ALBERT

Description: 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.

Description: Growing clinical demands for platelet transfusions combined with supply limitations have created shortages which are trending toward a global crisis. Major efforts have been taken to address key issues of platelet sources, storage, and utilization. Recent progress in ex vivo culture-based production of megakaryocytes (Mk) and platelets, "pharming," has highlighted the potential for novel, donor-independent sources amenable to antigenic editing and cryo-stockpiling. Such cultures can be easily initiated from umbilical cord blood (CB) progenitors, induced pluripotent stem cells (iPSC), or directly re-programmed somatic cells. The major roadblock associated with these Mk sources consists of their fetal ontogenic status, which is beneficial for expansion but severely limits platelet production. The ability to elicit in pre-expanded Mk an adult program of morphogenesis (polyploidization, enlargement, and proplatelet formation) would enable circumvention of this scalability barrier. A master regulator of adult Mk morphogenesis consists of the transcriptional coactivator MKL1 which undergoes nuclear translocation in response to RhoA-mediated actin polymerization, stimulated by thrombopoietin and environmental mechano-sensing. Nuclear MKL1 associates with the transcription factor SRF1 to upregulate cytoskeletal remodeling factors, including filamin A and Hic-5, that act as morphogenesis effectors. Our previous studies identified in infantile CB Mk a failure in MKL1 upregulation resulting from repression by the oncofetal RNA-binding factor IGF2BP3. Pharmacologic suppression of IGF2BP3 with BET inhibitors rescued MKL1 expression and improved platelet production but caused cycle arrest preventing polyploidization. As an alternative approach to abrogate the fetal blockade in Mk morphogenesis, we sought to promote MKL1 activity by targeting a kinase, Dyrk1a, which had been shown to restrain MKL1 from nuclear translocation. Treatment of infantile CB Mk with a variety of Dyrk1-selective inhibitors including harmine and EHT 1610 strongly enhanced polyploidization (p = 0.015 and 0.009 respectively), enlargement (p 〈 0.005) , and in vitro platelet release (2 fold each, p = 0.001 and 0.007 respectively), attaining levels seen with adult Mk. When xenotransplanted into NSG mice, harmine-treated CB Mk demonstrated enhanced capability for in vivo platelet release (about 5 fold, p = 0.016). CB stem cells expanded with the AHR antagonist SR1 and an iPSC-Mk cell line also responded to Dyrk1 inhibition with robustly increased morphogenesis. Several findings implicated MKL1 in this response: 1) induction of nuclear translocation by the inhibitors, 2) induction of target genes (filamin A and Hic-5) by the inhibitors, and 3) loss of response to inhibitors in Mkl1-ko murine progenitors. Supporting Dyrk1a as a relevant target, mice with Mk-specific loss of one Dyrk1a allele (Dyrk1aflox/wt;Pf4-Cre) displayed increases in platelet counts (p = 0.037) and marrow Mk polyploidization (p = 0.02). In addition, retroviral expression in human progenitors of a dominant negative Dyrk1a mutant K188R promoted Mk enlargement (p = 0.014). shRNA knockdowns could not be obtained due to toxicity of 〉 ~60% loss of Dyrk1a. To determine mechanisms for Dyrk1a control of morphogenesis, we analyzed the actin cytoskeleton, a key regulator of MKL1. Dyrk1 inhibition in all types of Mk progenitors (adult, infantile, and iPSC) induced assembly of cortical filamentous actin (F-actin), as detected by Alexa594-phalloidin staining. Prior studies showed cytoskeletal binding by Dyrk1a and direct phosphorylation of F-actin regulators N-WASP and Ablim1. A survey of human marrow expression patterns for candidate Dyrk1a substrates (The Human Protein Atlas) identified Ablim2, as showing a Mk-specific, cortical staining pattern. Dyrk1 inhibition increased Ablim2 levels ~5-fold in CB Mk (p 〈 0.005), and immunofluorescence displayed a cortical distribution similar to F-actin. Lentiviral shRNA knockdown of Ablim2 abrogated all effects of Dyrk1 inhibition, blocking: F-actin formation, MKL1 nuclear translocation, activation of the MKL1 targets, and Mk morphogenesis. These findings thus delineate a novel Dyrk1a-Ablim2-MKL1 regulatory module in Mk morphogenesis that can be manipulated to address the problem of scaling ex vivo production and might also serve as a future in vivo therapeutic target for thrombocytopenia. Disclosures Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.