometry panel for broad immunophenotyping of leukocytes from bronchoalveolar lavage from rhesus macaques. Using this panel, we were able to identify myeloid populations such as macrophages, neutrophils, monocytes, myeloid and plasmacytoid DCs, basophils and lymphoid cell lineages including B cells, natural killer (NK) cells, mucosal associated invariant T (MAIT) cells, γδ T cells, CD4 T cells, CD8 β T cells, CD8 T cells, and innate lymphoid cells (ILCs). We also included markers for defining memory, differentiation (CCR7, CD28, CD45RA), homing potential (CXCR3), cytotoxic potential (perforin, granzyme B, granzyme K), cell activation/differentiation (HLA-DR, CD69, IgD) and effector function (CD154, IFN-γ, TNF, IL-2, IL-17A, IL-6, IL-1β, CCL4 and CD107a). This panel was optimized on cryopreserved, bronchoalveolar lavage and splenocytes collected from rhesus macaques. The antibodies selected in this panel are human-specific antibodies that have been shown to cross-react with non-human primates except for CD45 clone D058-1283 which is specific for non-human primates.

BHLHE41, a transcriptional repressor involved in physiological processes and tumor development.

BHLHE41 is a nuclear transcriptional repressor that belongs to the basic helix-loop-helix protein superfamily. BHLHE41 expression tends to be restricted to specific tissues and is regulated by environmental cues and biological events. BHLHE41 homodimerizes or heterodimerizes with various partners, influencing its transcription factor function. BHLHE41 is involved in the regulation of many physiological processes implicated in tissue/organ homeostasis, such as myogenesis, adipogenesis, circadian rhythms and DNA repair. At cellular level, BHLHE41 is involved in the regulation of mesenchymal stem cell properties, tissue-specific macrophage functions and lymphoid lineage physiology. In several cancer types, BHLHE41 modulates the expression of different transcriptional programs influencing cell cycle control, apoptosis, invasiveness, epithelial to mesenchymal transition and hypoxia response in the tumor environment. Depending on the cancer cell type, BHLHE41 can act as a tumor suppressor or an oncogene, and could be a target for innovative therapies. This review summarizes the available knowledge on BHLHE41 structure, biological functions, regulation and potential partners, as well as its role in physiological processes, and its implication in major cancer steps.

Efficient generation of human NOTCH ligand-expressing haemogenic endothelial cells as infrastructure for in vitro haematopoiesis and lymphopoiesis

Arterial endothelial cells (AECs) are the founder cells for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single-cell RNA-sequencing reveals the dynamic evolution of haematopoiesis and lymphopoiesis, generating cell types with counterparts present in early human embryos, including stages marked by the pre-haematopoietic stem cell genes MECOM/EVI1, MLLT3 and SPINK2. DLL4+ AECs robustly support lymphoid differentiation, without the requirement for exogenous NOTCH ligands. Using this system, we find IL7 acts as a morphogenic factor determining the fate choice between the T and innate lymphoid lineages and also plays a role in regulating the relative expression level of RAG1. Moreover, we document a developmental pathway by which human RAG1+ lymphoid precursors give rise to the natural killer cell lineage. Our study describes an efficient method for producing lymphoid progenitors, providing insights into their endothelial and haematopoietic ontogeny, and establishing a platform to investigate the development of the human blood system.

Lin-CD117+CD34+FcεRI+ progenitor cells are increased in chronic spontaneous urticaria and predict clinical responsiveness to anti-IgE therapy.

Chronic spontaneous urticaria (CSU) is a common, debilitating skin disorder characterized by recurring episodes of raised, itchy and sometimes painful wheals lasting longer than 6 weeks. CSU is mediated by mast cells which are absent from peripheral blood. However, lineage-CD34hiCD117int/hiFcεRI+ cells in blood have previously been shown to represent a mast cell precursor. We enumerated FcεRI-, FcεRI+ and FcεRIhi lineage-CD34+CD117+ cells using flow cytometry in blood of patients with CSU (n = 55), including 12 patients receiving omalizumab and 43 not receiving omalizumab (n = 43). Twenty-two control samples were studied. Disease control and patient response to omalizumab was evaluated using the urticaria control test. We performed single-cell RNA sequencing (scRNA-Seq) on lineage-CD34hiCD117hi blood cells from a subset of patients with CSU (n = 8) and healthy controls (n = 4). CSU patients had more lineage-CD34+CD117+FcεRI+ blood cells than controls. Lineage-CD34+CD117+FcεRI+ cells were significantly higher in patients with CSU who had an objective clinical response to omalizumab when compared to patients who had poor disease control 90 days after initiation of omalizumab. scRNA-Seq revealed that lineage-CD34+CD117+FcεRI+ cells contained both lymphoid and myeloid progenitor lineages, with omalizumab responsive patients having proportionally more myeloid progenitors. The myeloid progenitor lineage contained small numbers of true mast cell precursors along with more immature FcεRI- and FcεRI+ myeloid progenitors. Increased blood CD34+CD117+FcεRI+ cells may reflect enhanced bone marrow egress in the setting of CSU. High expression of these cells strongly predicts better clinical responses to the anti-IgE therapy, omalizumab.

Lysosomal membrane protein TMEM106B modulates hematopoietic stem and progenitor cell proliferation and differentiation by regulating LAMP2A stability.

Hematopoietic stem and progenitor cells (HSPCs) are successfully employed for hematological transplantations, and impaired HSPC function causes hematological diseases and aging. HSPCs maintain the lifelong homeostasis of blood and immune cells through continuous self-renewal and maintenance of the multilineage differentiation potential. TMEM106B is a transmembrane protein localized on lysosomal membranes and associated with neurodegenerative and cardiovascular diseases; however, its roles in HSPCs and hematopoiesis are unknown. Here, we established tmem106bb-/- knockout (KO) zebrafish and showed that tmem106bb KO reduced the proliferation of HSPCs during definitive hematopoiesis. The differentiation potential of HSPCs to lymphoid lineage was reduced, whereas the myeloid and erythroid differentiation potentials of HPSCs were increased in tmem106bb-/- zebrafish. Similar results were obtained with morpholino knockdown of tmem106bb. Mechanistically, TMEM106B interacted with LAMP2A, the lysosomal associated membrane protein 2A, impaired LAMP2A-Cathepsin A interaction, and enhanced LAMP2A stability; tmem106bb KO or TMEM106B knockdown caused LAMP2A degradation and impairment of chaperone-mediated autophagy (CMA). Knockdown of lamp2a caused similar phenotypes to that in tmem106bb-/- zebrafish, and overexpression of lamp2a rescued the impaired phenotypes of HSPCs in tmem106bb-/- embryos. These results uncover a novel molecular mechanism for the maintenance of HSPC proliferation and differentiation through stabilizing LAMP2A via TMEM106B-LAMP2A interaction.

KMT2A oncoproteins induce epigenetic resistance to targeted therapies.

Chromosomal translocations involving the Lysine-Methyl-Transferase-2A ( KMT2A ) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative KMT2A -rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose KMT2A oncoproteins promote lineage-switching events through dynamic chromatin binding and can induce epigenetic lesions, marked by ENL, that support resistance to targeted therapies.

Genetic evidence for predisposition to acute leukemias due to a missense mutation (p.Ser518Arg) in ZAP70 kinase: a case-control study

BackgroundThe apparent lack of additional missense mutations data on mixed-phenotype leukemia is noteworthy. Single amino acid substitution by these non-synonymous single nucleotide variations can be related to many pathological conditions and may influence susceptibility to disease. This case-control study aimed to unravel whether the ZAP70 missense variant (rs104893674 (C > A)) underpinning mixed-phenotype leukemia.MethodsThe rs104893674 was genotyped in clients who were mixed-phenotype acute leukemia-, acute lymphoblastic leukemia- and acute myeloid leukemia-positive and matched healthy controls, which have been referred to all major urban hospitals from multiple provinces of country- wide, IRAN, from February 11’ 2019 to June 10’ 2023, by amplification refractory mutation system-polymerase chain reaction method. Direct sequencing for rs104893674 of the ZAP70 gene was performed in a 3130 Genetic Analyzer.ResultsWe found that the AC genotype of individuals with A allele at this polymorphic site (heterozygous variant-type) contribute to the genetic susceptibility to acute leukemia of both forms, acute myeloid leukemia and acute lymphoblastic leukemia as well as with a mixed phenotype. In other words, the ZAP70 missense variant (rs104893674 (C > A)) increases susceptibility of distinct cell populations of different (myeloid and lymphoid) lineages to exhibiting cancer phenotype. The results were all consistent with genotype data obtained using a direct DNA sequencing technique.ConclusionOf special interest are pathogenic missense mutations, since they generate variants that cause specific molecular phenotypes through protein destabilization. Overall, we discovered that the rs104893674 (C > A) variant chance in causing mixed-phenotype leukemia is relatively high.

Interference with PLA2G16 promotes cell cycle arrest and apoptosis and inhibits the reprogramming of glucose metabolism in multiple myeloma cells by modulating the Hippo/YAP signaling pathway.

Multiple myeloma, which is a clonal plasma cell tumor, derives from a postmitotic lymphoid B-cell lineage and remains untreatable. Group XVI phospholipase A2 (PLA2G16) can either be a tumor suppressor or an oncogene in different types of cancer. This study was intended to explore the role of PLA2G16 in multiple myeloma and to reveal the reaction mechanism. The mRNA and protein expressions of PLA2G16 in human bone marrow stromal cell line HS-5 and multiple myeloma cells were assessed using reverse transcription-quantitative PCR and western blot. The transfection efficacy of sh-PLA2G16 and oe-YAP was examined using reverse transcription-quantitative PCR and western blot. Through cell counting kit-8 assay and 5-ethynyl-2'- deoxyuridine staining, multiple myeloma cell viability and proliferation were detected. Flow cytometry was used to measure cell apoptosis and cell cycle distribution. Oxygen consumption rate, the activities of mitochondrial respiratory chain complexes I-V, and the activity of caspase-3 were estimated with Seahorse XF24 analyzer, oxidative phosphorylation activity assay kit, and caspase-3 assay kit, respectively. Lactate production and glucose consumption were evaluated usingcorresponding assay kits. Western blot was employed to meaure proteins associated with cell cycle, glycolysis, pentose phosphate pathway as well as Hippo/YAP signaling pathway. In this study, PLA2G16 expression was greatly increased in multiple myeloma cells and PLA2G16 silence inhibited cell proliferation, promoted cell apoptosis, facilitated cell cycle arrest, and suppressed the reprogramming of glucose metabolism in multiple myeloma. It was also identified that PLA2G16 depletion inhibited the Hippo/YAP signaling pathway. Further experiments revealed that the overexpression of YAP partially reversed the inhibitory effects of PLA2G16 silence on multiple myeloma cell malignant development and the reprogramming of glucose metabolism. Collectively, PLA2G16 silence impeded multiple myeloma progression and inhibited glucose metabolism reprogramming by blocking the Hippo/YAP signaling pathway.

DAP12 interacts with RER1 and is retained in the secretory pathway before assembly with TREM2

DNAX-activating protein of 12 kDa (DAP12) is a transmembrane adapter protein expressed in lymphoid and myeloid lineage cells. It interacts with several immunoreceptors forming functional complexes that trigger intracellular signaling pathways. One of the DAP12 associated receptors is the triggering receptor expressed on myeloid cells 2 (TREM2). Mutations in both DAP12 and TREM2 have been linked to neurodegenerative diseases. However, mechanisms involved in the regulation of subcellular trafficking and turnover of these proteins are not well understood. Here, we demonstrate that proteasomal degradation of DAP12 is increased in the absence of TREM2. Interestingly, unassembled DAP12 is also retained in early secretory compartments, including the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC), thereby preventing its transport to the plasma membrane. We also show that unassembled DAP12 interacts with the retention in ER sorting receptor 1 (RER1). The deletion of endogenous RER1 decreases expression of functional TREM2-DAP12 complexes and membrane proximal signaling, and resulted in almost complete inhibition of phagocytic activity in THP-1 differentiated macrophage-like cells. These results indicate that RER1 acts as an important regulator of DAP12 containing immunoreceptor complexes and immune cell function.Graphical