Individuals with Down syndrome (DS; also known as trisomy 21) have a markedly increased risk of leukemia in childhood but a decreased risk of solid tumors in adulthood. Acquired mutations in the transcription factor-encoding GATA1 gene are observed in nearly all individuals with DS who are born with transient myeloproliferative disorder (TMD), a clonal preleukemia, and/or who develop acute megakaryoblastic leukemia (AMKL). Individuals who do not have DS but bear germline GATA1 mutations analogous to those detected in individuals with TMD and DS-AMKL are not predisposed to leukemia. To better understand the functional contribution of trisomy 21 to leukemogenesis, we used mouse and human cell models of DS to reproduce the multistep pathogenesis of DS-AMKL and to identify chromosome 21 genes that promote megakaryoblastic leukemia in children with DS. Our results revealed that trisomy for only 33 orthologs of human chromosome 21 (Hsa21) genes was sufficient to cooperate with GATA1 mutations to initiate megakaryoblastic leukemia in vivo. Furthermore, through a functional screening of the trisomic genes, we demonstrated that DYRK1A, which encodes dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A, was a potent megakaryoblastic tumor-promoting gene that contributed to leukemogenesis through dysregulation of nuclear factor of activated T cells (NFAT) activation. Given that calcineurin/NFAT pathway inhibition has been implicated in the decreased tumor incidence in adults with DS, our results show that the same pathway can be both proleukemic in children and antitumorigenic in adults. IntroductionTrisomy 21 is the most common cytogenetic abnormality observed at birth (about 1 out of 700 individuals) and one of the most recurrent aneuploidies seen in leukemia. As an acquired clonal chromosomal change, its incidence varies between 4.1% and 14.8% in hematological disorders and malignant lymphomas (1). Supporting the link between trisomy 21 and abnormal hematopoiesis, epidemiological studies have shown that individuals with Down syndrome (DS) have an increased frequency of leukemia but a lower incidence of solid tumors (2). Whereas recent studies implicated a subset of trisomic genes, including Erg, Ets2, Adamts1, and Dscr1, in tumor growth inhibition, in part through an altered angiogenesis (3-6), the role of trisomy 21, the initiating event in DS leukemogenesis, and the functional implication of specific genes at dosage imbalances that predispose to and/or participate in leukemogenesis remain unclear.Children with DS are at an elevated risk of both acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL) (7). Moreover, epidemiological studies showed that approximately 4%-5% of children with DS are born with transient myeloproliferative disorder (TMD), a clonal preleukemia characterized by an accumulation of immature megakaryoblasts in the fetal liver and peripheral blood (8,9). Although TMD spontaneously disappears in most cases, TMD clones reemerge as AMKL in 20% of cases within 4 to 5 y...

Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy.

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant (MRSA) infection.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin 1 receptor (IL-1R)-dependent caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6 ΔPMN ) is sufficient to initiate IL-1R-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigated the mechanisms controlling IL-1α/β release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8, and either Ripk3 or Mlkl, strongly protected Ptpn6 ΔPMN mice. Ptpn6 ΔPMN neutrophils displayed increased p38 MAP kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor (TNF) production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 MAP kinase activation to control TNF and IL-1α/β expression, and in maintaining Ripk1 function to prevent caspase-8-and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/β release.