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Summary: Faithful recapitulation of human bone marrow complexity has been a major challenge for the sci­entific community for many years. In this issue of Cancer Discovery, Khan and colleagues present an improved induced pluripotent stem cell differentiation protocol that generates bone marrow organoids re-creating key characteristics of human marrow. See related article by Khan et al., p. 364 (8).

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B–selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein–expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3′-deoxy-3′-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)–CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

Supplementary Figure from LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms

Supplementary Figure S1 shows the cellular effects of AKIs and anti-leukemic agents across a panel of human AML cell lines. Supplementary Figure S2 shows expression of aurora-A and aurora-B protein levels in four AML cell lines. Supplementary Figure S3 shows AMG 900 induces a dose-dependent increase in polyploidy, apoptosis, and p53 protein levels in MOLM-13 cells. Supplementary Figure S4 shows AMG 900 plus Ara-C combination matrix and CI determination in MOLM-13 cells. Supplementary Figure S5 shows AMG 900 induced apoptosis is attenuated by peptide inhibitors of caspases in MOLM-13 cells. Supplementary Figure S6 shows FC gating strategy for annexin-V coupled JC-1 assay. Supplementary Figure S7 shows the anti-proliferative effects of AMG 900 and AZD1152-hQPA on primary human bone marrow mononuclear cells in culture. Supplementary Figure S8 shows a moderate reduction in mouse body weight after AMG 900 treatment.

Supplementary Table from LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs.Significance:Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.This article is highlighted in the In This Issue feature, p. 1307

Efficacy of conventional chemotherapeutic agents alone or in combination with Trametinib in PDX B-ALL cells in vitro

Material and Methods, Figure legends

Purpose:The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear.Experimental Design:We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage–committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN.Results:JAK2V617F-mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis.Conclusions:Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Supplementary Table 1

Supplementary Table 2

List of the SNVs identified through RNA-sequencing

Differentially expressed genes in the human B-ALL cohort

Characteristics of the B-ALL cohort

Supplementary figures and figure legends

Reagents and Resources

Supplementary Figs. 1-17 with legends

Purpose:Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis.Patients and Methods:Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis.Results:In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients.Conclusions:Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis.See related commentary by Piszczatowski and Steidl, p. 4868

Figure S1: Alisertib treatment did not result in a consistent cytokine response. Figure S2: Alisertib did not have a consistent effect on TGF-b levels. Figure S3: Alisertib induced GATA1 expression in the SET2 megakaryocytic cell line. Figure S4: AURKA inhibition induces megakaryocytic gene expression. Figure S5: Alisertib increases GATA1 expression in the bone marrow of patients on therapy. Table S1: Correlation of Genotype and prior JAK inhibitor therapy with response and adverse events Table S2: Summary of Treatment Related Adverse Events Table S3: Association between GATA1 staining, degree of fibrosis and clinical response

Purpose:Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL.Experimental Design:To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents.Results:Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRASG12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine.Conclusions:Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia.

List of the SNVs identified through whole exome sequencing

List of fusion transcripts identified through RNA-sequencing

Purpose:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes.Experimental Design:To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models.Results:We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo.Conclusions:These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.

List of datasets enriched in murine B cell precursors (GSEA analyses)

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.

KMT2A-rearranged (KMT2A-R) B cell acute lymphoblastic leukemia (ALL) is a high-risk disease in children and adults that is often chemotherapy resistant. To identify non-cytotoxic approaches to therapy, we performed a domain-specific kinome-wide CRISPR screen in KMT2A-R cell lines and patient derived xenograft samples (PDX) and identified dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a potential target. Pharmacologic inhibition of the KMT2A-fusion transcriptional co-regulator Menin released the KMT2A-fusion complex from the DYRK1A promoter thereby lowering DYRK1A expression levels confirming DYRK1A as a direct target of the KMT2A fusion oncogene. Direct pharmacologic inhibition of DYRK1A decreased cell proliferation of KMT2A-R ALL, thereby confirming the requirement of DYRK1A in this ALL subtype. To further understand the biologic function of DYRK1A in KMT2A-R ALL, we leveraged pharmacologic DYRK1A inhibitors in KMT2A-R PDX and cell line models. DYRK1A inhibition consistently led to upregulation of MYC protein levels, and hyperphosphorylation of ERK, which we confirmed via in vivo treatment experiments. Furthermore, DYRK1A inhibition decreased ALL burden in mice. Our results further demonstrate that DYRK1A inhibition induces the proapoptotic factor BIM, but ERK hyperphosphorylation is the driving event that induces cell cycle arrest. In contrast, combined treatment of KMT2A-R ALL cells in vitro and in vivo with DYRK1A inhibitors and the BCL2 inhibitor, venetoclax, synergistically decreases cell survival and reduced the leukemic burden in mice. Taken together these results demonstrate a unique function of DYRK1A specially in KMT2A-R ALL. Synergistic inhibition of DRYK1A and BCL2 may provide a low-toxic approach to treat this high risk ALL subtype.

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.

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. Significance: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression. This article is highlighted in the In This Issue feature, p. 1307

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.

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) is a serine/threonine kinase that belongs to the DYRK family of proteins, a subgroup of the evolutionarily conserved CMGC protein kinase superfamily. Due to its localization on chromosome 21, the biological significance of DYRK1A was initially characterized in the pathogenesis of Down syndrome (DS) and related neurodegenerative diseases. However, increasing evidence has demonstrated a prominent role in cancer through its ability to regulate biologic processes including cell cycle progression, DNA damage repair, transcription, ubiquitination, tyrosine kinase activity, and cancer stem cell maintenance. DYRK1A has been identified as both an oncogene and tumor suppressor in different models, underscoring the importance of cellular context in its function. Here, we review mechanistic contributions of DYRK1A to cancer biology and its role as a potential therapeutic target.

Primary myelofibrosis (PMF) is one of three myeloproliferative neoplasms (MPN) that are morphologically and molecularly inter-related, the other two being polycythemia vera (PV) and essential thrombocythemia (ET). MPNs are characterized by JAK-STAT–activating JAK2, CALR, or MPL mutations that give rise to stem cell–derived clonal myeloproliferation, which is prone to leukemic and, in case of PV and ET, fibrotic transformation. Abnormal megakaryocyte proliferation is accompanied by bone marrow fibrosis and characterizes PMF, while the clinical phenotype is pathogenetically linked to ineffective hematopoiesis and aberrant cytokine expression. Among MPN-associated driver mutations, type 1–like CALR mutation has been associated with favorable prognosis in PMF, while ASXL1, SRSF2, U2AF1-Q157, EZH2, CBL, and K/NRAS mutations have been shown to be prognostically detrimental. Such information has enabled development of exclusively genetic (GIPSS) and clinically integrated (MIPSSv2) prognostic models that facilitate individualized treatment decisions. Allogeneic stem cell transplantation remains the only treatment modality in MF with the potential to prolong survival, whereas drug therapy, including JAK2 inhibitors, is directed mostly at the inflammatory component of the disease and is therefore palliative in nature. Similarly, disease-modifying activity remains elusive for currently available investigational drugs, while their additional value in symptom management awaits controlled confirmation. There is a need for genetic characterization of clinical observations followed by in vitro and in vivo preclinical studies that will hopefully identify therapies that target the malignant clone in MF to improve patient outcomes.

International audience; Research focused on Down syndrome has increased in the last several years to advance understanding of the consequences of trisomy 21 (T21) on molecular and cellular processes and, ultimately, on individuals with Down syndrome. The Trisomy 21 Research Society (T21RS) is the premier scientific organization for researchers and clinicians studying Down syndrome. The Third International Conference of T21RS, held June 6–9, 2019, in Barcelona, Spain, brought together 429 scientists, families, and industry representatives to share the latest discoveries on underlying cellular and molecular mechanisms of T21, define cognitive and behavioral challenges and better understand comorbidities associated with Down syndrome, including Alzheimer’s disease and leukemia. Presentation of cutting-edge results in neuroscience, neurology, model systems, psychology, cancer, biomarkers and molecular and phar­ma­cological therapeutic approaches demonstrate the compelling interest and continuing advancement in all aspects of understanding and ameliorating conditions associated with T21.

GATA1 is an essential regulator of erythroid cell gene expression and maturation. In its absence, erythroid progenitors are arrested in differentiation and undergo apoptosis. Much has been learned about GATA1 function through animal models, which include genetic knockouts as well as ones with decreased levels of expression. However, even greater insights have come from the finding that a number of rare red cell disorders, including Diamond Blackfan Anemia, are associated with GATA1 mutations. These mutations affect the amino-terminal zinc finger (N-ZF) and the amino-terminus of the protein, and in both cases can alter the DNA binding activity, which is primarily conferred by the third functional domain, the carboxyl-terminal zinc finger (C-ZF). Here we discuss the role of GATA1 in erythropoiesis with an emphasis on the mutations found in human patients with red cell disorders.

Purpose: The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. Experimental Design: We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage–committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. Results: JAK2V617F-mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. Conclusions: Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.