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Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor β and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF.

GATA1 mutations that result in loss of the N-terminal 83 amino acids are a feature of myeloid leukemia in children with Down syndrome (ML-DS), rare familial cases of dyserythropoietic anemia, and a subset of cases of Diamond-Blackfan Anemia (DBA). The Gata1s mouse model, which expresses only the short GATA1 isoform that begins at methionine 84, has been shown to have a defect in hematopoiesis, specially impaired erythropoiesis with expanded megakaryopoiesis, during gestation. However, the mice were reported to not show any post-natal phenotype. Here we demonstrate that Gata1s mutant mice display macrocytic anemia and features of aberrant megakaryopoiesis throughout life, culminating in profound splenomegaly and bone marrow fibrosis. These data support the use of this animal model for studies of GATA1 deficiencies.

Megakaryocytes give rise to platelets, which have a wide variety of functions in coagulation, immune response, inflammation, and tissue repair. Dysregulation of megakaryocytes is a key feature of in the myeloproliferative neoplasms, especially myelofibrosis. Megakaryocytes are among the main drivers of myelofibrosis by promoting myeloproliferation and bone marrow fibrosis. In vivo targeting of megakaryocytes by genetic and pharmacologic approaches ameliorates the disease, underscoring the important role of megakaryocytes in myeloproliferative neoplasms. Here we review the current knowledge of the function of megakaryocytes in the JAK2, CALR, and MPL-mutant myeloproliferative neoplasms.

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.

DNA is compacted into higher order structures that have major implications in gene regulation. These structures allow for long-range interactions of DNA elements, such as the association of promoters with their cognate enhancers. In recent years, mutations in genes that control these structures, including the cohesin-complex and the insulator-binding protein CTCF, have been found in a spectrum of hematologic disorders, and especially in acute leukemias. Cohesin and CTCF are critical for mediating looping and establishing boundaries within chromatin. Cells that harbor mutations in these genes display aberrant chromatin architecture and resulting differences in gene expression that contribute to leukemia initiation and progression. Here, we provide detailed discussion of the nature of 3D interactions and the way that they are disrupted in acute leukemia. Continued research in this area will provide new insights into the mechanisms of leukemogenesis and may shed light on novel treatment strategies.

Hematologists have long been fascinated by how platelet production occurs, and our understanding continues to evolve as technology advances and as more and more genetic causes for thrombocytopenia are discovered. In this Review Series on megakaryopoiesis and platelet production edited by Associate Editor John Crispino, experts in the field discuss the latest insights into megakaryopoiesis in fetal and adult life, the varied roles that megakaryocytes are now recognized to play, the mechanics of platelet creation, and how all of this new knowledge underpins clinical disorders requiring diagnosis and management.

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. Abstract: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

Children with Down syndrome are at an elevated risk of leukemia, especially myeloid leukemia (ML-DS). This malignancy is frequently preceded by transient abnormal myelopoiesis (TAM), which is self-limited expansion of fetal liver derived megakaryocyte progenitors. An array of international studies has led to consensus in treating ML-DS with reduced intensity chemotherapy leading to excellent outcomes. In addition, studies performed in the past 20 years, have revealed many of the genetic and epigenetic features of the tumors, including GATA1 mutations which are arguably associated with all cases of both TAM and ML-DS. Despite these advances in understanding the clinical and biological aspects of ML-DS, little is known about the mechanisms of relapse. Upon relapse, patients face a poor outcome, and there is no consensus on treatment. Future studies need to be focused on this challenging aspect of leukemia in children with DS.

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder against platelets characterized by a low platelet count and increased bleeding risk. ITP is likely to rise from defective immune tolerance in addition to a triggering event, such as vaccination. COVID-19 vaccination is associated with a small increased risk of development of de novo ITP. In patients historically diagnosed with ITP, relapse of thrombocytopenia after COVID-19 vaccination has been described. However, the precise platelet dynamics in previously diagnosed ITP patients after COVID-19 vaccination is unknown Aims: To investigate the effect of the COVID-19 vaccine on platelet count, the occurrence of severe bleeding complications and necessity of rescue medication in patients historically diagnosed with ITP. Methods: Platelet counts of ITP patients and healthy controls were collected immediately before, 1 and 4 weeks after the first and second vaccination. Linear mixed effects modelling was applied to analyse platelet count dynamics over time. Results: We included 218 ITP patients (50.9% women) with a mean (SD) age of 58 (17) years and 200 healthy controls (60.0% women) with a mean (SD) age of 58 (13) years. Healthy controls and ITP patients had similar baseline characteristics (Table 1). 201/218 (92.2%)ITP patients received the mRNA-1273 vaccine, 16/218 (7.3%) the BNT162b vaccine and 1/218 (0.46%) the Vaxzevria vaccine. All healthy controls received the mRNA-1273 vaccine. Fifteen (6.8%) patients needed rescue medication (Table 1). Significantly more ITP patients who needed rescue medication were on ITP treatment prior COVID-19 vaccination compared to patients without exacerbation (56.2% (7/16) vs 27.4% (55/202), p=0.016). We found a significant effect of vaccination on platelet count over time in both ITP patients and healthy controls (Figure 1A). Platelet counts of ITP patients decreased 7.9% between baseline and 4 weeks after second vaccination (p=0.045). Rescue medication and prior treatment significantly increased platelet count over time (p=0.042 and p=0.044). Healthy controls decreased 4.5% in platelet count (p150x10 9/L had a significant decrease of platelet count 4 weeks after second vaccination compared to baseline (median platelet count (IQR) 205 (94) vs 203 x10 9/L (109) p=0.001). No significant decrease was seen in ITP patients with a baseline platelet count

Deregulation of key regulators of histone modification is important in the initiation and progression of human leukemia. Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) participates in histone acetylation and its role in acute myeloid leukemia remains unclear. Here we observed significant upregulation of ANP32A in primary AML cells, which was essential for AML cell proliferation, survival, and colony formation. Integrative analysis of the genome-wide histone H3 acetylation and gene expression demonstrated that ANP32A deficiency reduced histone H3 acetylation, in accordance with changes in gene expression. Notably, significant histone H3 acetylation enrichment was associated with mRNA changes in lipid-related genes, including APOC1, PCSK9, P2RX1, and LPPR3. Indeed, over-expression of APOC1 partially compensated the proliferation-defect phenotype in ANP32A deficient AML cells while APOC1 knockdown alone mimicked the effect of ANP32A deficiency. Collectively, our data indicate that ANP32A is a novel regulator of histone H3 acetylation and promotes leukemogenesis.

Background: KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 75% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. We performed a kinome-wide CRISPR screen and identified that DYRK1A is specifically required for the survival of KMT2A-R ALL cell. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as negatively regulator of cell proliferation. Results: We performed a kinome-wide CRISPR screen in human ALL cell lines and PDX models and identified DYRK1A as a novel target in KMT2A-R ALL. DYRK1A is a serine-threonine kinase with a proposed, but poorly defined role in cell cycle regulation. We performed a meta-analysis of multiple ChIP-Seq experiments and identified that oncogenic KMT2A fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses of KMT2A-R ALL cells treated with a menin inhibitor (MI-503) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A, indicating that DYRK1A is directly regulated by the KMT2A fusion complex. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced potent leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells. Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.

Pre-fibrotic-primary myelofibrosis PMF (Pre-PMF) is an indolent form of PMF that frequently progresses to overt-PMF. While both stages of the disease are characterized by the presence of dysplastic megakaryocytes, progression to fulminant disease is associated with significantly increased fibrosis in the bone marrow. We have previously shown that megakaryocyte maturation in overt-PMF is impaired due to a GATA1 deficiency. However, in Pre-PMF patients most megakaryocytes express GATA1. This raises the possibility that alterations in megakaryocyte development occur during the progression of the disease and may contribute to fibrosis. This progression in megakaryocyte defects is likely driven not only by the aberrant JAK/STAT signaling but also microenvironmental factors. To identify such factors, we performed studies in two mouse models of PMF (driven by JAK2V617 and MPLW515L mutations) before and after development of fibrosis. Using an unbiased approach, we measured the levels of different cytokines in the bone marrow, plasma, and spleen. In addition, we performed single cell RNAseq in bone marrow populations. We observed extensive changes in the level of cytokines in the bone marrow of the MPLW515L mouse model compared to the JAK2V617F model. We initially focused on those cytokines that are elevated in the bone marrow of both murine models, including IL13 (Figure 1A), because previous studies have shown that IL13 is elevated in PMF patients and that JAK2 inhibitors do not decrease IL13 (1-3). Moreover, elevated IL13 has been identified in patients who progress to secondary AML (2). How IL13 may contribute to the progression of the disease has not been investigated. We assayed the effect of IL13 on megakaryocytes in vitro and discovered that it promoted megakaryocyte differentiation in the absence of thrombopoietin (TPO) and potentiated the effect of TPO. This effect was observed in cultures of both wild-type and MPLW515L megakaryocytes. Next, we assayed for expression of the IL13 receptor (Il13ra1) in the bone marrow of JAK2V617F and MPLW515L mutant mice and found that it was highly upregulated compared to wild-type animals. IL13ra1 expression was particularly intense in the megakaryocyte lineage, and its expression increased with disease progression (Figure 1B). Next, we asked whether IL13 is essential for myeloproliferative neoplasm (MPN) development in vivo. To study this, we transplanted bone marrow cells from Il4/13 f/f Mx1-Cre mice expressing MPLW515L to irradiated recipients, waited until MPN developed, and then excised by pIpC injection. This experiment revealed that loss of IL13 and IL4 led to a profound reduction in disease burden (Figure 1C), decreased splenomegaly, and diminished degree of bone marrow fibrosis. Moreover, loss of IL13 and IL4 decreased the levels of pro-inflammatory cytokines in the bone marrow and spleen (Figure 1D). We attribute this effect to deletion of IL13 because IL4 was only moderately increased in the bone marrow of the MPLW515L mouse model, and because IL4 has been reported to not be altered in the MPNs. Finally, we performed single cell RNA-seq on bone marrow cells from mice transplanted with JAK2V617F or control progenitor cells early and late in the disease process (Figure 1E). Our results revealed that there was decreased myeloid progenitors but an enhancement in the mast cell lineage that tracked with the degree of fibrosis. We confirmed the presence of elevated numbers of mast cells in the bone marrow by immunohistochemistry (Figure 1F). Mast cells produce IL13, and therefore they are the likely source for the increased IL13. Finally, consistent with the observation that IL13 signaling is primarily mediated through STAT6, we found enrichment of STAT6 target genes in megakaryocyte progenitors from the late timepoint in our scRNAseq data (Figure 1G). In summary, our data demonstrate that IL13 is involved in the progression of PMF and that inhibition of the IL13 signaling pathway should be investigated as a therapeutic option in PMF. 1. Tefferi A, et al. J Clin Oncol (2011) 2. Fisher DAC, et al. Leukemia (2019) 3. Chen P, et al. Front Med (2021) Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy.

A mayor pathobiological role for interleukin 8 in the etiology of myelofibrosis has been suggested by observations indicating that megakaryocytes expanded in culture from these patients express great levels of interleukin 8 1 and that the plasma levels of this cytokine are predictive of poor prognosis 2. In preliminary experiments we demonstrated that the megakaryocytes from the bone marrow of the Gata1 low model of myelofibrosis express not only high levels of TGF-β, but also levels greater than normal of lipokalin-2, a known inducer of IL-8 production, and of CXCL1, the murine equivalent of IL-8. In addition, these megakaryocytes express also high levels of the CXCL1 receptors CXCLR1 and CXCR2 and the bone marrow from these mice express an CXCR1/CXCR2 activated signature. Using these data as a foundation, we tested here the effects of treatment of Gata1 low mice with the CXCR1/R2 inhibitor reparixin on the myelofibrosis phenotype expressed by this models. To these aim, Gata1 low mice (8-month old) were treated either with vehicle (3 males and 3 females) or with reparixin (formerly referred to as repertaxin) 3 (5 males and 5 females) for either 20 or 37 days. The drug was administered by minipumps implanted subcutaneously in the dorsal region set to deliver 7.5mg of drug/hr/Kg of body weight. The mice receiving the drug for 37 days had the minipumps replaced by day 17. The efficiency of drug delivery decreased over time since the plasma levels of reparixin were 13.90±4.18 and 6.71±4.18ug/mL at day 20 and 37, respectively (p References: 1) Emadi S et al. Blood. 2005;105:464; 2) Tefferi et al, J Clin Oncol. 2011;29:1356; 3) Bertini R et al, PNAS 2004; 101:11791 Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Massucci: Dompe Farmaceutici Spa R&D: Current Employment. Brandolini: Dompe farmaceutici Spa R&D: Current Employment. Giorgio: Dompe farmaceutici Spa R&D: Current Employment. Allegretti: Dompe farmaceutici Spa R&D: Current Employment. Migliaccio: Dompe farmaceutici Spa R&D: Other: received funding for reserach .

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.

GATA family proteins play essential roles in development of many cell types, including hematopoietic, cardiac, and endodermal lineages. The first three factors, GATAs 1, 2, and 3, are essential for normal hematopoiesis, and their mutations are responsible for a variety of blood disorders. Acquired and inherited GATA1 mutations contribute to Diamond-Blackfan anemia, acute megakaryoblastic leukemia, transient myeloproliferative disorder, and a group of related congenital dyserythropoietic anemias with thrombocytopenia. Conversely, germ line mutations in GATA2 are associated with GATA2 deficiency syndrome, whereas acquired mutations are seen in myelodysplastic syndrome, acute myeloid leukemia, and in blast crisis transformation of chronic myeloid leukemia. The fact that mutations in these genes are commonly seen in blood disorders underscores their critical roles and highlights the need to develop targeted therapies for transcription factors. This review focuses on hematopoietic disorders that are associated with mutations in two prominent GATA family members, GATA1 and GATA2.

The myeloproliferative neoplasms (MPNs) are somatic mutation-driven hematologic malignancies characterized by bone marrow fibrosis and the accumulation of atypical megakaryocytes with reduced polyploidization in the primary myelofibrosis subtype of the MPNs. Increasing evidence points to a dominant role of abnormal megakaryocytes in disease initiation and progression. Here we review the literature related to kinase signaling pathways relevant to megakaryocyte differentiation and proliferation, including Aurora A kinase, RhoA/ROCK, and JAK/STAT, as well as the activities of their targeted inhibitors in models of the disease. Some of these pathway inhibitors selectively induce megakaryocyte differentiation, suppress malignant proliferation, and promote polyploidization and proplatelet formation. Moreover, combining sets of these inhibitors may be an effective approach to treat and potentially cure MPN patients. For example, preclinical studies reported significant synergistic effects of the combination of an Aurora A inhibitor and JAK1/2 inhibitor, in a murine model of the primary myelofibrosis. Future basic and clinical research into the contributions of these signaling pathways to aberrant megakaryopoiesis may lead to novel effective treatments for MPN patients.

Background: KMT2A-rearranged (R) ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in children and adults with ALL. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. We performed a kinome-wide CRISPR screen and identified DYRK1A as required for KMT2A-R ALL cell survival, but not in other high risk ALL genetic subtypes. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncoprotein in a murine Down syndrome model of megakaryoblastic leukemia. DYRK1A negatively regulates cell proliferation and induces quiescence. Paradoxically, genetic deletion or pharmacological inhibition of DYRK1A upregulates the cell cycle regulator CCND3 and increased numbers of B cells in S-phase, yet also significantly reduces cell proliferation. The specific role of DYRK1A in ALL has not been reported. Results: We assessed the importance of DYRK1A deletion in a focused screen of 14 previously identified kinases. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines and a human KMT2A-Aff1-FLAG transduced ALL model demonstrated direct binding of both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-fusion to the DYRK1A promoter. To assess if KMT2A fusion directly regulates DYRK1A expression, we treated SEM cells with the menin-KMT2A disrupter MI-503 and identified that the KMT2A fusion protein is a positive regulator of DYRK1A. Pharmacologic inhibition of DYRK1A with EHT1610 demonstrated potent leukemic cell growth inhibition, demonstrating that DYRK1 could be a new therapeutic target in KMT2A-R ALL. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which resulted in accumulation of CCND3 as expected. In addition, we detected upregulation of the positive cell cycle regulator MYC and the replication stress response molecule CHK1. In a second experiment, we validated the upregulation of MYC and identified significant upregulation of the proapoptotic protein BIM. Strikingly, meta-analysis of gene expression data from Dyrk1a-deleted murine pre-B cells isolated from a conditional Dyrk1a knockout mouse model also demonstrated increased levels of MYC and CHK1, validating that the EHT1610 mediated upregulation of MYC or CHK1 is a specific effect induced by DYRK1A inhibition. Western blot analysis demonstrated that KMT2A-R ALL cell lines have constitutive activation of pH2AX. Based on these data, we hypothesize that DYRK1A-mediated upregulation of CCND3 and MYC forces the cells to proliferate, which significantly increases replication stress and causes apoptosis, as evident by upregulation of CHK1 and BIM. To test if targeting the interaction of BIM with BCL2 will have an increased apoptotic effect when combined with EHT1610, we treated two KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, we observed a synergistic effect with both drugs, suggesting that combining these inhibitors has superior anti-leukemic activity. Conclusions: DYRK1A and MYC are positively regulated by the KMT2A fusion protein in KMT2A-R ALL and negatively regulate each other. Pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells due to increased MYC and CHK1 proteins that induce replication stress. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for KMT2A-R ALL that is translatable to the clinic for patients with these high-risk leukemias. Disclosures Tasian: Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Research Funding.

Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.

Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.

Children with Down syndrome (DS) are at a 20-fold increased risk for acute lymphoblastic leukemia (DS-ALL). Although the etiology of this higher risk of developing leukemia remains largely unclear, the recent identification of CRLF2 (cytokine receptor like factor 2) and JAK2 mutations and study of the effect of trisomy of Hmgn1 and Dyrk1a (dual-specificity tyrosine phosphorylation-regulated kinase 1A) on B-cell development have shed significant new light on the disease process. Here we focus on the clinical features, biology and genetics of ALL in children with DS. We review the unique characteristics of DS-ALL on both the clinical and molecular levels and discuss the differences in treatments and outcomes in ALL in children with DS compared with those without DS. The identification of new biological insights is expected to pave the way for novel targeted therapies.

Nearly 20% of patients with myelofibrosis progress to blast phase disease; an aggressive form of acute myeloid leukemia. Although previous studies have implicated loss of TP53 or JARID2 in progression, by and large the genetic events that lead to conversion to blast phase remain unknown. To identify genes whose loss drives progression, we performed a focused CRISPR/Cas9 screen in which murine Jak2V617F bone marrow cells expressing Cas9 were transduced with two separate sgRNA libraries of known tumor suppressor genes and subjected to colony replating assays. Transduction of one of the two libraries led to serial replating and enhanced self-renewal of the Jak2V617F cells. Subsequent DNA sequencing revealed enrichment of all four guides targeting STK11, the gene that encodes LKB1 which regulates a number of key cellular pathways including energy utilization by activation of AMPK. To confirm that loss of Stk11 is the event that leads to increased clonogenicity, we collected cells from Jak2V617F/Vav-Cre+ and control Vav-Cre+ mice and induced Stk11 knockout by electroporating Cas9-Stk11 sgRNA ribonucleoprotein complexes. Consistent with the screening results, only Jak2V617F Vav-Cre+ cells with Cas9-Stk11 sgRNA showed serial replating. To determine whether Stk11 is required for growth of cells with a different driver of enhanced JAK/STAT signaling, we doubly transduced Stk11 homozygous floxed bone marrow cells with MPLW515L-mCherry and Cre-GFP to delete Stk11. As expected, cells with both MPLW515L and Cre recombinase showed enhanced self-renewal, while singly infected control cells failed to replate. These results demonstrate that activation of JAK/STAT signaling can overcome the requirement for Stk11 in normal hematopoiesis and suggest that STK11 loss may be a strong driver of malignant transformation in combination with enhanced JAK-STAT signaling. We next investigated the mechanism by which loss of STK11 cooperates with enhanced JAK/STAT signaling to promote leukemia. RNA-sequencing of wild-type, Stk11+/+/ MPLW515L, and Stk11-/-/MPLW515L hematopoietic cells revealed enrichment of a number of pathways related to hypoxia, oxidative phosphorylation and mitochondrial translation in cells lacking LKB1. Western blot assays confirmed activation of mTOR signaling as well as HIF1a stabilization and pathway activation, both of which have been reported to lie downstream of LKB1 loss. We also performed a number of studies to determine the relevance of reduced LKB1 expression to leukemic progression. First, we induced deletion of Stk11 in mice that were transplanted with HSPCs expressing MPLW515L after development of the MPN phenotype. Loss of Stk11 caused a rapid lethality that was associated with enhanced bone marrow fibrosis and osteosclerosis. We also observed accumulation of leukemic blasts in small clusters consistent with AML transformation arising in the spent phase MPN. Additionally, we deleted STK11 by CRISPR/Cas9 in primary MPN patient samples and monitored their engraftment in immunocompromised mice. We observed enhanced engraftment and increased reticulin fibrosis and osteosclerosis in mice that received the STK11 edited cells compared to those with non-targeted sgRNA. Third, we compared the expression of STK11 in paired blast and chronic phase myelofibrosis patient samples by RT-PCR. Consistent with the hypothesis that loss of STK11 facilitates leukemia, we found that its expression was decreased by more than 50% in five of seven paired post-MPN AML patient samples, with two having STK11 levels below 20%. We further validated downregulation of LKB1 by immunohistochemistry on paired chronic and blast phase MPN specimens and observed little staining in the blast phase specimens. Finally, to further show that the mechanism of in vitro enhanced self-renewal is related to leukemia progression, we stained the paired marrows for HIF1a and saw a dramatic increase in staining at the AML phase. We also analyzed RNA-seq data of paired chronic versus blast phase MPNs specimens and observed that there is a strong congruence of enriched pathways that are associated with the in vitro mouse HSPC phenotype and the human blast phase progression, such as oxidative phosphorylation and hypoxia. Together, our study demonstrates that loss of LKB1/STK11 promotes transformation of cells with activated JAK/STAT signaling and that STK11 is a prominent candidate tumor suppressor gene in post-MPN AML. Disclosures Gurbuxani: UpToDate: Honoraria. Hoffman:Dompe: Research Funding; Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees. Levine:Astellas: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Rampal:Galecto: Consultancy; Incyte: Consultancy, Research Funding; Constellation: Research Funding; Stemline: Consultancy, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; CTI Biopharma: Consultancy; Abbvie: Consultancy; Pharmaessentia: Consultancy; Promedior: Consultancy; Blueprint: Consultancy.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by ineffective clonal hematopoiesis, splenomegaly, bone marrow fibrosis, and the propensity for transformation to acute myeloid leukemia. The discovery of mutations in JAK2, CALR, and MPL have uncovered activated JAK-STAT signaling as a primary driver of MF, supporting a rationale for JAK inhibition. However, JAK inhibition alone is insufficient for long-term remission and offers modest, if any, disease-modifying effects. Given this, there is great interest in identifying mechanisms that cooperate with JAK-STAT signaling to predict disease progression and rationally guide the development of novel therapies. This review outlines the latest discoveries in the biology of MF, discusses current clinical management of patients with MF, and summarizes the ongoing clinical trials that hope to change the landscape of MF treatment.

Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1A (DYRK1A) is a serine/threonine kinase that regulates diverse pathways such as splicing, cell cycle, differentiation, apoptosis, and transcription. DYRK1A is encoded within the Down syndrome (DS) critical region of chromosome 21, underlying its importance in DS-related pathologies, such as Alzheimer's disease. Children with DS have an increased risk of developing hematologic malignancies, namely acute megakaryoblastic leukemia (DS-AMKL) and B-cell acute lymphoblastic leukemia (DS-ALL). We previously reported that DYRK1A promotes DS-AMKL by regulating subcellular localization of its substrate NFAT. In a subsequent study, we examined its role in normal hematopoiesis and found that DYRK1A is necessary for B and T cell development through phosphorylation and destabilization of Cyclin D3. Dyrk1a-deficient large pre-B cells and double negative thymocytes are unable to enter quiescence for maturation. Despite elevated levels of Cyclin D3, however, these cells lose proliferative capacity due to a block at the G2-M transition. This observation suggests that DYRK1A inhibition may exhibit anti-tumor activity in lymphocytes by first stimulating exit from quiescence but then blocking repeated rounds of cell division. Notably, DYRK1A is overexpressed in acute leukemias, including both T-ALL and B-ALL, relative to normal hematopoietic counterparts. Moreover, overexpression of dominant-negative DYRK1A-K188R impairs proliferation in human B-ALL cell lines, suggesting that DYRK1A kinase activity is required for B-ALL growth. In order to assess the physiologic relevance of targeting DYRK1A in vivo, we generated a murine model of B-ALL with a floxed Dyrk1a allele and observed significant survival advantages with homozygous (p=0.0045) and heterozygous deletion (p=0.0015). Additionally, both B-ALL cell lines and patient samples were sensitive to EHT1610, a potent and selective DYRK1 inhibitor. Relevant to the localization of DYRK1A on chromosome 21, DS-ALL samples were especially sensitive to kinase inhibition. EHT1610 also conferred synergistic growth inhibition of B-ALL cells when combined with cytotoxic chemotherapy drugs used in traditional ALL treatment regimens, such as dexamethasone, methotrexate and cytarabine. We next aimed to elucidate the mechanism by which DYRK1A inhibition cause a failure of G2-M progression. Using global and directed phosphoproteomic studies, we identified several DYRK1A substrates in pre-B cells that are involved in cell cycle, splicing, transcriptional regulation, and RNA metabolism. In addition to Cyclin D3, a notable substrate is FOXO1, an indispensable transcription factor in B lymphopoiesis. We observed that inhibition of DYRK1A led to an accumulation of FOXO1 in the nucleus of large pre-B cells despite intact PI3K/Akt signaling, which is the predominant negative regulator of FOXO1. Treatment of pre-B cells with AS1842856, an inhibitor of FOXO1 nuclear translocation, rescued the G2-M blockade and proliferative impairment induced by EHT1610 treatment. Despite FOXO1 acting as a tumor suppressor in normal lymphocytes, B-ALL cell lines and patient samples were paradoxically sensitive to FOXO1 inhibition, suggesting a unique requirement in the survival of B-ALL cells. This may be due to regulation of DNA damage, as DYRK1A inhibition alone led to negligible changes in gamma-H2AX foci, whereas FOXO1 inhibition increased DNA damage. When DYRK1A and FOXO1 were inhibited in combination, we observed a synergistic accumulation of DNA damage along with cell death in B-ALL cell lines. Finally, as both EHT1610 and AS1842856 are potent inhibitors of B-ALL cell growth in vitro, we assessed their in vivo efficacy. Both EHT1610 and AS1842856 significantly increased survival in xenograft models of B-ALL (p=0.0002 and p=0.001, respectively). We therefore conclude that both DYRK1A and its substrate FOXO1 are therapeutic targets in B-ALL. Importantly, EHT1610 represents the first selective DYRK1A inhibitor with suitable in vivo activity. Ultimately, we have determined that the DYRK1A pathway is integral to the maintenance of normal and malignant B-lymphopoiesis, the latter which can be effectively targeted through 1) a primary proliferative impairment, 2) sensitization to cell cycle-dependent chemotherapy, and 3) downstream inhibition of DYRK1A substrates such as FOXO1. Disclosures Lee: AbbVie: Employment. Bourquin:Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Ras mutations are commonly observed in juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). JMML and CMML transform into acute myeloid leukemia (AML) in about 10% and 50% of patients, respectively. However, how additional events cooperate with Ras to promote this transformation are largely unknown. We show that absence of the ubiquitin-specific peptidase 22 (USP22), a component of the Spt-Ada-GCN5-acetyltransferase chromatin-remodeling complex that is linked to cancer progression, unexpectedly promotes AML transformation in mice expressing oncogenic KrasG12D/+. USP22 deficiency in KrasG12D/+ mice resulted in shorter survival compared with control mice. This was due to a block in myeloid cell differentiation leading to the generation of AML. This effect was cell autonomous because mice transplanted with USP22-deficient KrasG12D/+ cells developed an aggressive disease and died rapidly. The transcriptome profile of USP22-deficient KrasG12D/+ progenitors resembled leukemic stem cells and was highly correlated with genes associated with poor prognosis in AML. We show that USP22 functions as a PU.1 deubiquitylase by positively regulating its protein stability and promoting the expression of PU.1 target genes. Reconstitution of PU.1 overexpression in USP22-deficient KrasG12D/+ progenitors rescued their differentiation. Our findings uncovered an unexpected role for USP22 in Ras-induced leukemogenesis and provide further insights into the function of USP22 in carcinogenesis.

Acute myeloid leukemia (AML) patients with NPM1 mutations demonstrate a superior response to standard chemotherapy treatment. Our previous work has shown that these favorable outcomes are linked to the cytoplasmic relocalization and inactivation of FOXM1 driven by mutated NPM1. Here, we went on to confirm the important role of FOXM1 in increased chemoresistance in AML. A multiinstitution retrospective study was conducted to link FOXM1 expression to clinical outcomes in AML. We establish nuclear FOXM1 as an independent clinical predictor of chemotherapeutic resistance in intermediate-risk AML in a multivariate analysis incorporating standard clinicopathologic risk factors. Using colony assays, we show a dramatic decrease in colony size and numbers in AML cell lines with knockdown of FOXM1, suggesting an important role for FOXM1 in the clonogenic activity of AML cells. In order to further prove a potential role for FOXM1 in AML chemoresistance, we induced an FLT3-ITD–driven myeloid neoplasm in a FOXM1-overexpressing transgenic mouse model and demonstrated significantly higher residual disease after standard chemotherapy. This suggests that constitutive overexpression of FOXM1 in this model induces chemoresistance. Finally, we performed proof-of-principle experiments using a currently approved proteasome inhibitor, ixazomib, to target FOXM1 and demonstrated a therapeutic response in AML patient samples and animal models of AML that correlates with the suppression of FOXM1 and its transcriptional targets. Addition of low doses of ixazomib increases sensitization of AML cells to chemotherapy backbone drugs cytarabine and the hypomethylator 5-azacitidine. Our results underscore the importance of FOXM1 in AML progression and treatment, and they suggest that targeting it may have therapeutic benefit in combination with standard AML therapies., Nuclear FOXM1 confers resistance to standard acute myeloid leukemia chemotherapy and may represent a potential therapeutic target.

Introduction: GATA1, the founding member of a family of transcription factors, plays important roles in the development of hematopoietic cells of several lineages. Although loss of GATA1 has been known to impair hematopoiesis in animal models for nearly 25years, the link between GATA1 defects and human blood diseases has only recently been realized. Areas covered: Here the current understanding of the functions of GATA1 in normal hematopoiesis and how it is altered in disease is reviewed. GATA1 is indispensable mainly for erythroid and megakaryocyte differentiation. In erythroid cells, GATA1 regulates early stages of differentiation, and its deficiency results in apoptosis. In megakaryocytes, GATA1 controls terminal maturation and its deficiency induces proliferation. GATA1 alterations are often found in diseases involving these two lineages, such as congenital erythroid and/or megakaryocyte deficiencies, including Diamond Blackfan Anemia (DBA), and acquired neoplasms, such as acute megakaryocytic leukemia (AMKL) and the myeloproliferative neoplasms (MPNs). Expert commentary: Since the first discovery of GATA1 mutations in AMKL, the number of diseases that are associated with impaired GATA1 function has increased to include DBA and MPNs. With respect to the latter, we are only just now appreciating the link between enhanced JAK/STAT signaling, GATA1 deficiency and disease pathogenesis.

Children with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS.

GATA1 is a master transcriptional regulator of the differentiation of several related myeloid blood cell types, including erythrocytes and megakaryocytes. Germ-line mutations that cause loss of full length GATA1, but allow for expression of the short isoform (GATA1s), are associated with defective erythropoiesis in a subset of patients with Diamond Blackfan Anemia. Despite extensive studies of GATA1s in megakaryopoiesis, the mechanism by which GATA1s fails to support normal erythropoiesis is not understood. In this study, we used global gene expression and chromatin occupancy analysis to compare the transcriptional activity of GATA1s to GATA1. We discovered that compared to GATA1, GATA1s is less able to activate the erythroid gene expression program and terminal differentiation in cells with dual erythroid-megakaryocytic differentiation potential. Moreover, we found that GATA1s bound to many of its erythroid-specific target genes less efficiently than full length GATA1. These results suggest that the impaired ability of GATA1s to promote erythropoiesis in DBA may be caused by failure to occupy erythroid-specific gene regulatory elements.

Most heritable anemias are caused by mutations in genes encoding globins, red blood cell (RBC) membrane proteins, or enzymes in the glycolytic and hexose monophosphate shunt pathways. A less common class of genetic anemia is caused by mutations that alter the functions of erythroid transcription factors (TFs). Many TF mutations associated with heritable anemia cause truncations or amino acid substitutions, resulting in the production of functionally altered proteins. Characterization of these mutant proteins has provided insights into mechanisms of gene expression, hematopoietic development, and human disease. Mutations within promoter or enhancer regions that disrupt TF binding to essential erythroid genes also cause anemia and heritable variations in RBC traits, such as fetal hemoglobin content. Defining the latter may have important clinical implications for de-repressing fetal hemoglobin synthesis to treat sickle cell anemia and β thalassemia. Functionally important alterations in genes encoding TFs or their cognate cis elements are likely to occur more frequently than currently appreciated, a hypothesis that will soon be tested through ongoing genome-wide association studies and the rapidly expanding use of global genome sequencing for human diagnostics. Findings obtained through such studies of RBCs and associated diseases are likely generalizable to many human diseases and quantitative traits.

The transcription factor “nuclear factor erythroid 2” (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2(V617F)-positive cell lines.

Myelodysplastic syndrome (MDS) is a heterogenous myeloid lineage malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of secondary transformation to acute myeloid leukemia (sAML). Genomic sequencing of large MDS cohorts has led to the identification of recurrent genetic abnormalities that carry independent prognostic significance and overlap with mutational changes in sAML. However, no set of mutations is sufficient to predict the transformation of MDS raising the question of how an identical genotype produces MDS in one patient and sAML in another? We hypothesize there are therapeutically targetable cellular processes altered by the initiating genetic changes in MDS that predict transformation to sAML. To uncover novel cellular pathways involved in MDS transformation, we performed an unbiased genome-wide CRISPR/Cas9 in the human MDS-L cell line. MDS-L was established from bone marrow mononuclear cells in a 52-year-old male patient and requires IL3-containing media for growth in vitro (Figure 1A). GFP expressing MDS-L cells were transduced with Cas9 and a sgRNA against GFP to confirm functional Cas9 expression in MDS-L (Figures 1B and C). In Cas9 expressing MDS-L cells, we then transduced the Brunello sgRNA CRISPR library and subjected the cells to IL-3 starvation for 4 weeks. Cells surviving IL-3 starvation were then expanded and harvested for genomic DNA. High throughput sequencing of the barcoded DNA produced raw reads that were analyzed using the PinAPL-Py web-based software. sgRNAs appearing in duplicate with absolute read counts over 1000 or in triplicate over 100 were considered significant. We identified 5 genes that conferred resistance to IL-3 starvation, which included FBXO11 (Figure 1D). The Fbox protein FBXO11 is a component of the SCF ubiquitin ligase complex and regulates its substrates via ubiquitination and proteasomal degradation. FBXO11 is mutated in up to 20% of diffuse large B-cell lymphomas and its loss in breast cancer models leads to increased metastases. Therefore, we hypothesized FBXO11 may also function as a tumor suppressor in the transformation of MDS to AML. We confirmed in the Bloodspot gene expression database that there are decreased levels of FBXO11 in a variety of AML samples, including complex karyotype, compared to normal HSCs. To validate the results of the screen, we synthesized two sgRNAs targeting FBXO11, transduced these into MDS-L cells, and detected reduced FBXO11 expression (Figure 1E). Loss of FBXO11 expression promoted survival in IL-3 free media, confirming the selection readout of the screen (Figure 1F). We then designed a silent mutation in the shorter isoform of FBXO11 (FBXO11v1sm1) that rendered resistance to CRISPR/Cas9 (Figure 1G) and observed that overexpression of FBXO11v1sm1 re-sensitized cells to cytokine starvation (Figure 1H). Whether there are different functions between FBXO11 variant 1 and 4 are currently being explored. We are actively performing RNA sequencing and ubiquitin proteomics in FBXO11 knockout cells to identify its downstream targets and assaying for reduced expression of FBXO11 in primary patient MDS and AML samples. Based on our studies, we predict that SCF ubiquitin ligase component FBXO11 is a tumor suppressor regulating the transformation of MDS to secondary AML. Figure 1 Disclosures Crispino: Sierra Oncology: Consultancy; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

Acute megakaryoblastic leukemia (AMKL) is more frequently observed in Down syndrome (DS) patients, in whom it is often preceded by a transient myeloproliferative disorder (TMD). The development of DS-TMD and DS-AMKL requires not only the presence of the trisomy 21 but also that of GATA1 mutations. Despite extensive studies into the genetics of DS-AMKL, the importance of epigenetic deregulation in this disease has been unexplored. We performed DNA methylation profiling at different stages of development of DS-AMKL and analyzed the dynamics of the epigenetic program. Early genome-wide DNA methylation changes can be detected in trisomy 21 fetal liver mononuclear cells, prior to the acquisition of GATA1 mutations. These early changes are characterized by marked loss of DNA methylation at genes associated with developmental disorders, including those affecting the cardiovascular, neurological, and endocrine systems. This is followed by a second wave of changes detected in DS-TMD and DS-AMKL, characterized by gains of methylation. This new wave of hypermethylation targets a distinct set of genes involved in hematopoiesis and regulation of cell growth and proliferation. These findings indicate that the final epigenetic landscape of DS-AMKL is the result of sequential and opposing changes in DNA methylation occurring at specific times in the disease development.

We used high resolution mate-pair sequencing (HRMPS) in 15 patients with primary myelofibrosis (PMF): eight with normal karyotype and seven with PMF-characteristic cytogenetic abnormalities, including der(6)t(1;6)(q21–23;p21.3) (n = 4), der(7)t(1;7)(q10;p10) (n = 2), del(20)(q11.2q13.3) (n = 3), and complex karyotype (n = 1). We describe seven novel deletions/translocations in five patients (including two with normal karyotype) whose breakpoints were PCR-validated and involved MACROD2, CACNA2D4, TET2, SGMS2, LRBA, SH3D19, INTS3, FOP (CHTOP), SCLT1, and PHF17. Deletions with breakpoints involving MACROD2 (lysine deacetylase; 20p12.1) were recurrent and found in two of the 15 study patients. A novel fusion transcript was found in one of the study patients (INTS3-CHTOP), and also in an additional non-study patient with PMF. In two patients with der(6)t(1;6)(q21–23;p21.3), we were able to map the precise translocation breakpoints, which involved KCNN3 and GUSBP2 in one case and HYDIN2 in another. This study demonstrates the utility of HRMPS in uncovering submicroscopic deletions/translocations/fusions, and precise mapping of breakpoints in those with overt cytogenetic abnormalities. The overall results confirm the genetic heterogeneity of PMF, given the low frequency of recurrent specific abnormalities, identified by this screening strategy. Currently, we are pursuing the pathogenetic relevance of some of the aforementioned findings. Am. J. Hematol. 88:741–746, 2013. © 2013 Wiley Periodicals, Inc.

The requirement that leukemic Gata1 mutations be present in cells harboring trisomy 21 led to the discovery that overexpression of ERG drives aberrant megakaryopoiesis. Given that constitutive PI3K/AKT signaling is a frequent component of hematologic malignancies and the relationship between AKT and Notch in this lineage, we studied the crosstalk between AKT signaling and ERG in megakaryopoiesis. We discovered that constitutive AKT signaling is associated with a dramatic increase in apoptosis of WT megakaryocytes (MKs), but that overexpression of ERG blocks AKT-induced death. We further found that Gata1 mutations protect MKs from activated AKT-induced apoptosis. As a consequence, however, the enhanced signaling inhibits differentiation of Gata1 mutant, but not WT, MKs. Gata1 mutant cells that overexpress ERG with hyperactive AKT are characterized by diminished FOXO1/3a expression and an increased dependency on the c-Jun pathway similar to that seen in acute megakaryoblastic leukemia (AMKL) cell lines, acute myeloid leukemia (AML) with knockdown of FOXO3a, or AML with expression of myristoylated Akt. Additionally, we found that the AKT allosteric inhibitor MK2206 caused reduced cell viability and proliferation of AMKL cell lines. The contribution of aberrant AKT signaling during the ontogeny of Down syndrome-transient myeloproliferative disorder/AMKL indicates that AKT is a therapeutic target in this form of AML.

Megakaryocytes are among the largest and rarest cells in the body, accounting for approximately 0.01% of nucleated cells in the bone marrow. Their differentiation involves a progression from hematopoietic stem cell to the megakaryocyte progenitor and finally to platelets.[1][1] A key step in their

Imetelstat (GRN163L) is a specific telomerase inhibitor that has demonstrated clinical activity in patients with myeloproliferative neoplasms (MPN) and in patients with solid tumors. The antitumor effects were associated with the development of thrombocytopenia, one of the common side effects observed in patients treated with imetelstat. The events underlying these adverse effects are not apparent. In this report, we investigated the potential mechanisms that account for imetelstat's beneficial effects in MPN patients and the manner by which imetelstat treatment leads to a reduction in platelet numbers. Using a well-established system of ex vivo megakaryopoiesis, we demonstrated that imetelestat treatment affects normal megakaryocyte (MK) development by exclusively delaying maturation of MK precursor cells. By contrast, additional stages along MPN MK development were affected by imetelstat resulting in reduced numbers of assayable colony-forming unit MK and impaired MK maturation. In addition, treatment with imetelstat inhibited the secretion of fibrogenic growth factors by malignant but not by normal MK. Our results indicate that the delay observed in normal MK maturation may account for imetelstat-induced thrombocytopenia, while the more global effects of imetelstat on several stages along the hierarchy of MPN megakaryopoiesis may be responsible for the favorable clinical outcomes reported in MPN patients.

Every 2 years since 1978, an international group of scientists, physicians, and other researchers meet to discuss the latest developments in the underlying etiology, mechanisms of action, and developmental acquisition of cellular and systemic defects exhibited and elicited by the most common inherited human disorders, the hemoglobinopathies. The 19th Hemoglobin Switching Conference, held in September 2014 at St. John's College in Oxford, once again exceeded all expectations by describing cutting edge research in cellular, molecular, developmental, and genomic advances focused on these diseases. The conference comprised about 60 short talks over 3 days by leading investigators in the field. This meeting report describes the highlights of the conference.

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal as this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of polycomb repressive complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying that both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations.

There are many examples of transcription factor families whose members control gene expression profiles of diverse cell types. However, the mechanism by which closely related factors occupy distinct regulatory elements and impart lineage specificity is largely undefined. Here we demonstrate on a genome wide scale that the hematopoietic GATA factors GATA-1 and GATA-2 bind overlapping sets of genes, often at distinct sites, as a means to differentially regulate target gene expression and to regulate the balance between proliferation and differentiation. We also reveal that the GATA switch, which entails a chromatin occupancy exchange between GATA2 and GATA1 in the course of differentiation, operates on more than one-third of GATA1 bound genes. The switch is equally likely to lead to transcriptional activation or repression; and in general, GATA1 and GATA2 act oppositely on switch target genes. In addition, we show that genomic regions co-occupied by GATA2 and the ETS factor ETS1 are strongly enriched for regions marked by H3K4me3 and occupied by Pol II. Finally, by comparing GATA1 occupancy in erythroid cells and megakaryocytes, we find that the presence of ETS factor motifs is a major discriminator of megakaryocyte versus red cell specification.

Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that inhibition of survivin in patient-derived ALL can eradicate leukemia. Targeting survivin with shRNA in combination with chemotherapy resulted in no detectable minimal residual disease in a xenograft model of primary ALL. Similarly, pharmacologic knock-down of survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide, in combination with chemotherapy eliminated drug-resistant ALL cells. These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL.

Erythroid cells and megakaryocytes are derived from a common precursor, the megakaryocyte-erythroid progenitor. Although these 2 closely related hematopoietic cell types share many transcription factors, there are several key differences in their regulatory networks that lead to differential gene expression downstream of the megakaryocyte-erythroid progenitor. With the advent of next-generation sequencing and our ability to precisely define transcription factor chromatin occupancy in vivo on a global scale, we are much closer to understanding how these 2 lineages are specified and in general how transcription factor complexes govern hematopoiesis.

Myeloid leukemia of Down syndrome (ML-DS) is a tractable human model of acute myeloid leukemia. A preleukemia phase, transient abnormal myelopoiesis (TAM) and silent TAM, occurs in 28% of neonates with Down Syndrome (Roberts et al. Blood 2013). TAM is caused by trisomy 21 and acquired mutations in GATA1 that result in a N-terminal truncated protein, GATA1s, in hematopoietic stem and progenitor cells (HSPCs) of fetal origin. ML-DS evolves from TAM by acquisition of additional genetic lesions. The nature of these lesions and the mechanism of transformation are incompletely understood. We performed exome sequencing and targeted resequencing of 141 ML-DS and 111 TAM patients to characterize the evolving mutational landscape from TAM to ML-DS. On average 1.6 acquired mutations were detected in ML-DS (in addition to GATA1 mutations), significantly more than in TAM (0.4 mutations per sample). Additional anticipated loss-of-function mutations acquired in ML-DS mainly affected cohesin components including CTCF (43% of patients), PRC2 components (13%), KANSL1 and other epigenetic regulators (14%). Conversely, anticipated gain-of-function mutations were most prevalent in signaling pathways, e.g. JAK kinases, MPL, KIT and RAS family members (40%). Importantly, we detected a novel recurrent hotspot mutation in 4% of patients (6/141 cases) in CSF2RB encoding the IL3-, IL5-, GM-CSF-receptor common beta chain. To test if the A455D/T variant in the CSF2RB transmembrane domain is a putative oncogenic driver, we ectopically expressed CSF2RBA455D in TF1 cells. Cells expressing CSF2RBA455D exhibited cytokine independent growth and STAT5 autonomous phosphorylation. In a CD34+-HSPC megakaryocytic differentiation assay, CSF2RBA455D blocked terminal megakaryocytic differentiation whilst increasing proliferation by 30-fold (P=0.046). Moreover, the median survival of NSG mice transplanted with CSF2RBA455DTF1 cells was shortened by 30 days compared to wild type TF1 cells (23 days compared to 53 days, P=0.0097). To experimentally test the potential of loss-of-function mutations to transform TAM to ML-DS, we performed an in vivo murine isogenic transplantation screen using Gata1s expressing fetal hematopoietic cells from Cas9-knockin mice. We tested variants in 22 genes, recurrently detected in ML-DS, with a pool of prevalidated gRNAs. This resulted in short latency (n=18 mice; median survival 36 days) and high penetrance (100%) leukemia. Leukemia was not detected in mice infected with control gRNAs. Leukemias had a typical ML-DS megakaryoblastic phenotype (CD117+ and CD41a+). Amplicon sequencing revealed on average 2.9 gRNAs per leukemia and high representation (61% of all leukemias) of gRNAs directed to the tumor suppressor Trp53, which was alone sufficient to induce leukemia with 100% penetrance. When excluding the Trp53 gRNA from pools, leukemic cells from moribund mice contained gRNAs against negative regulators of the RAS and JAK-STAT signaling cascade, such as Nf1, Cbl and Sh2b3 (70% of the mice), Ezh2, Asxl1, Kdm6a,Bcor and other epigenetic modifiers (85%) or Ctcf (15%), closely resembling the mutational landscape of ML-DS. In contrast to ML-DS, gRNAs targeting cohesion components, such as Rad21 and Stag2, were not present in any of the leukemias. In summary, we performed the largest genetic analysis of transforming events in ML-DS that cooperate with trisomy 21 and GATA1s and uncovered a previously undescribed activating mutation in CSR2B. We experimentally validated many of the loss-of-function mutations in a novel murine fetal leukemia assay for ML-DS. The field is now well-placed to study mechanisms of oncogenic cooperativity and identify novel therapeutic approaches for this leukemia. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Mutations in GATA1 are seen in rare cases of dyserythropoietic anemia and in a subset of patients with Diamond Blackfan Anemia (DBA). Of note the truncation mutations in DBA, known as GATA1s, closely resemble those that are more commonly associated with acute megakaryoblastic leukemia in children with Down syndrome (DS). Studies with a mouse model of the Gata1s mutation revealed that replacement of the full-length protein by the shortened isoform led to a marked yet transient enhancement in megakaryopoiesis, similar in some respects to transient myeloproliferative disorder in DS. Furthermore, these mutant mice displayed impaired embryonic erythropoiesis but ostensibly no defects in adult hematopoiesis. In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in the Gata1s mouse strain. We observed a striking impairment in erythropoiesis in fetuses at E10.5 though E12.5, but saw improvement as the animals progressed through E14.5 and beyond. Defects included impaired terminal maturation and reduced numbers of erythroid progenitors, likely at the expense of expanded megakaryopoiesis. RNA-sequencing revealed that both erythroid genes and megakaryocytic genes were altered by the Gata1s mutation. Epiproteomic histone modification analysis further revealed there was an accumulation of H3K27 methylation in the R3 (CD71hiTer119hi) erythroid progenitor population, which suggests that GATA1 has a link to the epigenetic machinery that is altered in Gata1s mutant cells. Despite a global increase in H3K27me3, critical Gata2 regulatory elements in Gata1s mutant erythroid progenitors were marked by substantially less H3K27me3 than in wild-type littermates. Given that overexpression of GATA2 has been reported to impair erythropoiesis, we investigated whether reducing the GATA2 levels would ameliorate the phenotype. Indeed, we observed that haploinsufficiency for Gata2 rescued the erythroid defects of Gata1s fetuses. Next, to comprehensively study the effect of absence of the GATA1 N-terminus genome-wide, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with H3K27me3, GATA1 or GATA1s antibodies on wild-type versus Gata1s expressing fetal erythroid cells. Our data indicated that there is a substantial reduction in H3K27me3 along regulatory elements of the Runx1 gene at the late stage (R3) of fetal erythropoiesis in Gata1s mice. Along with an increase in Runx1 expression we observed strong downregulation of Klf1, a repressive target of RUNX1. Thus, failure of GATA1s to facilitate trimethylation of Runx1 and Gata2 regulatory elements appears to cause the defects in erythroid cell and megakaryocyte development. In parallel, we performed an in-depth analysis of the phenotype of adult Gata1s mice and discovered that they have reduced red cell counts, lower hemoglobin and hematocrit, increased extramedullary hematopoiesis and impaired stress erythropoiesis compared to control littermates. Although there were significantly more megakaryocyte erythrocyte progenitors (MEPs, Lin-c-Kit+Sca-1-CD34-FcgR-) in Gata1s mouse bone marrow, there were fewer pre-colony-forming unit erythroid cells (preCFU-E, Lin-c-Kit+Sca-1-CD41-FcgR-CD150hiCD105hi), likely at the expense of expanded megakaryocyte progenitors (MkP, Lin-c-Kit+Sca-1-CD41+CD150hi).Gata1s mice also developed an MDS-like disease with age. Together, our integrated genomic analysis of transcriptome, GATA1/GATA1s chromatin binding profile and chromatin signature reveal that, although Gata1s mice do not precisely model DBA, they provide novel insights into the role of the N-terminus of GATA1 in gene transcriptional regulation, lineage determination and red blood cell maturation. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that mediates signal transduction from the extracellular surface to the nucleus. Canonically, STAT3 is phosphorylated at Tyrosine 705 (Y705) by JAK family kinases, which promotes its dimerization and subsequent localization to the nucleus. However, the role of Serine 727 (S727) phosphorylation in regulating STAT3 activity varies across cell types and remains unclear in hematopoietic tissues particularly. Several studies indicate that phosphorylation at S727 is critical for optimal STAT3 function. For example, astrogliogenesis is regulated by enhancing STAT3 activity by phosphorylation of S727 by DYRK1A. Of note, DYRK1A is overexpressed in Down syndrome-acute lymphoblastic leukemia (DS-ALL), and has previously been found to phosphorylate substrates in order to prime them for downstream phosphorylation events. Given these findings, we hypothesized that the DYRK1A phosphorylation of STAT3 at S727 is critical for promoting DS-ALL. Furthermore, certain subtypes of ALL have high rates of JAK2 activation, namely DS-ALL and Philadelphia-like ALL (Ph-like ALL); we propose that STAT3 can effectively be targeted specifically in these subtypes. In order to elucidate the role of DYRK1A phosphorylation of STAT3, we treated cytokine-deprived murine pre-B cells with EHT1610, a selective DYRK1 inhibitor, or vehicle and then pulsed the cells with JAK-STAT activating cytokines. EHT1610-treated cells had diminished S727 phosphorylation compared to vehicle, regardless of cytokine pulse; however, only vehicle-treated cells regained Y705 phosphorylation after cytokine pulse. This suggests that S727 phosphorylation is cytokine-independent and is critical for maintenance of Y705 phosphorylation. We then generated flag-tagged STAT3 S727 phospho-mimetic (S727D/E) and phospho-deficient (S727A) alleles and transduced them into pre-B cells. We observed that the degree of Y705 phosphorylation is dependent on S727, as cells expressing S727A have reduced Y705 phosphorylation compared to wild-type STAT3. Additionally, overexpression of the phospho-deficient allele conferred a significant proliferative impairment compared to the phospho-mimetic alleles. As DS-ALL and Ph-like ALL often have JAK2-activating mutations, we next aimed to determine if loss of S727 phosphorylation would decrease ALL cell growth. Indeed, two human Ph-like ALL cell lines, MHH-CALL4 and MUTZ5, displayed decreased proliferation when overexpressing the S727A mutant. These cell lines were also sensitive to treatment with C188-9, a small molecule STAT3 inhibitor that is in clinical trials for various solid tumors. Additionally, we treated primary patient ALL samples with amplification of HSA21 segments ex vivo and found that DS-ALL samples were preferentially sensitive to STAT3 inhibition compared to HD-ALL or iAMP-ALL, suggesting that STAT3 is specifically a target in JAK2-activated ALL. Our study provides new and significant insights into the regulation of STAT3 by DYRK1A, and presents a new therapeutic target for ALL cells with JAK2 activating mutations. Disclosures Bourquin: Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.