Chimeric Antigen Receptor-Natural Killer Cell (CAR-NK) is a promising next-generation immunotherapy. Persistence of CAR-NK cell therapy is considered a key hurdle limiting its full therapeutic potential. This review highlighted the causes of poor CAR-NK persistence and summarized strategies developed to improve CAR-NK persistence in both hematologic malignancies and solid tumors. These strategies included cytokine and cytokine signaling mediated strategies; optimization of NK cell source and CAR design; intrinsic and extrinsic checkpoint disruption and metabolic reprogramming; and lymphodepletion, alloevasion and fratricide evasion strategies. Multiple strategies that aim at improving in vivo persistence should prove useful in enhancing therapeutic efficacy of CAR-NK.

Hematopoietic stem cells (HSCs) are defined by their remarkable self-renewal and differentiation capacities, giving rise to progenitors that generate diverse blood and immune cell lineages through the tightly regulated process of hematopoiesis. This process is governed by key transcription factors that control gene expression programs essential for cellular homeostasis, differentiation, and lineage commitment. The transcription factor Lyl1 plays a critical role in regulating HSC function and the lineage specification of hematopoietic progenitors. To investigate its role in adult hematopoiesis, we developed a novel conditional Lyl1 knockout (KO) mouse model. Conditional deletion of Lyl1 in adult mice resulted in reduced HSC and lymphoid progenitor populations, consistent with findings from constitutive Lyl1 KO models. Despite these reductions, mature B- and T-lymphocyte numbers remained unchanged, in contrast to previous reports from embryonically deleted Lyl1 models. Competitive transplantation assays further confirmed that Lyl1 is essential for the generation of adult HSC-derived lymphoid progenitors that support adaptive T- and B-cell production. Our new conditional Lyl1 KO model provides a valuable tool for dissecting Lyl1 function across developmental stages and uncovering the mechanisms that maintain postnatal hematopoietic homeostasis.

T-lineage acute lymphoblastic leukemia (ALL) is an aggressive cancer comprising diverse subtypes that are challenging to stratify using conventional immunophenotyping. To gain insights into subset-specific therapeutic vulnerabilities, we performed an integrative multiomics analysis of bone marrow samples from newly diagnosed T cell ALL, early T cell precursor ALL, and T/myeloid mixed phenotype acute leukemia. Leveraging cellular indexing of transcriptomes and epitopes in conjunction with T cell receptor sequencing, we identified a subset of patient samples characterized by activation of inflammatory and stem gene programs. These inflammatory T-lineage samples exhibited distinct biological features compared with other T-lineage ALL samples, including the production of proinflammatory cytokines, prevalence of mutations affecting cytokine signaling and chromatin remodeling, an altered immune microenvironment, and poor treatment responses. Moreover, we found that, although inflammatory T-lineage ALL samples were less sensitive to dexamethasone, they exhibited unique sensitivity to a BCL-2 inhibitor, venetoclax. To facilitate classification of patients with T-lineage ALL, we developed a computational inflammatory gene signature scoring system, which stratified patients and was associated with disease prognosis in three additional patient cohorts. By identifying a high-risk T-lineage ALL subtype on the basis of an inflammatory score, our study provides a framework for targeted therapeutic approaches for these challenging-to-treat cancers.

Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) is an immature subtype of T-cell acute lymphoblastic leukemia (T-ALL) commonly show deregulation of the LMO2-LYL1 stem cell transcription factors, activating mutations of cytokine receptor signaling, and poor early response to intensive chemotherapy. Previously, studies of the Lmo2 transgenic mouse model of ETP-ALL identified a population of stem-like T-cell progenitors with long-term self-renewal capacity and intrinsic chemotherapy resistance linked to cellular quiescence. Here, analyses of Lmo2 transgenic mice, patient-derived xenografts, and single-cell RNA-sequencing data from primary ETP-ALL identified a rare subpopulation of leukemic stem cells expressing high levels of the cytokine receptor FLT3. Despite a highly proliferative state, these FLT3-overexpressing cells had long-term self-renewal capacity and almost complete resistance to chemotherapy. Chromatin immunoprecipitation and assay for transposase-accessible chromatin sequencing demonstrated FLT3 and its ligand may be direct targets of the LMO2 stem-cell complex. Media conditioned by Lmo2 transgenic thymocytes revealed an autocrine FLT3-dependent signaling loop that could be targeted by the FLT3 inhibitor gilteritinib. Consequently, gilteritinib impaired in vivo growth of ETP-ALL and improved the sensitivity to chemotherapy. Furthermore, gilteritinib enhanced response to the BCL2 inhibitor venetoclax, which may enable “chemo-free” treatment of ETP-ALL. Together, these data provide a cellular and molecular explanation for enhanced cytokine signaling in LMO2-driven ETP-ALL beyond activating mutations and a rationale for clinical trials of FLT3 inhibitors in ETP-ALL.

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4-CD8-CD25+CD44-) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a- thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

IL-7 supports the growth and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL), particularly the early T-cell precursor subtype (ETP-ALL), which frequently has activating mutations of IL-7 signaling. STAT5 is an attractive therapeutic target because it is almost universally activated in ETP-ALL, even in the absence of mutations of upstream activators such as the IL-7R, JAK and FLT3. To examine the role of activated STAT5 in ETP-ALL, we have used a Lmo2-transgenic (Lmo2Tg) mouse model in which we can monitor chemoresistant pre-leukemia (pre-LSCs) and leukemia stem cells (LSCs) that drive T-ALL development and relapse following chemotherapy. Using IL-7R-deficient Lmo2Tg mice, we show that IL-7 signaling was not required for the formation of pre-LSCs but essential for their expansion and clonal evolution into LSCs to generate T-ALL. Activated STAT5B was sufficient for the development of T-ALL in IL-7R; Lmo2Tg mice, indicating that inhibition of STAT5 is required to block the supportive signals provided by IL-7. To further understand the role of activated STAT5 in LSCs of ETP-ALL, we developed a new transgenic mouse that enables T-cell specific and doxycycline-inducible expression of the constitutively activated STAT5B1*6 mutant. Expression of STAT5B1*6 in T-cells had no effect alone but promoted expansion and chemoresistance of LSCs in Lmo2Tg mice. Pharmacologic inhibition of STAT5 with Pimozide induced differentiation and loss of LSCs, whilst enhancing response to chemotherapy. Furthermore, Pimozide significantly reduced leukemia burden in vivo and overcame chemoresistance of patient-derived ETP-ALL xenografts. Overall, our results demonstrate that STAT5 is an attractive therapeutic target for eradicating LSCs in ETP-ALL.

Nogo receptor 1 is the high affinity receptor for the potent myelin-associated inhibitory factors that make up part of the inflammatory extracellular milieu during experimental autoimmune encephalomyelitis. Signalling through the Nogo receptor 1 complex has been shown to be associated with axonal degeneration in an animal model of multiple sclerosis, and neuronal deletion of this receptor homologue, in a disease specific manner, is associated with preserving axons even in the context of neuroinflammation. The local delivery of Nogo receptor(1-310)-Fc, a therapeutic fusion protein, has been successfully applied as a treatment in animal models of spinal cord injury and glaucoma. As multiple sclerosis and experimental autoimmune encephalomyelitis exhibit large numbers of inflammatory cell infiltrates within the CNS lesions, we utilized transplantable haematopoietic stem cells as a cellular delivery method of the Nogo receptor(1-310)-Fc fusion protein. We identified CNS-infiltrating macrophages as the predominant immune-positive cell type that overexpressed myc-tagged Nogo receptor(1-310)-Fc fusion protein at the peak stage of experimental autoimmune encephalomyelitis. These differentiated phagocytes were predominant during the extensive demyelination and axonal damage, which are associated with the engulfment of the protein complex of Nogo receptor(1-310)-Fc binding to myelin ligands. Importantly, mice transplanted with haematopoietic stem cells transduced with the lentiviral vector carrying Nogo receptor(1-310)-Fc and recovered from the peak of neurological decline during experimental autoimmune encephalomyelitis, exhibiting axonal regeneration and eventual remyelination in the white matter tracts. There were no immunomodulatory effects of the transplanted, genetically modified haematopoietic stem cells on immune cell lineages of recipient female mice induced with experimental autoimmune encephalomyelitis. We propose that cellular delivery of Nogo receptor(1-310)-Fc fusion protein through genetically modified haematopoietic stem cells can modulate multifocal experimental autoimmune encephalomyelitis lesions and potentiate neurological recovery.

Addressing therapy failure and relapse is critical in improving outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Relapse is thought to represent a failure to effectively eliminate leukemia-initiating cells (L-ICs), which survive chemotherapy and self-renew, regenerating the full complement of leukemic cell populations. Results from mouse T-ALL models show that the double negative-3 (DN3) thymic progenitors are enriched for L-IC activity; however, the L-IC remains an undefined subset of this population. We used single cell RNA-sequencing (scRNA-seq) to profile leukemic development in vivo, with a focus on uncovering DN3 heterogeneity. Integrated UMAP clustering of scRNA-seq libraries from wild type (WT), Tal1/Lmo2 preleukemic (PL), and Tal1/Lmo2 T-ALL (Leuk) thymi revealed an expansion of DN3 progenitors in PL and Leuk samples with decreases in mature (single positive) thymocytes. Unlike WT thymocytes, where Notch1 target gene expression was restricted to DN3 cells, Notch1-Myc pathway activation was maintained throughout thymocyte development in Tal1/Lmo2 transgenic mice. Re-clustering of DN3 cells revealed the presence of dormant and proliferative DN3 cells in PL and Leuk thymus. Dormant DN3 signatures resembled quiescent, Notch1-active DN3a (pre-β-selection) thymocytes and proliferative DN3 cells were transcriptionally similar to DN3b thymocytes. Dormant mouse DN3 cells were enriched for signatures of quiescent, therapy resistant ALL patient cells and cells obtained from ALL patients at minimal residual disease (Ebinger et al., Cancer Cell 2016), suggesting that dormant leukemic DN3 cells resemble human L-ICs that mediate drug resistance and relapse. To test this, we employed in vivo nucleosome labeling to profile dormant DN3 cells throughout leukemia development and functionally evaluate their response to chemotherapy. To assess cell divisional history, WT, Tal1, Lmo2, or Tal1/Lmo2 mice harboring the tet-inducible H2B-GFP reporter allele were subjected to in vivo doxycycline pulse (labeling), followed by a two-week chase. Analysis of pulse-chased thymocytes revealed low frequencies of label retaining (GFPHI) DN3 cells in WT mice. However, label retention was significantly increased in Lmo2 DN3 thymocytes (2.9-fold), consistent with other Lmo2 models (Tremblay et al., Nat. Commun. 2018). Tal1 expression also promoted DN3 label retention (6.0-fold) and Tal1/Lmo2 co-expression resulted in further increases in DN3 GFPHI cells (25-fold), indicating that Tal1 and Lmo2 cooperate to promote DN3 dormancy. DN3 GFPHI cells were present at similar frequency in mice with overt T-ALL and in line with scRNA-seq data, GFPHI cells were observed nearly exclusively as a subset of the leukemic DN3a population. Treatment with Vincristine, DeXamethasone, and L-asparaginase (VXL) led to a further increase in GFPHI DN3 frequency in Lmo2 and Tal1 preleukemic mice (3.1- and 9.7-fold, respectively), suggesting that cell cycle restricted L-ICs may evade conventional chemotherapy and mediate relapse. We then tested whether dormancy is a feature of the L-IC in T-ALL patients. Analysis of relapsed pediatric T-ALL cells revealed that the CD7+CD1a- L-IC population (Chiu et al., Blood 2010) is cell cycle restricted compared to CD7+CD1a+ leukemic cells and that VXL treatment enriched for CD7+CD1a- cells in xenografted mice, suggesting that cell cycle restriction may be an important feature of both mouse and human T-ALL L-ICs. Analysis of transcriptomes to reveal mediators of dormancy that may be exploited to chemosensitize L-ICs will be discussed.

Introduction: The epigenome of T-cell acute lymphoblastic leukemia (T-ALL) is characterized by DNA hypermethylation at CpG islands and altered chromatin accessibility. However, it remains unclear when and how these changes of the epigenome contribute to the development of T-ALL. Methods: To better understand their role in T-ALL, we have examined the epigenome at three stages of T-cell leukemogenesis using the Lmo2 transgenic (Lmo2Tg) mouse model of human early thymocyte progenitor (ETP)-ALL (Fig. 1). 1. Pre-leukemic stem cells (pre-LSCs) isolated from 6-week-old mice, in which double negative 3 (DN3) thymocytes have long-term self-renewal enabling the development of T-ALL but with a long latency of 6 months. 2. Leukemic stem cells (LSCs) from 6-month-old mice, in which DN3 thymocytes have acquired the additional (epi) genetic mutations required for rapidly generating T-ALL when transplanted into recipient mice. 3. DN3 cells from 10 to 12-month-old mice that have succumbed to proliferative T-ALL. DN3 cells from each of these stages of T-cell leukemogenesis together with age-matched wild-type controls were FACS-purified for gene expression (RNA-seq), chromatin accessibility (Assay of Transposase Accessible Chromatin sequencing, ATAC-seq), DNA methylation (Enhanced Reduced Representation Bisulfite Sequencing, ERRBS). Abnormalities in the epigenome directly related to Lmo2 binding were investigated by integration with CHIP-seq. Results: Overall, principal component analyses demonstrated distinct trajectories for chromatin, DNA methylation and gene expression changes during leukemogenesis (Fig. 2). In pre-LSCs, chromatin was more accessible in CpG open seas enriched for the heptad transcription factor (TF) binding sites of hematopoietic stem cells (Runx1, Fli1, Tal1, Lyl1, Gata2, Lmo2, Erg), and less accessible in CpG islands enriched for Klf15 binding sites. Lmo2 directly bound to 1,831 more accessible chromatin regions and was associated with gene activation of the stem cell heptad TFs. In addition to this 'stem-cell’ chromatin pattern, pre-LSCs had DNA hypermethylation in CpG open seas enriched for Runx1 and Fli1 binding. There was also the beginning of the more classic DNA hypermethylation of GpG islands reported for human T-ALL but this was not associated with gene repression. Instead, GpG islands with increased heterogeneity of DNA methylation had closed chromatin and associated gene repression (R=-0.16, p=0.01). In LSCs, chromatin changes remained relatively stable, with increased accessibility at heptad TF binding regions and decreased accessibility in Klf15 binding sites. In contrast, there was a marked increase in DNA hypermethylation of bivalent promoters that was strongly associated with gene repression of several putative tumour suppressors including Klf4 and Tet2 (R=-0.31, p<0.01). Importantly, many of these hypermethylated regions (41%) in LSCs were preceded by increased heterogeneity in pre-LSCs. The most striking observations were those in the transition from LSCs to proliferative T-ALL, where there was reversal of many of the preceding chromatin changes, including closure of the stem cell heptad TF binding sites and re-opening at Klf15 binding in CpG islands. These changes were associated with gene expression changes characterised by loss of the stem cell program and increase in T-cell differentiation. While DNA hypermethylation at promoters was maintained, there was new DNA hypomethylation in lamina-associated late replicating regions. Conclusion: This temporal analysis of purified cell populations facilitates integration of epigenomic changes with gene expression, which is a powerful approach to address the 'chicken-or-egg’ dilemma of the cancer epigenome. Our studies of the epigenome during T-cell leukemogenesis reveal a far more dynamic process than previously recognized and impossible to discern by analysis of T-ALL alone. Specifically, heterogeneity of DNA methylation precedes DNA hypermethylation at bivalent promoters while DNA hypomethylation at late replicating domains occurs much later. In contrast, we show that the T-cell oncogene establishes an early stem cell-like chromatin structure that is reversed in overt T-ALL. We postulate that DNA hypermethylation and gene repression of tumour suppressors in LSCs allows a 'relaxation’ of the chromatin to enable additional adaptive changes required for the rapid growth of T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The IκB kinase complex, consisting of IKK1, IKK2 and the regulatory subunit NEMO, is required for NF-κB signalling following the activation of several cell surface receptors, such as members of the Tumour Necrosis Factor Receptor superfamily and the Interleukin-1 Receptor. This is critical for haematopoietic cell proliferation, differentiation, survival and immune responses. To determine the role of IKK in the regulation of haematopoiesis, we used the Rosa26Cre-ERT2 Cre/lox recombination system to achieve targeted, haematopoietic cell-restricted deletion of the genes for IKK1 or IKK2 in vivo. We found that the IKK complex plays a critical role in haematopoietic cell development and function. Deletion of IKK2, but not loss of IKK1, in haematopoietic cells led to an expansion of CD11b/Gr-1-positive myeloid cells (neutrophilia), severe anaemia and thrombocytosis, with reduced numbers of long-term haematopoietic stem cells (LT-HSCs), short-term haematopoietic stem cells (ST-HSCs) and multipotential progenitor cells (MPPs), increased circulating interleukin-6 (IL-6) and severe gastrointestinal inflammation. These findings identify distinct functions for the two IKK catalytic subunits, IKK1 and IKK2, in the haematopoietic system.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21688-1

Endocytosis entails selective packaging of cell surface cargos in cytoplasmic vesicles, thereby controlling key intrinsic cellular processes as well as the response of normal and malignant cells to their microenvironment. The purpose of this review is to outline the latest advances in the development of endocytosis-targeting therapeutic strategies in hematological malignancies.

The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding hematopoietic system activity in health and disease and have had a major impact on how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single-cell transplantation, lentiviral barcoding, and Sleeping Beauty barcoding. This perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar.

Supplemental Digital Content is available in the text.

Hematopoietic stem cells (HSCs) originate from a subset of endothelium in the embryo known as hemogenic endothelium.Hemogenic endothelium must undergo an endothelial to hematopoietic transition (EHT) to form HSCs. EHT requires the activity of the transcription factor (TF) RUNX1.Ectopic pan-endothelial expression of RUNX1 in mouse embryos is able to specify embryonic endothelium as hemogenic.However, as compared to embryonic endothelium, RUNX1 is not as effective at specifying fetal endothelium as hemogenic.To understand the difference in the competency of embryonic versus fetal endothelium to be specified as hemogenic, we examined differences in chromatin at these two developmental stages.ATAC-seq revealed that RUNX1 appears to be specifying embryonic endothelium through a canonical EHT program.To orchestrate this EHT transcriptional program, RUNX1 participates with other hematopoietic TFs, such as FLI1, ETS1, SPI1, and GFI1.Following RUNX1 induction in embryonic endothelium, immune genes and motility related genes are upregulated.In fetal endothelium, however, RUNX1 induction results in a more complex picture.To determine if these differences were due to underlying chromatin differences between fetal and embryonic endothelium, we examined control embryonic and fetal endothelium using stochastic optical reconstruction microscopy (STORM).STORM imaging suggests that there are global differences in chromatin structure between fetal and embryonic endothelium.Fetal endothelium appears to have more densely packed chromatin that is located more peripherally than embryonic endothelium.Taken together, these data show that the reduced specification efficiency by RUNX1 observed between embryonic and fetal endothelium is due, at least in part, to chromatin structure.

The stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.

Abstract Background: Myeloproliferative neoplasms (MPN) are a diverse group of hematopoietic stem cell disorders. JAK2V617F gain-of-function is the most prevalent mutation, accounting for more than 60% of MPNs. PRMT5 was initially identified as a JAK-binding protein. Its enzymatic function catalyses the symmetric di-methylation of arginine on a variety of substrates, including histones and proteins of the splicing apparatus. It has been proposed that mutant JAK2 can phosphorylate PRMT5, leading to loss of methylation activity and promotion of erythropoiesis (Liu F. et al. Cancer Cell 2011). Based upon this study, it was proposed that enhancing PRMT5 activity may be a useful therapeutic measure (Skoda RC et al. Cancer Cell 2011). Aim: To determine the role of PRMT5 in JAK2V671F mutant hematopoiesis. Hypothesis: Inhibition of PRMT5 will exacerbate JAK2V617F hematopoiesis R esults: Using a conditional null allele, we deleted Prmt5 in embryonic development with the hematopoietic-specific VavCre transgene. This led to embryonic lethality at E9.5 due to absence of erythropoiesis but not other lineages. Similar embryonic lethality was observed using the erythroid specific EpoRCre transgene. Following a 350,000-compound library screen, we developed a potent and selective SAM-dependent inhibitor (CTx034) of PRMT5 similar to that reported by Chan-Penebre E. at al. Nat. Chem. Biol. 2015. Consistent with the genetic evidence that PRMT5 is most important for erythropoiesis, CTx034 was a potent inhibitor of erythropoiesis in cultures derived from healthy human CD34+ cells. This suppression of erythropoiesis was associated with activation of p53. However, progenitor assays of bone marrow cells from patients with MPN showed that JAK2V617F erythropoiesis was more sensitive to CTx034 than normal erythropoiesis. We established JAK2V617F bone marrow chimeric mice to directly compare the in vivo effects of PRMT5 inhibition on mutant and wild-type erythropoiesis within the same animal. Remarkably, these studies showed normalization of spleen size and erythropoiesis, comparable to the current standard of care, Ruxolitinib (Figure 1A-B). Importantly, CTx034 was well tolerated in healthy animals with no suppression of hematopoiesis. One of the major therapeutic challenges for MPN is the eradication of the malignant clone, which is rarely achieved with Ruxolitinib. The addition of MDM2 inhibitors, which activate p53, are currently in trial. Importantly, CTx034 not only suppressed JAK2-mutant erythropoiesis but also activated p53 in JAK2-mutant progenitors, unlike Ruxolitinib (Figure 1C). This result strengthens the therapeutic rationale for PRMT5 inhibitors in MPN. To understand how CTx034 inhibits erythropoiesis, we initially considered direct methylation effects on JAK-STAT signalling and p53. Challenging previous reports, we could find no evidence that JAK alters PRMT5 activity, no evidence that PRMT5 inhibition perturbs JAK-STAT signalling and no evidence that PRMT5 methylates p53. To look more broadly, we performed RNA-seq analysis of CD34+ cells following 72 hours exposure to CTx034. Globally, this demonstrated a potent 'starvation' signal with suppression of protein synthesis despite activation of the upstream mTOR signalling pathway. This suppression of protein synthesis could be linked to three mechanisms. First, CTx034 inhibited methylation of the Sm core complex of the spliceosome, leading to alternate splicing (skipped exons and retained introns) affecting the elongation initiation factor 2 (EIF2) pathway. Second, PRMT5 directly interacts with the translation initiation complex (eIF4A, eIF4B, eIF4E and the poly(A)-binding protein 1, PABP1. Moreover, mass spectrometry identified PABP1 as a new target of PRMT5. Treatment with CTx034 did not alter protein abundance of any of these factors but decreased the RNA binding capacity of PABP1, thereby preventing the correct formation of the initiation of translation complex. Finally, CTx034 perturbed polysome formation with loss of methylation of RPS10. C onclusion: Challenging previous reports, we show that PRMT5 inhibitors are an attractive and novel therapeutic for JAK2V617F MPN by targeting initiation of translation, ribosome biogenesis and activation of p53. Disclosures Sonderegger: CRC Cancer Therapeutics: Research Funding. Cerruti:CRC Cancer Therapeutics: Research Funding. Toulmin:CRC Cancer Therapeutics: Research Funding. Lane:Novartis: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Stupple:CRC Cancer Therapeutics: Employment. Street:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Employment, Patents & Royalties. Jane:CRC Cancer Therapeutics: Patents & Royalties. Altura:MERCK: Employment. Nicholson:MERCK: Employment. Curtis:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Patents & Royalties, Research Funding.