Qiang Jeremy Wen

Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition

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.Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleen of patients are full of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines including TGF-β. Although the JAK inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or significantly reverse fibrosis. Here we show through pharmacologic and genetic studies that, apart from JAK2, Aurora kinase A (AURKA) is a novel therapeutic target in PMF. MLN8237, a selective AURKA inhibitor promoted polyploidization and differentiation of PMF megakaryocytes and displayed potent anti-fibrotic and anti-tumor activity in vivo. We also reveal that loss of one allele of AURKA is sufficient to ameliorate fibrosis and other PMF phenotypes in vivo. Our data suggest that megakaryocytes are drivers of fibrosis and that targeting them with AURKA inhibitors will provide therapeutic benefit in PMF.

Downregulation of GATA1 drives impaired hematopoiesis in primary myelofibrosis

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 is required for hematopoiesis but is dispensable for murine megakaryocyte endomitosis and differentiation

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.

Kinase signaling and targeted therapy for primary myelofibrosis

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.

An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia

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.

AKT activation is a feature of CALR mutant myeloproliferative neoplasms

Among the driver mutations in the MPNs, the mechanism by which CALR mutations activate JAK/STAT signaling is unique in that the novel C-terminus of CALR mutant proteins binds directly to MPL leading to constitutive activation [1, 2]. CALR mutants have also been shown to activate the MAPK pathway [3]. Whether CALR mutants further activate PI3K/AKT signaling is controversial, with several studies reporting a lack of (or modest) activation [2, 4], but another demonstrating potent activation [5]. The former studies relied upon transduction of Ba/F3 TpoR cells with CALR mutant alleles whereas the latter probed the pathway in cells lines with megakaryocytic potential. Moreover, the extent to which PI3K/AKT signaling is a target in CALR mutant MPNs has been debated, with at least one study reporting that inhibitors of this pathway do not synergize with ruxolitinib [2]. To address the discrepancy, we assayed for activation of JAK/STAT and AKT in multiple settings. First, we overexpressed the CALR type 1 (del52) and type 2 (ins5) mutants in primary murine c-kit bone marrow progenitors and cultured the cells for 48 h. We observed enhanced activation of both STAT5 and AKT (Fig. 1a). Next we transplanted bone marrow transduced with CALRdel52 mutant or empty vector to irradiated recipient mice and assessed AKT activation. Both intracellular flow and western blots of spleen cells revealed increase in p-AKT (Figs. 1b, c). Finally, we assayed for total and phosphoAKT in primary CALR mutant MPN samples. We observed robust increase in AKT phosphorylation in patient derived CD34 cells (Fig. 1d and Supplementary Table 1). This was accompanied by an increase in cyclinD3 consistent with the increased proliferation of the CALR mutant cells. These cells also expressed MPL, consistent with the manner in which CALR mutants activate signaling (Fig. 1d). We previously reported that inhibition of AKT activity with the selective AKT inhibitor MK-2206 resulted in reduced growth of MPLW515L expressing cells both in vitro and in vivo, suggesting that this pathway is a therapeutic target in ET and PMF [6]. To determine whether CALR mutant expressing cells are similarly dependent on PI3K/AKT signaling, we treated CD34+ cells from PMF patients or healthy individuals with MK-2206. We found that CALR mutant cells were susceptible to AKT inhibition and more sensitive than healthy progenitor cells in colony assays (Figs. 1e, f). Of note, the differential effect was much more significant for megakaryocyte colonies (Fig. 1f) than for myeloid colonies (Fig. 1e), consistent with a reliance of megakaryocytes on MPL signaling. Finally, while the level of p-AKT was higher in bone marrow cells from mice transplanted with CALRdel52 expressing cells compared to those with empty vector, the degree of inhibition was similar (Fig. 1g). MK-2206 was well tolerated in healthy C57Bl/6 mice with no evidence of myelosuppression or impaired hematopoiesis [6]. To determine the extent to which the drug suppressed thrombocytosis and megakaryopoiesis induced by mutant CALR, we transplanted CALRdel52 mutant expressing hematopoietic progenitor cells into irradiated

Loss of pleckstrin-2 reverts lethality and vascular occlusions in JAK2V617F-positive myeloproliferative neoplasms

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.

Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

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.