Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of ...individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-β. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit.
NSD2, a histone methyltransferase specific for methylation of histone 3 lysine 36 (H3K36), exhibits a glutamic acid to lysine mutation at residue 1099 (E1099K) in childhood acute lymphocytic leukemia ...(ALL), and cells harboring this mutation can become the predominant clone in relapsing disease. We studied the effects of this mutant enzyme in silico, in vitro, and in vivo using gene edited cell lines. The E1099K mutation altered enzyme/substrate binding and enhanced the rate of H3K36 methylation. As a result, cell lines harboring E1099K exhibit increased H3K36 dimethylation and reduced H3K27 trimethylation, particularly on nucleosomes containing histone H3.1. Mutant NSD2 cells exhibit reduced apoptosis and enhanced proliferation, clonogenicity, adhesion, and migration. In mouse xenografts, mutant NSD2 cells are more lethal and brain invasive than wildtype cells. Transcriptional profiling demonstrates that mutant NSD2 aberrantly activates factors commonly associated with neural and stromal lineages in addition to signaling and adhesion genes. Identification of these pathways provides new avenues for therapeutic interventions in NSD2 dysregulated malignancies.
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/
led to enhanced
self-renewal of murine MPN cells. Deletion of
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
. 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
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/
leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.
.
Constitutive JAK-STAT signaling drives the proliferation of most myeloproliferative neoplasms (MPN) and a subset of acute myeloid leukemia (AML), but persistence emerges with chronic exposure to JAK ...inhibitors. MPN and post-MPN AML are dependent on tyrosine phosphorylation of STATs, but the role of serine STAT1 phosphorylation remains unclear. We previously demonstrated that Mediator kinase inhibitor cortistatin A (CA) reduced proliferation of JAK2-mutant AML in vitro and in vivo and also suppressed CDK8-dependent phosphorylation of STAT1 at serine 727. Here we report that phosphorylation of STAT1 S727 promotes the proliferation of AML cells with JAK-STAT pathway activation. Inhibition of serine phosphorylation by CA promotes growth arrest and differentiation, inhibits colony formation in MPN patient samples and reduces allele burden in MPN mouse models. These results reveal that STAT1 pS727 regulates growth and differentiation in JAK-STAT activated neoplasms and suggest that Mediator kinase inhibition represents a therapeutic strategy to regulate JAK-STAT signaling.
•CDK8/19 inhibitor cortistatin A synergizes with FDA-approved JAK1/2 ruxolitinib and inhibits ruxolitinib-persistent cells.•CDK8/19 phosphorylation of STAT1 S727 promotes growth and suppresses differentiation.•Cortistatin A upregulates expression of STAT1 pS727- and SE-associated genes.
Previously, it was known that cancer cells with activated JAK-STAT signaling are driven by oncogenic actions of JAK2 and tyrosine-phosphorylated STAT3 and STAT5. The FDA-approved JAK inhibitor ruxolitinib targets these dependencies, but significant challenges remain in the clinic, especially for leukemia patients. We show here that JAK2-mutant leukemia cells that become resistant to ruxolitinib are sensitive to CDK8/19 inhibitor CA and that CA synergizes with ruxolitinib, indicating that CDK8/19 inhibitors may be an effective therapeutic strategy for these cancers. Further, our studies provide insights into the mechanistic role of STAT1 serine phosphorylation by CDK8/19 in JAK2-activated leukemia.
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.
We used
transgenic mice to drive expression of
in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in
and
BMT models of MPN.
-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
. Furthermore, depletion of megakaryocytes in the
and
BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis.
Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance
. 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
allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.
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.
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.
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.
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.
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Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute ...myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the RPS14-deficient gene signature, which is associated with defective ribosomal protein function and linked to the erythroid lineage in 5q deletion myelodysplastic syndrome. Surprisingly, reduced GATA1 expression and impaired differentiation were limited to megakaryocytes, consistent with a proproliferative effect of a GATA1 deficiency on this lineage. Importantly, expression of GATA1 effectively rescued maturation of PMF megakaryocytes. Together, these results suggest that ribosomal deficiency contributes to impaired megakaryopoiesis in myeloproliferative neoplasms.
Aurora kinase A (AURKA) is a therapeutic target in acute megakaryocytic leukemia. However, its requirement in normal hematopoiesis and megakaryocyte development has not been extensively ...characterized. Based on its role as a cell cycle regulator, we predicted that an Aurka deficiency would lead to severe abnormalities in all hematopoietic lineages. Here we reveal that loss of Aurka in hematopoietic cells causes profound cell autonomous defects in the peripheral blood and bone marrow. Surprisingly, in contrast to the survival defects of nearly all hematopoietic lineages, deletion of Aurka was associated with increased differentiation and polyploidization of megakaryocytes both in vivo and in vitro. Furthermore, in contrast to other cell types examined, megakaryocytes continued DNA synthesis after loss of Aurka. Thus, like other cell cycle regulators such as Aurkb and survivin, Aurka is required for hematopoiesis, but is dispensable for megakaryocyte endomitosis. Our work supports a growing body of evidence that the megakaryocyte endomitotic cell cycle differs significantly from the proliferative cell cycle.
•AURKA is essential for adult hematopoiesis.•AURKA is dispensable for megakaryocyte polyploidization and differentiation.
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.
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