ERK2 Substrate Binding Domains Perform Opposing Roles in Pathogenesis of a JAK2V617F-Driven Myeloproliferative Neoplasm

The interaction between ERK2 and its substrates is critically mediated by two domains, the common docking (CD) D-domain and DEF-binding pocket (DBP) domain. Previous studies have suggested that ERK2 is not only necessary to drive hematopoietic and myelo-erythroid development, but it is also important for the pathogenesis of hematological cancers, as revealed by recurrent ERK2 somatic mutations in many types of lymphoma and leukemia. Here we show that the activation of ERK2 in JAK2V617F-driven myeloproliferative neoplasm (MPN) enhance polycythemia vera (PV) progression from erythrocytosis to myelofibrosis when ERK2-DBP binding capacity is disabled. Conversely, targeting the ERK2 D-domain preserves ERK2 catalytic function while reducing outgrowth and proliferation of human and murine MPN cell lines. To determine whether ERK2 binding domains play a role in the pathogenesis of JAK2V617F-driven MPN, we generated an ERK2-Y261A (Erk2 Y261A) mutant knockin mouse model in which the ERK2-DBP domain was inactivated, but ERK2 kinase activity and D-domain function was preserved. We observed splenomegaly in Erk2 Y261A/Y261A, characterized by a 10-fold expansion of CD44+/Ter119+ primitive erythroblasts and Ter119+CD44+/ FSC hi immature erythroid progenitors. We then transplanted Jak2 V617F-expressing ERK2 wildtype (WT), knockout (KO) or DBP hematopoietic progenitors (HSPCs) into irradiated, immunodeficient mice (Rag2 −/−Il2rg −/−) and observed disease resembling human PV, with elevated RBC counts, hematocrit, and hemoglobin levels. ERK2-DBP mutant recipients exhibited mild but sustained erythrocytosis with a prevalence of circulating GFP+ monocytes and neutrophils. At 12-weeks post-transplantation, ERK2-DBP mutant recipients had significant splenic burden and decreased bone marrow cellularity relative to WT and KO recipients. Reticulin staining confirmed progression towards myelofibrosis with interstitial infiltration in both the BM and the spleen of ERK2 DBP mutant recipients. To understand the molecular basis of ERK-DBP domain in JAK2V617F-driven MPN, we performed in vitro colony forming unit assay to assess the clonogenic potential of JAK2V617F-expressing HSPCs in cytokine supplemented-methylcellulose supporting myeloid or erythroid progenitor development. Ectopic JAK2V617F expression markedly reduced colony formation by both ERK2 wildtype and knockout HSPCs. On the other hand, JAK2V617F expression did not reduce colony formation by ERK2-DBP HSPCs. These differences were not evident in methylcellulose cultures that supported erythroid development, suggesting that disabling ERK-DBP binding capacity primarily affects the myeloid lineage. Furthermore, we found that JAK2V617F increased senescence associated β-galactosidase (SA-βGa) activity in ERK2-WT HSPCs. SA-βGal activity was modestly affected by ERK deficiency but was strongly attenuated by the ERK2-DBP mutation, supporting the notion that ERK2-DBP domain promotes oncogene-induced senescence (OIS). Previously, we identified transcription factor, Early growth response 1 (Egr1) which interacts with ERK2-DBP domain and plays a critical role as a tumor suppressor in myeloid neoplasms. We observed that JAK2V617F expression failed to suppress colony formation and senescence by Egr1-deficient HSPCs. The ability of EGR1 to restore these effects is abrogated by inactivating the DEF motif (EGR1-Y252A). Together, this data demonstrates that the interaction between ERK2-DBP and Egr1 is required for JAK2V617F-mediated colony suppression and senescence induction. Finally, to determine whether ERK-D domain acts to promote progression by targeting substrates distinct from those that bind to the DBP domain, we developed an inhibitor (#76) of the ERK-D domain. Indeed, 76 spared ERK phosphorylation and EGR1 induction but impaired RSK phosphorylation, a critical D-domain substrate involved in cancer progression. Moreover, 76 suppressed colony formation by JAK2V61F-expressing HSPCs akin to the active site MEK/ERK inhibitor, U0126. By altering the 4-ethoxybenylidene moiety of 76, we were able to enhance anti-proliferative effects of the parent compound against human MPN cell lines, SET-2, UKE-1, and Ku812. Our findings identify ERK2-domain specific roles in the pathogenesis of JAK2V617F driven MPN and supports a novel therapeutic approach to targeting JAK2 and MAPK dependent MPN. Disclosures Levine: Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Lilly: Honoraria; Janssen: Consultancy; Astellas: Consultancy; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Imago: Membership on an entity's Board of Directors or advisory committees. Wiest: Janssen Pharmaceuticals: Research Funding.

Requirement for antiapoptotic MCL-1 during early erythropoiesis

Although BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other antiapoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1 deletion results in embryonic lethality beyond embryonic day 13.5 as a result of severe anemia caused by a lack of mature red blood cells (RBCs). Mcl1-deleted embryos exhibit stunted growth, ischemic necrosis, and decreased RBCs in the blood. Furthermore, we demonstrate that MCL-1 is only required during early definitive erythropoiesis; during later stages, developing erythrocytes become MCL-1 independent and upregulate the expression of BCL-xL. Functionally, MCL-1 relies upon its ability to prevent apoptosis to promote erythroid development because codeletion of the proapoptotic effectors Bax and Bak can overcome the requirement for MCL-1 expression. Furthermore, ectopic expression of human BCL2 in erythroid progenitors can compensate for Mcl1 deletion, indicating redundancy between these 2 antiapoptotic family members. These data clearly demonstrate a requirement for MCL-1 in promoting survival of early erythroid progenitors.

The Interplay between Drivers of Erythropoiesis and Iron Homeostasis in Rare Hereditary Anemias: Tipping the Balance

Rare hereditary anemias (RHA) represent a group of disorders characterized by either impaired production of erythrocytes or decreased survival (i.e., hemolysis). In RHA, the regulation of iron metabolism and erythropoiesis is often disturbed, leading to iron overload or worsening of chronic anemia due to unavailability of iron for erythropoiesis. Whereas iron overload generally is a well-recognized complication in patients requiring regular blood transfusions, it is also a significant problem in a large proportion of patients with RHA that are not transfusion dependent. This indicates that RHA share disease-specific defects in erythroid development that are linked to intrinsic defects in iron metabolism. In this review, we discuss the key regulators involved in the interplay between iron and erythropoiesis and their importance in the spectrum of RHA.

Bcor deficiency perturbs erythro-megakaryopoiesis and cooperates with Dnmt3a loss in acute erythroid leukemia onset in mice

Recurrent loss-of-function mutations of BCL6 co-repressor (BCOR) gene are found in about 4% of AML patients with normal karyotype and are associated with DNMT3a mutations and poor prognosis. Therefore, new anti-leukemia treatments and mouse models are needed for this combinatorial AML genotype. For this purpose, we first generated a Bcor−/− knockout mouse model characterized by impaired erythroid development (macrocytosis and anemia) and enhanced thrombopoiesis, which are both features of myelodysplasia/myeloproliferative neoplasms. We then created and characterized double Bcor−/−/Dnmt3a−/− knockout mice. Interestingly, these animals developed a fully penetrant acute erythroid leukemia (AEL) characterized by leukocytosis secondary to the expansion of blasts expressing c-Kit+ and the erythroid marker Ter119, macrocytic anemia and progressive reduction of the thrombocytosis associated with loss of Bcor alone. Transcriptomic analysis of double knockout bone marrow progenitors revealed that aberrant erythroid skewing was induced by epigenetic changes affecting specific transcriptional factors (GATA1-2) and cell-cycle regulators (Mdm2, Tp53). These findings prompted us to investigate the efficacy of demethylating agents in AEL, with significant impact on progressive leukemic burden and mice overall survival. Information gained from our model expands the knowledge on the biology of AEL and may help designing new rational treatments for patients suffering from this high-risk leukemia.

Regulation of GATA-1-Controlled Genes By O-Glcnacylation in Erythroid Cells

The role of post-translational modification (PTM) of proteins in regulating developmental and differentiation processes is understudied, but recently we established that O-GlcNAcylation regulates erythropoiesis. O-GlcNAc regulates numerous cellular functions including stress response, transcription, and cell cycle progression. O-GlcNAc is a single O-linked β-N-acetyl-D-glucosamine moiety added to serine/threonine amino acids of nuclear, cytoplasmic, and mitochondrial proteins. O-GlcNAc transferase (OGT), which adds the modification, and O-GlcNAcase (OGA), which removes the modification, are responsible for the dynamic processing of the PTM. Newly developed erythroid-specific OGT conditional knockout mice show that OGT is essential for fetal definitive erythropoiesis, although during primitive erythropoiesis, erythrocytes exhibit hallmarks of ineffective erythropoiesis. In addition, using G1E-ER cells, we have shown that GATA-1 interacts with OGT and OGA and delivers OGT and OGA to GATA-1 regulated genes. When we perturb this process, we observe erythroid defects, most strikingly, a shift in commitment towards other hematopoietic lineages. We hypothesize that at the onset of erythroid lineage commitment, GATA-1 functions as an adaptor protein to deliver these enzymes to erythroid-specific cis-regulatory DNA elements, where the O-GlcNAc status of bound protein complexes is modified to direct transcriptional networks necessary for normal erythroid development and terminal differentiation. Two key proteins, LRF/ZBTB7A and BCL11A, silence the γ-globin promoter during adult erythropoiesis by recruiting the penultimate NuRD repressor complex to these genes. Loss of either of these proteins leads to partial restoration of γ-globin expression. Our previous work demonstrated that O-GlcNAc plays a role in γ-globin gene transcription. GATA-1 recruits FOG-1 and the NuRD complex to silence γ-globin by binding GATA sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin transcription start sites, respectively. OGT and OGA interact with GATA-1, FOG-1 and CHD4, a key component of the NuRD repressor complex and these three proteins are O-GlcNAcylated. Specifically, O-GlcNAcylation modulates the formation of the multi-protein NuRD repressor complex. OGT adds O-GlcNAc to CHD4 stimulating the formation of this repressor complex, whereas removal of this PTM by OGA results in the activation of γ-globin gene expression. In addition, the GATA-1 binding protein, GATAD2A, interacts with CHD4 and organizes it within the NuRD complex. The domain of GATAD2A that interacts with CHD4 is O-GlcNAcylated. Finally, the NuRD complex proteins RBBP7, HDAC1, and MTA2 are also modified by O-GlcNAc. We hypothesize that O-GlcNAcylation controls NuRD repressor assembly by directing subunit interactions. To ascertain the function of OGT and OGA at GATA-1 regulated genes, we developed novel CRISPR/Cas9 targeting tools in K562 cells. We fused OGT and OGA to catalytically dead Cas9 endonuclease (dCas9). Negative controls were generated by mutating a catalytic residue of OGT (H558F) or OGA (D174A). The CBP/p300 acetyltransferase core was fused to dCas9 as a positive control. As a proof of principle, we targeted the γ-globin promoters. Using the UCSC Genome Browser, single guide RNAs (sgRNAs) that do not disrupt endogenous cis-regulatory elements were selected, synthesized, and subsequently introduced into K562 cells to establish stably expressing monoclonal cell lines. Interestingly, when OGA-dCas9 was targeted to the -1 to -223 bp region relative to the mRNA start site of the γ-globin promoters, we observed a significant increase in γ-globin expression. OGT-dCas9 had a significant repressive effect when targeted to regions around the transcriptional start site. All controls behaved as predicted. To validate these results, we are performing chromatin immunoprecipitation (ChIP) to demonstrate occupancy of the targeted regions by our constructs and the presence or absence of O-GlcNAc. Our findings highlight a new post-translational mechanism regulating gene transcription during erythroid development, which affects γ-globin expression and erythropoiesis. Our data will provide insight into how O-GlcNAc controls erythroid differentiation and reveal O-GlcNAcylated protein targets that may be manipulated for the treatment of hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

Developmental constraint shaped genome evolution and erythrocyte loss in Antarctic fishes following paleoclimate change

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change.

p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis shows that ribosome biogenesis is abruptly interrupted by the drop of rDNA transcription and the collapse of ribosomal protein neo-synthesis. Its premature arrest by RNA polI inhibitor, CX-5461 targets the proliferation of immature erythroblasts. We also show that p53 is activated spontaneously or in response to CX-5461 concomitantly to ribosome biogenesis arrest, and drives a transcriptional program in which genes involved in cell cycle arrest, negative regulation of apoptosis and DNA damage response were upregulated. RNA polI transcriptional stress results in nucleolar disruption and activation of ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation are crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold of ribosome biogenesis down-regulation could be prematurely reached and together with pathological p53 activation prevents a normal expansion of erythroid progenitors.