ALBERT

Description: Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments.

Description: Epo-induced endocytosis of EpoR plays important roles in the down-regulation of EpoR signaling and is the primary means that regulates circulating Epo concentrations. Here we show that cell-surface EpoR is internalized via clathrin-mediated endocytosis. Both JAK2 kinase activity and EpoR cytoplasmic tyrosines are important for ligand-dependent EpoR internalization. Phosphorylated Y429, Y431, and Y479 in the EpoR cytoplasmic domain bind p85 subunit of PI3 kinase on Epo stimulation and individually are sufficient to mediate Epo-dependent EpoR internalization. Knockdown of p85α and p85β or expression of their dominant-negative forms, but not inhibition of PI3 kinase activity, dramatically impaired EpoR internalization, indicating that p85α and p85β may recruit proteins in the endocytic machinery on Epo stimulation. Furthermore, mutated EpoRs from primary familial and congenital polycythemia (PFCP) patients lacking the 3 important tyrosines do not bind p85 or internalize on stimulation. Addition of residues encompassing Y429 and Y431 to these truncated receptors restored p85β binding and Epo sensitivity. Our results identify a novel PI3 kinase activity-independent function of p85 in EpoR internalization and support a model that defects of internalization in truncated EpoRs from PFCP patients contribute to Epo hypersensitivity and prolonged signaling.

Description: Abstract 2868 The Janus tyrosine kinase 2 (JAK2) plays an important role in hematopoiesis of multiple lineages. A gain-of-function JAK2 mutation, V617F, is the major determinant in myeloproliferative neoplasms (MPNs), a phenotypically diverse group of hematological diseases in which cells of the myelo-erythroid lineage are overproduced. JAK2 kinase inhibitors showed hematological toxicity in treating MPNs, calling for novel therapeutics that can target only the affected lineage while sparing others. This task is hindered by lack of understanding in how JAK2 signaling differentially regulates the generation of different blood cells. We performed an unbiased screen for residues essential for JAK2 auto-inhibition, and identified a panel of novel gain-of-function JAK2 mutations in addition to V617F (1). Surprisingly, three activating JAK2 mutants with similar kinase activities in vitro elicited distinctive hematopoietic abnormalities in mice. Specifically, JAK2(K539I) results primarily in erythrocytosis, JAK2(N622I) predominantly granulocytosis, and JAK2(V617F) in both. These phenotypes are consistent with clinical data showing that patients with the V617F mutation exhibit erythrocytosis and granulocytosis, whereas those with mutations in exon 12 (where K539 resides) exhibit erythrocytosis only (2). Quantification of the hematopoietic stem and progenitor populations in mice expressing wild-type JAK2 or JAK2 mutants showed significant granulocytic skewing by JAK2(V617F) and JAK2(N622I) both in the bone marrow and spleen. In contrast, erythroid skewing by JAK2(K539I) was observed. Consistent with these results, qualitative and quantitative differences were observed in signaling events downstream of JAK2 in stem and progenitor cells from mice expressing different JAK2 mutants. JAK2 mutants also caused redistribution of hematopoietic stem and progenitors from the bone marrow to spleen. In later more differentiated compartments, JAK2(K539I) and JAK2(V617F) expanded erythroid precursor cells, including proerythroblasts and later precursors, to cause erythrocytosis, while JAK2(V617F) and JAK2(N622I) expanded myeloid precursors to cause granulocytosis. The expansion of these later compartments was at least in part due to a decrease in apoptosis. Together, our results showed that JAK2 mutants differentially skew early stem and progenitor compartments toward the erythroid or granulocytic lineage, and expand distinct precursor subsets to cause erythrocytosis or granulocytosis in mice. These results provide mechanistic basis for the phenotypic diversity observed in mice expressing different JAK2 mutants. Our results show that differential JAK2 signaling regulates hierarchically early and late progenitor compartments to drive erythropoiesis vs. granulopoiesis. These results shed light on MPN biology and may facilitate the design of novel and more effective therapeutic agents that specifically target affected lineage without compromising other lineages. Disclosures: No relevant conflicts of interest to declare.

Description: The mechanisms underlying the development of erythropoietin (EPO)-refractory anemia in the setting of chronic inflammatory states are largely unknown. Elevated levels of the classical inflammatory mediators decrease red cell output. However, pathologic concentrations of many of these molecules do not persist beyond the acute phase, indicating that specific mediators are likely to play a role in the anemia associated with chronic inflammation. High mobility group box protein 1 (HMGB1) is a potent alarmin able to induce tissue injury during the acute and chronic phases of inflammation, and recently, shown to contribute to anemia in a murine model of sepsis. Here, we show that HMGB1 directly inhibits erythropoiesis by modulating EPO signal transduction in human erythroid cells through a newly identified HMGB1 receptor, which is surprisingly the erythropoietin receptor (EPOR). Surface plasmon resonance (SPR) reveals that HMGB1 binds the extracellular domain of EPOR (Kd = 130nM) with an affinity comparable to that of EPO. Cysteine residues contained within the A- and B-box domains of HMGB1 that have previously been shown to mediate HMGB1-receptor interactions are also responsible for the EPOR-HMGB1 interaction since a mutant form of HMGB1 lacking these cysteine residues (i.e. 3S HMGB1) fails to bind the EPOR. Cell-based assays suggest that the direct binding of HMGB1 to the EPOR and the subsequent degradation of EPOR accounts for altered EPO signaling by HMGB1. Biologically, HMGB1 reduces the phosphorylation of intracellular EPO effectors including JAK2 (2-fold reduction), STAT5 (4-fold), and ERK1/2 (4-fold). Decreased effector phosphorylation is not due to the increased activity of SHP1/2 phosphatases further implicating inhibition at the receptor level. Loss of EPO signaling due to HMGB1 binding results in decreased erythroid proliferation of differentiated CD34+ cells at the EPO-dependent stages of erythropoiesis: Day 14: 1.03x108 ± 4.67x107 cells/mL vs 1.87x106 ± 9.70x105 cells/mL, vehicle vs HMGB1, respectively. In addition, HMGB1 decreases the numbers of colony forming unit-erythroid (CFU-E) progenitors by 60%, and these progenitors fail to undergo terminal erythroid differentiation with a block at the basophilic erythroblast stage and apoptosis of late-stage erythroblasts as determined by flow cytometric analysis of annexin V staining. To understand the consequences of HMGB1-EPOR interactions on the EPO-induced transcriptome, RNA-sequencing was performed on purified human CFU-E dosed with HMGB1 and EPO. HMGB1 reduces the expression of known EPO target genes (ERFE, CISH, EGR1), and concomitantly, upregulates a number of unique transcripts (ETS2, VMP1, NFKBIZ) suggesting that HMGB1-EPOR interactions may alter receptor conformation in manner that differentially activates the EPOR and consequently, gene expression. Finally, in a mouse model of sepsis survival, bone marrow-derived erythroid precursor cells contain diminished phosphorylated STAT5 levels at a time when elevated HMGB1 plasma concentrations are observed, thereby demonstrating that the loss of EPO signal transduction also occurs in vivo. Taken together, our work identifies HMGB1 as a novel inhibitor of EPO signaling through its interaction with the EPOR, and strongly implicates HMGB1 as a previously undiscovered effector of EPO-refractory anemia associated with chronic inflammation. Disclosures No relevant conflicts of interest to declare.

Description: Myeloproliferative neoplasms (MPNs) are a phenotypically diverse group of pre-leukemic diseases characterized by overproduction of one or more of the myeloid cell lineages. Gain-of -function mutations in the Janus tyrosine kinase 2 (JAK2) are major determinants in MPNs, These include the V617F mutation and mutations in exon 12. Interestingly, MPN phenotype in patients with exon 12 mutations is distinct from that of patients with the V617F mutation. Mechanisms underlying the phenotypic differences are not well understood. We performed an unbiased screen for residues essential for JAK2 auto-inhibition, and identified a panel of novel gain-of-function mutations. Interestingly, three of them with similar kinase activities in vitro elicited distinctive hematopoietic abnormalities in mice. Specifically, JAK2(K539I) results primarily in erythrocytosis, JAK2(N622I) predominantly granulocytosis, and JAK2(V617F) in both. These phenotypes are consistent with clinical data showing that patients with the V617F mutation exhibit erythrocytosis and granulocytosis, whereas those with mutations in exon 12 (where K539 resides) exhibit erythrocytosis only. To determine the mechanisms underlying the phenotypic differences by different JAK2 mutants, we characterized hematopoietic progenitors and precursor subsets in these mice for their proliferation, apoptosis and differentiation. Quantification of the hematopoietic stem and progenitor population showed an increased percentage of granulocyte-monocyte progenitors (GMP) and skewing of differentiation towards the granulocytic lineage in JAK2(V617F) and JAK2(N622I) mice compared to JAK2(K539I) or wild-type JAK2 mice. Because no difference was observed in the proliferation or apoptosis of bone marrow progenitors from JAK2 mutant mice, differentiation of the common myeloid progenitors (CMP) was likely skewed towards GMP by JAK2(V617F) and JAK2(N622I). Consistent with this hypothesis, similar results were observed in colony forming assays from sorted CMP populations. In the spleen, a decrease in GMP apoptosis and an increase in apoptosis of the megakaryocyte-erythrocyte progenitors (MEP) also contributed to the skewing towards the granulocytic lineage in JAK2(N622I) mice. Similar to MPN patients, mice expressing JAK2 mutants exhibited splenomegaly. We found that JAK2 mutants caused redistribution of hematopoietic stem and progenitors from the bone marrow to spleen. As a result, more differentiated precursors were expanded in the spleens of JAK2 mutants mice compared to mice expressing wild-type JAK2. Consistent with their phenotypes, the percentage of Annexin V＋7AAD－erythroblasts in JAK2(K539I) and JAK2(V617F) mice was significantly less than in JAK2(N622I) or wild-type JAK2 mice. On the other hand, both proliferation and apoptosis contribute to the differential degrees of granulocytosis among mice expressing different JAK2 mutants. In line with the different effects elicited by different JAK2 mutants in progenitor and precursor cells, signal transduction pathways were differentially activated downstream of different JAK2 mutants. In summary, our results showed that JAK2 mutants differentially skew differentiation in early stem and progenitor compartments, and also regulate apoptosis and proliferation of distinct precursor subsets to cause erythrocytosis or granulocytosis in mice. These results provide the mechanistic basis for the phenotypic diversity observed in MPNs with different JAK2 mutants. Disclosures: No relevant conflicts of interest to declare.

Description: Homozygous E6V alleles in the beta globin gene cause disease in the majority of sickle cell patients and the use of gene editing technologies to correct this mutation in hematopoietic stem cells offers the potential to cure disease. Approaches utilizing viral transduction or exogenous nucleases to facilitate gene editing merit consideration, but also invoke a number of safety and manufacturing concerns that are avoided through use of peptide-nucleic acids (PNAs) for triplex gene editing. In our approach, nanoparticle delivery of a synthetic PNA that binds the beta globin gene near the E6V mutation triggers high-fidelity endogenous gene repair pathways. Inclusion of a synthetic DNA correction oligo subsequently directs correction of the mutation. This methodology has previously been shown to facilitate gene repair in vivo in several murine models including sickle disease, beta-thalassemia, and cystic fibrosis (Quijano et al., [ASGCT abstr.] 2019, Bahal et al., 2016, McNeer et al., 2015). To date, there has been limited exploration of the structure-activity relationship of PNA designs and the resultant gene editing. Here we describe the design, synthesis, and structure-activity relationship of a hit-to-lead series of more than 300 PNAs and show with certain design features we can improve gene editing efficiency in both murine and human hematopoietic stem and progenitor cells. Our first round of PNA design and synthesis consisted of 45 PNAs within 200 nucleotides of the E6V allele, of which 18/45 (40 %) showed 〉/= 10% allele correction to wild-type HBB allele when tested exvivo in total bone marrow cultures derived from humanized sickle disease "Townes mice". The best PNA in this series consistently demonstrated ~ 20% editing with an ~ED50 of ~1-3 ug/ml of donor DNA or PNA. Editing observed in total mouse bone marrow was also robust in separate cultures of both Townes mouse c-kit(+) HSPCs and healthy donor human CD34(+) HSPCs. In our second round of PNA designs, considerations encompassed numerous factors including whether the PNA binds to the transcribed versus non-transcribed chromosomal strand, distance and location of the PNA relative to the mutation site and relative to the donor template sequence, substitutions on gamma-carbons of the PNA polyamide backbone, structure of the linker joining PNA nucleobase segments, incorporation of novel bases to eliminate off-target base pairing or aggregation to due self-complementarity, and inclusion of lysine residues to facilitate kinetics and affinity of the association with the poly-anionic backbone of target DNA sequences. At the time of this submission we have synthesized and assessed 〉250 additional PNA designs and identified 6 modifications that increase editing 2-4 fold over the initial designs. We will share data on the design and activity of these molecules, data on impact of combining design features, and next generation designs in the presentation. This is the first extensive study on the structure-activity relationship of PNAs for use in gene editing. Our data demonstrate a number of PNA parameters significantly improve gene editing activity. The combination of these findings with additional technological improvements offers the prospect of high-fidelity gene editing without use of any biologics (e.g., viruses or nucleases) that have inherent clinical risk and manufacturing challenges which may limit or prevent their implementation as a cure for sickle patients. Figure Disclosures Ebens: Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Curd:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Sim:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Quijano:Yale University: Patents & Royalties; Trucode Gene Repair, Inc.: Consultancy, Research Funding. Tam:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Coull:Trucode Gene Repair, Inc.: Consultancy, Equity Ownership, Patents & Royalties. Huang:Trucode Gene Repair, Inc.: Employment, Equity Ownership. Hayes:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Fordyce:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Pearson:Trucode Gene Repair, Inc.: Employment. Whoriskey:Trucode Gene Repair, Inc.: Consultancy, Equity Ownership, Patents & Royalties. Srivastava:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Li:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties.

Description: Key Points Epo-induced EpoR internalization is mediated through a novel Cbl/p85/epsin-1 pathway. Mutated EpoR in primary familial and congenital polycythemia patients cannot activate this pathway, exhibiting excessive Epo signaling.