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177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) is used clinically to treat metastasized or unresectable neuroendocrine tumors (NETs). Although 177Lu-DOTATATE is mostly well tolerated in patients, bone marrow suppression and long-term renal toxicity are still side effects that should be considered. Amino acids are often used to minimize renal radiotoxicity, however, they are associated with nausea and vomiting in patients. α1-microglobulin (A1M) is an antioxidant with heme- and radical-scavenging abilities. A recombinant form (rA1M) has previously been shown to be renoprotective in preclinical models, including in PRRT-induced kidney damage. Here, we further investigated rA1M’s renal protective effect in a mouse 177Lu-DOTATATE model in terms of administration route and dosing regimen and as a combined therapy with amino acids (Vamin). Moreover, we investigated the protective effect of rA1M on peripheral blood and bone marrow cells, as well as circulatory biomarkers. Intravenous (i.v.) administration of rA1M reduced albuminuria levels and circulatory levels of the oxidative stress-related protein fibroblast growth factor-21 (FGF-21). Dual injections of rA1M (i.e., at 0 and 24 h post-177Lu-DOTATATE administration) preserved bone marrow cellularity and peripheral blood reticulocytes. Administration of Vamin, alone or in combination with rA1M, did not show any protection of bone marrow cellularity or peripheral reticulocytes. In conclusion, this study suggests that rA1M, administered i.v. for two consecutive days in conjunction with 177Lu-DOTATATE, may reduce hematopoietic and kidney toxicity during PRRT with 177Lu-DOTATATE.

During red blood cell (RBC) lysis hemoglobin and heme leak out of the cells and cause damage to the endothelium and nearby tissue. Protective mechanisms exist; however, these systems are not sufficient in diseases with increased extravascular hemolysis e.g. hemolytic anemia. α1-microglobulin (A1M) is a ubiquitous reductase and radical- and heme-binding protein with antioxidation properties. Although present in the circulation in micromolar concentrations, its function in blood is unclear. Here, we show that A1M provides RBC stability. A1M-/- mice display abnormal RBC morphology, reminiscent of macrocytic anemia conditions, i.e. fewer, larger and more heterogeneous cells. Recombinant human A1M (rA1M) reduced in vitro hemolysis of murine RBC against spontaneous, osmotic and heme-induced stress. Moreover, A1M is taken up by human RBCs both in vitro and in vivo. Similarly, rA1M also protected human RBCs against in vitro spontaneous, osmotic, heme- and radical-induced hemolysis as shown by significantly reduced leakage of hemoglobin and LDH. Addition of rA1M resulted in decreased hemolysis compared to addition of the heme-binding protein hemopexin and the radical-scavenging and reducing agents ascorbic acid and Trolox (vitamin E). Furthermore, rA1M significantly reduced spontaneous and heme-induced fetal RBC cell death. Addition of A1M to human whole blood resulted in a significant reduction of hemolysis, whereas removal of A1M from whole blood resulted in increased hemolysis. We conclude that A1M has a protective function in reducing hemolysis which is neither specific to the origin of hemolytic insult, nor species specific.

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to

Cyclin-dependent kinase 8 (CDK8) is a mediator complex-associated transcriptional regulator that acts depending on context and cell type. While primarily under investigation as potential cancer therapeutics, some inhibitors of CDK8-and its paralog CDK19-have been reported to affect the osteoblast lineage and bone formation. This study investigated the effects of two selective CDK8/19 inhibitors on osteoclastogenesis and osteoblasts in vitro, and further evaluated how local treatment with a CDK8/19 inhibitor affects cancellous bone healing in rats. CDK8/19 inhibitors did not alter the proliferation of neither mouse bone marrow-derived macrophages (BMMs) nor primary mouse osteoblasts. Receptor activator of nuclear factor κΒ (NF-κB) ligand (RANKL)-induced osteoclastogenesis from mouse BMMs was suppressed markedly by inhibition of CDK8/19, concomitant with reduced tartrate-resistant acid phosphatase (TRAP) activity and C-terminal telopeptide of type I collagen levels. This was accompanied by downregulation of PU.1, RANK, NF-κB, nuclear factor of activated T-cells 1 (NFATc1), dendritic cell-specific transmembrane protein (DC-STAMP), TRAP, and cathepsin K in RANKL-stimulated BMMs. Downregulating RANK and its downstream signaling in osteoclast precursors enforce CDK8/19 inhibitors as anticatabolic agents to impede excessive osteoclastogenesis. In mouse primary osteoblasts, CDK8/19 inhibition did not affect differentiation but enhanced osteoblast mineralization by promoting alkaline phosphatase activity and downregulating osteopontin, a negative regulator of mineralization. In rat tibiae, a CDK8/19 inhibitor administered locally promoted cancellous bone regeneration. Our data indicate that inhibitors of CDK8/19 have the potential to develop into therapeutics to restrict osteolysis and enhance bone regeneration. Funding agencies: Vetenskapsradet [521-2013-2593, 2016-06097, K2015-99x-10363-23-4, 2016-01822]; Swedish Research Council

The nucleolar surveillance pathway (NSP) monitors nucleolar fidelity and responds to nucleolar stresses (i.e., inactivation of ribosome biogenesis) by mediating the inhibitory binding of ribosomal proteins (RPs) to mouse double minute 2 homolog (MDM2), a nuclear-localised E3 ubiquitin ligase, which results in p53 accumulation. Inappropriate activation of the NSP has been implicated in the pathogenesis of collection of human diseases termed “ribosomopathies”, while drugs that selectively activate the NSP are now in trials for cancer. Despite the clinical significance, the precise molecular mechanism(s) regulating the NSP remain poorly understood. Using genome-wide loss of function screens, we demonstrate the ribosome biogenesis (RiBi) axis as the most potent class of genes whose disruption stabilises p53. Furthermore, we identified a novel suite of genes critical for the NSP, including a novel mammalian protein implicated in 5S ribonucleoprotein particle (5S-RNP) biogenesis, HEATR3. By selectively disabling the NSP, we unexpectedly demonstrate that a functional NSP is required for the ability of all nuclear acting stresses tested, including DNA damage, to robustly induce p53 accumulation. Together, our data demonstrates that the NSP has evolved as the dominant central integrator of stresses that regulate nuclear p53 abundance, thus ensuring RiBi is hardwired to cellular proliferative capacity.

© 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA.

α1-microglobulin (A1M) is a small protein present in vertebrates including humans. It has several physiologically relevant properties, including binding of heme and radicals as well as enzymatic reduction, that are used in the protection of cells and tissue. Research has revealed that A1M can ameliorate heme and ROS-induced injuries in cell cultures, organs, explants and animal models. Recently, it was shown that A1M could reduce hemolysis in vitro, observed with several different types of insults and sources of RBCs. In addition, in a recently published study, it was observed that mice lacking A1M (A1M-KO) developed a macrocytic anemia phenotype. Altogether, this suggests that A1M may have a role in RBC development, stability and turnover. This opens up the possibility of utilizing A1M for therapeutic purposes in pathological conditions involving erythropoietic and hemolytic abnormalities. Here, we provide an overview of A1M and its potential therapeutic effect in the context of the following erythropoietic and hemolytic conditions: Diamond-Blackfan anemia (DBA), 5q-minus myelodysplastic syndrome (5q-MDS), blood transfusions (including storage), intraventricular hemorrhage (IVH), preeclampsia (PE) and atherosclerosis.

Diamond–Blackfan anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity and suppression of NLK expression both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data indicate that the effects of metformin on erythroid proliferation and differentiation are mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3′ untranslated region (3′UTR) to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treatment of DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1a short promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19-deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

Background: Myelodysplastic syndromes (MDS) are a group of malignant blood disorders characterized by ineffective hematopoiesis and cytopenias and frequent evolution to acute myeloid leukemia (AML). MDS results from the expansion of genetically and epigenetically changed clones with impaired differentiation and maturation. Primary clinical goals in MDS are to achieve remissions, alleviate symptoms associated with cytopenias, and minimize the transfusion burden. While supportive red blood cell transfusions, erythropoiesis-stimulating agents and novel targeted agents may lead to clinical improvement, an allogeneic bone marrow transplant (BMT) remains the only potential curative option for patients with MDS. RVU120 (SEL120) is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human Phase Ib clinical trial with RVU120 in patients with AML or high risk (HR)-MDS is currently ongoing. Preclinical studies indicated the strong antileukemic potential of RVU120 that was often associated with the multilineage commitment of CD34+ AML cells. Moreover, RVU120 could improve proliferation and induce erythroid differentiation of CD34+ cells derived from Diamond-Blackfan anemia (DBA) patients. Aims: Primary aim was to evaluate the erythroid differentiation potential of RVU120 in primary MDS and transformed cord blood CD34+ blood cells characterized with an early block in erythroid differentiation. Methods: Efficacy and mechanism of action of RVU120 and other CDK8 inhibitors were investigated in cord blood (CB) cells transduced with TLS-ERG, a fusion gene generated from t(16;21)(p11;q22). Transformed cells displayed an increased capacity for self-renewal, proliferation, and altered erythroid differentiation. Efficacy was further tested in CD34+ cells isolated from BM of MDS patients and established MDS cell lines. Cells were treated with RVU120 and global transcriptional changes and chromatin status were analyzed by RNA-seq, ATAC-seq, and ChIP-seq. Cell cycle, proliferation, and lineage-specific markers were studied by flow cytometry in liquid and semi-solid methylcellulose-based media. Results: RVU120 treatment leads to transcriptional reprogramming of transformed CD34+ CB cells. The most profound changes included decreased CDK8 occupancy followed by increased STAT5 and RNA Polymerase II loading at transcription start site and gene bodies. RVU120 treatment transcriptionally represses multiple genes associated with hematopoietic and leukemia stem cells such as CD34, FLI1, ENG and RGS18 and importantly induce the expression of genes involved in erythroid commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1, INHBA, GATA1/2 and hemoglobin genes. Detailed analysis of transformed CB and MDS CD34+ cells by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, leading to erythroid differentiation. Conclusions: Presented results indicate strong erythroid differentiation potential of RVU120 in CD34+ cells that acquired genetic abnormalities leading to arrested erythroid commitment, characteristics of many MDS and AML subtypes. Observed differentiation phenotype strikingly resembles effects of RVU120 in DBA cells caused by disruption of genes encoding ribosomal proteins. Detailed transcriptomic profiling strongly links the differentiation with enrichment of genes representing regulators of erythroid commitment and hemoglobin metabolism. Further studies are warranted to investigate the efficacy of RVU120 in chronic anemias associated with bone marrow failures in AML and MDS patients. Figure 1 Figure 1. Disclosures Pakulska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Obacz: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Goller: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Combik: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mazan: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Juszczynski: Ryvu Therapeutics: Current equity holder in publicly-traded company. Brzozka: Ardigen: Current Employment, Membership on an entity's Board of Directors or advisory committees; Selvita SA: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Dziedzic: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Angelosanto: Ryvu Therapeutics: Current Employment. Shamsili: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Background: CDK8 and its paralog CDK19 are part of the kinase module of the mediator complex, which functions as a bridge between enhancers and core promoters. The CDK8 module functions as a master regulator of transcription and lineage development, including regulation of various oncogenic programs and importantly also hematopoiesis and differentiation. The CDK8/CDK19 inhibitor RVU120 (SEL120) is being investigated in a Phase Ib clinical study (NCT04021368) in AML and HR-MDS patients. Preclinical data indicate the high efficacy of RVU120 in AML models, particularly in cells with stem cell-like characteristics, where the treatment leads to lineage commitment and eventually cell death. Results from the patient cohorts of the dose-escalation phase indicate signs of clinical efficacy, including a complete response (CR) in a relapsed/refractory (R/R) AML patient. Aim: It is now critical to establish the relationship between preclinical and clinical efficacy results and molecular characteristics to identify actionable biomarkers predicting response to CDK8/CDK19 inhibitors. Methods: Association between gene mutations and gene expression patterns with responses to RVU120 has been analyzed on 27 genetically annotated AML patient-derived cells (PDCs). The activity and efficacy of RVU120 were assessed in both non-differentiating and differentiating media followed by flow cytometry and bioinformatics analysis. DNMT3A mutant PDCs were implanted intravenously into NSG-SGM3 mice and after disease onset, animals were treated orally with RVU120. Profiling of transcriptional response to RVU120 in DMNT3A mutant cells has been performed by RNA-seq. The first-in-human Phase1b study CLI120-001 (NCT04021368) of RVU120 is currently enrolling R/R AML and HR-MDS patients, in a dose-escalation design aimed at exploring safety/tolerability and identifying the randomized Phase II dose (RPD2). RVU120 is administered orally, every other day (EOD), 7 total doses per cycle, in 21-day treatment cycles until disease progression/unacceptable toxicity. Local assessment of Bone Marrow (BM) and Peripheral Blood (PB) are performed at different time points according to investigator guidelines to define response to study drug according to Dohner 2017 response criteria. Results: Screening of 27 AML PDCs against RVU120 and other non-related CDK8/CDK19 inhibitors indicated high anti-cancer efficacy in >40% of tested samples (12 out of 27). Correlation of efficacy with the genetic profile of samples showed specific enrichment of DNMT3A mutants (8 out of 12) in the responder group (p=0.015). Notably, efficacy results were further corroborated in vivo in a disseminated PDX AML model, showing complete clearance of blasts positive for DNMT3A mutation and recovery of normal murine BM in animals treated with RVU120. Molecular profiling of responder cells indicated transcriptional reprogramming and lineage commitment. At the date of this abstract submission, 7 patients have been enrolled into the Phase 1b CLI120-001 trial: 5 AML and 2 HR-MDS patients, median age 73 years, failing 2 median previous lines of therapy. Notably, a 62 YO R/R AML patient that has achieved a CR was positive for DNMT3A R882C mutation. At study entry, this patient had progressed after venetoclax/decitabine, with pancytopenia and >50% BM monocytic blasts and skin leukemia. At the end of the first cycle of the study drug, BM showed complete clearance of blasts with hematological recovery and strong monocytic differentiation starting in cycle 2. Skin leukemia lesions improved gradually during treatment with a complete resolution in cycle 7, resulting in CR. After 1 month from CR patient progressed with 65% BM blasts with the same phenotype as at the study entry. Conclusion: AML PDCs with DNMT3 mutations show increased sensitivity to RVU120 treatment both in vitro and in vivo. The anti-cancer efficacy of RVU120 was strongly associated with transcriptomic reprogramming and lineage commitment. Preliminary evidence of response to RVU120 has also been shown in a R/R AML patient positive for DNMT3A mutation that has achieved a CR. Further molecular studies in more patients treated with RVU120 are ongoing and could provide evidence for predictive biomarkers of response to RVU120 in AML. Figure 1 Figure 1. Disclosures Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Pakulska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Burris: Boehringer Ingelheim: Consultancy, Other: research grant ; AstraZeneca: Consultancy, Other: research grant ; Bayer: Consultancy, Other: research grant ; Daiichi Sankyo: Consultancy; Grail: Consultancy; Incyte: Consultancy, Other: research support; Novartis: Consultancy, Other: research grant, Expert Testimony; Pfizer: Consultancy, Other: research grant ; Vincerix Pharma: Consultancy; Abbvie: Other: research grant ; Agios: Other: research grant ; ARMO Biosciences: Other: research grant ; Array BioPharma: Other: research grant ; BioAtla: Other: research grant ; BioMed Valley Discoveries: Other: research grant ; Boehringer Ingelheim: Other: research grant ; Bristol Myers Squibb: Other: research grant ; CALGB: Other: research grant ; CicloMed: Other: research grant ; eFFECTOR Therapeutics: Other: research grant ; Lilly: Other: research grant ; EMD Serono: Other: research grant ; Roche/Genetech: Other: research grant ; GlaxoSmithKline: Other: research grant ; Harpoon: Other: research grant ; Hengrui Therapeutics: Other: research grant ; Infinity Pharmaceuticals: Other: research grant ; Janssen: Other: research grant ; Jounce: Other: research grant ; Kymab: Other: research grant ; MacroGenics: Other: research grant ; MedImmune: Other: research grant ; Merck: Other: research grant ; Millennium Pharmaceuticals: Other: research grant ; Moderna: Other: research grant ; Foundation Medicine: Other: research grant ; Revolution Medicine: Other: research grant ; Seattle Genetics: Other: research grant ; Tesaro: Other: research grant ; TG Therapeutics: Other: research grant ; Verastem: Other: research grant ; Vertex Pharmaceuticals: Other: research grant ; XBiotech: Other: research grant ; Zymeworks: Other: research grant ; Arch Oncology: Other: research grant ; Arvinas: Other: research grant ; Coordination Pharmaceuticals: Other: research grant ; NGM Biopharmaceuticals: Other: research grant ; Gossamer Bio: Other: research grant ; Ryvu Therapeutics: Other: research grant ; BioTheryX: Other: research grant ; HCA Healthcare: Other: stock ownership. Obacz: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Goller: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Combik: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mazan: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Juszczynski: Ryvu Therapeutics: Current equity holder in publicly-traded company. Zawadzka: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Brzozka: Selvita SA: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ardigen: Current Employment, Membership on an entity's Board of Directors or advisory committees; Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Shamsili: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Angelosanto: Ryvu Therapeutics: Current Employment.

Diamond Blackfan Anemia (DBA) is associated with a hypoproliferative anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life with the majority of patients carrying mutations in one of at least 17 ribosomal proteins, with RPS19 being the most common. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions, chronic effects from long term corticosteroid use, or complications from stem cell transplantation. The serine threonine kinase Nemo-like Kinase (NLK) is an atypical member of the MAP kinase family of enzymes and has been shown to be chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as in erythroid progenitors from DBA patients. In RPS19-insufficient human hematopoietic stem and progenitor cells, genetic silencing of NLK by shRNA increased erythroid expansion by 220.3% (SD = 6.6%), indicating that aberrant NLK activation may contribute to the pathogenesis of the disease and is a potential target for DBA therapy. A number of clinically approved or advanced compounds have been developed to inhibit MAP kinases with various degrees of cross reactivity among its family members. We therefore screened a number of compounds that inhibit NLK as an off-target and found that these NLK inhibitors improved erythroid expansion in DBA models. Of these inhibitors, OTS-167 performed optimally, improving erythropoiesis by 2-fold at 300nM, with an EC50 of 146nM. Previous studies of OTS-167 in xenograft models of neuroblastoma for one month did not result in neutropenia, suggesting very little to no toxicity to myeloid cells. The goal in treating DBA patients with NLK inhibitors is to sufficiently raise the hemoglobin to prevent the need for chronic red cell transfusions or treatment with steroids. Our results suggest that pharmacologic inhibition of NLK is a potential approach to treat patients with DBA. We are currently investigating other NLK inhibitors in preclinical models for future clinical application. Disclosures Glader: Agios: Consultancy.

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.

During red blood cell (RBC) lysis hemoglobin and heme leak out of the cells and cause damage to the endothelium and nearby tissue. Protective mechanisms exist; however, these systems are not sufficient in diseases with increased extravascular hemolysis e.g. hemolytic anemia. α

SummaryCurrent sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41+, display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.

Summary Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development., Graphical Abstract, Highlights • Gata1, Tal1, Lmo2, and c-Myc reprogram fibroblasts to erythroid progenitors (iEPs) • iEP gene expression is more similar to that of primitive than definitive erythroblasts • Klf1 or Myb overexpression induces adult hemoglobin expression in iEPs, Capellera-Garcia et al. show that Gata1, Tal1, Lmo2, and c-Myc directly convert murine and human fibroblasts into erythroid progenitor/precursor cells. This finding defines a conserved transcriptional program instructing erythroid cell fate in mammals.

Nemo-like Kinase (NLK) is an atypical member of the Mitogen-activated protein (MAP) kinase family and shares a highly conserved kinase domain with other MAP kinases. For this reason it is challenging to design small molecules that specifically inhibit NLK. In hematopoiesis, NLK expression is suppressed in all lineages except erythroid progenitors. In non-erythroid progenitors, micro-ribonucleic acid (miR)-181 is upregulated and binds to the 3' untranslated region (UTR) of NLK transcripts, resulting in dramatic degradation of NLK transcripts. NLK is not activated or required for healthy erythropoiesis, but in Diamond Blackfan Anemia (DBA), NLK is chronically hyper-activated, which contributes to failed erythroid expansion and disease pathogenesis. Given the sensitivity of NLK transcripts to 3'UTR binding-mediated degradation, we hypothesized that targeting NLK degradation by upregulating miRNAs that bind the NLK 3'-UTR could provide an alternative approach to directly inhibiting the kinase to suppress aberrant NLK activity in bone marrow progenitors from DBA patients. One miRNA binding site that is present in the NLK-3'UTR is miR-26a. We determined that miR-26a is upregulated 2.4-fold (p=0.009) in CD235+ erythrocytes by the Type 2 diabetes drug metformin. Metformin suppressed NLK expression 62.4% (p=0.006) and improved erythroid expansion 4-fold (p=0.001) in human and 6-fold (p=0.042) in zebrafish models of DBA. This effect required an intact miR-26a binding site within the NLK 3'UTR. However, metformin failed to induce miR-26a in murine models of DBA, however the exogenous expression of miR-26a suppressed NLK 36.4% (p=0.028) and improved erythropoiesis 2-fold (p=0.017). In summary, targeted suppression of NLK through metformin-mediated upregulation of NLK transcript-binding miRNAs effectively improved red blood cell production from 5.6% up to 33.3% of controls in a preclinical model of DBA. These results suggest that metformin is a potential drug for treatment of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy. OffLabel Disclosure: metformin. This drug is given to differentiating HSPCs in vitro to examine effect on specific kinases.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Diamond-Blackfan anaemia (DBA) is a rare congenital erythroid hypoplastic anaemia characterised by severely reduced numbers of erythroid precursors in the bone marrow. This reduction of erythroid progenitors is partly due to p53-induced apoptosis during erythroid differentiation in response to nucleolar stress. Nucleolar stress can be caused by mutations in ribosomal proteins. Mutations in ribosomal proteins are found in over 50% of DBA patients and mutations in RPS19 are found in ∼25% of DBA patients. We hypothesise that preventing p53-induced apoptosis in DBA erythroid cells would lead to the survival and differentiation of erythroid progenitors. We performed a genome-wide, gain-of-function screen to identify factors that can modulate levels of p53 in RPS19 knockdown cells. For screening, we generated a human cell line with doxycycline-inducible shRNA-mediated RPS19 knockdown. Induction leads to a decrease in RPS19 protein and robust induction of p53. Using a lentiviral human ORF expression library containing ∼17000 genes in a high-throughput overexpression assay, we confirmed 36 high-confidence candidates that can modulate levels of p53 in the presence of perturbed ribosome biogenesis. To determine if overexpression of genes that led to a reduction of p53 (p53low hits) can regulate p53 within erythroid cells and thereby rescue the DBA phenotype, we utilised a doxycycline-inducible RPS19 knock-down mouse model. This mouse displays many characteristics of DBA such as bone marrow failure which can be rescued by RPS19 gene transfer or loss of Trp53. We tested if expression of p53low hits can rescue the differentiation and proliferation of shRPS19 erythroid progenitors from foetal livers of 14.5dpc embryos in vitro and bone marrow in vivo. By identifying factors that regulate p53, we aim to identify pathways involved in the disease and targets for novel DBA therapies.

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Nemo-like Kinase (NLK) is hyperactivated in erythroid progenitors in murine and human models as well as DBA patients. In an RPS19-insufficient human model, genetic silencing of NLK increased erythroid expansion by 2.2 fold, indicating that aberrant NLK activation contributes to disease pathogenesis. A high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increased erythroid expansion in murine (5.4 fold) and human (6.3 fold) models of DBA with no effect on wild type erythropoiesis (EC50: 0.7 µM). Virtually identical results were observed in CD34+ progenitors from 3 DBA patient bone marrow aspirates with 2.3, 1.9 and 2.1 fold increases in CD235+ erythroblast generation. In erythroid progenitors, RPS19 insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR activity by 82%. This was restored to basal levels upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. Probably due to NLK phosphorylation of raptor, DBA patients did not respond as anticipated. While leucine treatment mildly increased mTOR activity in both control and RPS19-insufficiency, combining leucine with NLK inhibition increased mTOR activity to 142% of control and significantly improved erythroid expansion. Identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA.

Stem cells and progenitors in many lineages undergo self-renewing divisions, but the extracellular and intracellular proteins that regulate this process are largely unknown. Glucocorticoids stimulate red blood cell formation by promoting self-renewal of early burst-forming unit–erythroid (BFU–E) progenitors. Here we show that the RNA-binding protein ZFP36L2 is a transcriptional target of the glucocorticoid receptor (GR) in BFU–Es and is required for BFU–E self-renewal. ZFP36L2 is normally downregulated during erythroid differentiation from the BFU–E stage, but its expression is maintained by all tested GR agonists that stimulate BFU–E self-renewal, and the GR binds to several potential enhancer regions of ZFP36L2. Knockdown of ZFP36L2 in cultured BFU–E cells did not affect the rate of cell division but disrupted glucocorticoid-induced BFU–E self-renewal, and knockdown of ZFP36L2 in transplanted erythroid progenitors prevented expansion of erythroid lineage progenitors normally seen following induction of anaemia by phenylhydrazine treatment. ZFP36L2 preferentially binds to messenger RNAs that are induced or maintained at high expression levels during terminal erythroid differentiation and negatively regulates their expression levels. ZFP36L2 therefore functions as part of a molecular switch promoting BFU–E self-renewal and a subsequent increase in the total numbers of colony-forming unit–erythroid (CFU–E) progenitors and erythroid cells that are generated., National Institutes of Health (U.S.) (Grant P01 HL 32262)

Diamond-Blackfan anemia is a congenital erythroid hypoplasia and is associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with Diamond-Blackfan anemia have mutations in a gene encoding ribosomal protein S19 (RPS19). Through overexpression of RPS19 using a lentiviral vector with the spleen focus-forming virus promoter, we demonstrated that the Diamond-Blackfan anemia phenotype can be successfully treated in Rps19-deficient mice. In our present study, we assessed the efficacy of a clinically relevant promoter, the human elongation factor 1α short promoter, with or without the locus control region of the β-globin gene for treatment of RPS19-deficient Diamond-Blackfan anemia. The findings demonstrate that these vectors rescue the proliferation defect and improve erythroid development of transduced RPS19-deficient bone marrow cells. Remarkably, bone marrow failure and severe anemia in Rps19-deficient mice was cured with enforced expression of RPS19 driven by the elongation factor 1α short promoter. We also demonstrate that RPS19-deficient bone marrow cells can be transduced and these cells have the capacity to repopulate bone marrow in long-term reconstituted mice. Our results collectively demonstrate the feasibility to cure RPS19-deficient Diamond-Blackfan anemia using lentiviral vectors with cellular promoters that possess a reduced risk of insertional mutagenesis.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.

This article reviews the regulation of production of RBCs at several levels. We focus on the regulated expansion of burst-forming unit-erythroid erythroid progenitors by glucocorticoids and other factors that occur during chronic anemia, inflammation, and other conditions of stress. We also highlight the rapid production of RBCs by the coordinated regulation of terminal proliferation and differentiation of committed erythroid colony-forming unit-erythroid progenitors by external signals, such as erythropoietin and adhesion to a fibronectin matrix. We discuss the complex intracellular networks of coordinated gene regulation by transcription factors, chromatin modifiers, and miRNAs that regulate the different stages of erythropoiesis.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

Using RNA-seq technology, we found that the majority of microRNAs (miRNAs) present in CFU-E erythroid progenitors are down-regulated during terminal erythroid differentiation. Of the developmentally down-regulated miRNAs, ectopic overexpression of miR-191 blocks erythroid enucleation but has minor effects on proliferation and differentiation. We identified two erythroid-enriched and developmentally up-regulated genes, Riok3 and Mxi1, as direct targets of miR-191. Knockdown of either Riok3 or Mxi1 blocks enucleation, and either physiological overexpression of miR-191 or knockdown of Riok3 or Mxi1 blocks chromatin condensation. Thus, down-regulation of miR-191 is essential for erythroid chromatin condensation and enucleation by allowing up-regulation of Riok3 and Mxi1.

MJ and JF provided equal contribution as senior authors Acute myeloid leukemia (AML) is associated with poor survival and characterized by an accumulation of immature myeloid blasts in the bone marrow. To efficiently target AML, new therapies directed to leukemia stem cells (LSCs), a self-renewing population that constitutes a chemo-resistant reservoir responsible for disease relapse, are warranted. Cyclin-dependent kinase 8 inhibitors' (CDK8i) anti-cancer activity has been demonstrated in human acute myeloid leukemia (AML) cell lines. Efficacy has been associated with activation of super enhancer regions and reduction of STAT5 S726 phosphorylation in sensitive cells (Pelish, Nature, 2015). SEL120-34A (Selvita, Poland), a selective low nanomolar CDK8/CDK19 inhibitor, has been shown to have antileukemic effect in a panel of AML cell lines (Rzymski, Oncotarget 2017). To evaluate whether primitive AML cells, enriched for LSCs, are sensitive to CDK8 inhibition, we tested the CDK8i SEL120-34A and Senexin B on TEX cells, an AML cell line that exhibits a hierarchical organization and can be used as a LSC model (Warner, Leukemia, 2005). Treatment of TEX cells with SEL120-34A or Senexin B resulted in an inhibition of cell growth (IC50 of 8 and 31 nM respectively at 10 days of culture), associated with reduced activation of STAT5 and STAT1. Both STAT proteins were previously identified as biomarkers for SEL120-34A activity (Rzymski, Oncotarget 2017). In addition, RNA sequencing followed by gene-set enrichment analysis (GSEA) revealed a loss of a LSC signature. To functionally address the effects of CDK8i on LSCs, we used a murine dsRed+ AML model driven by retroviral MLL-AF9 expression. This model has a well-defined LSC population and initiates AML with a short latency, enabling rapid follow-up experiments in syngeneic hosts. Treatment of c-Kit+ AML cells with SEL120-34A or Senexin B in vitro resulted in strong inhibition of leukemia cell growth (IC50 of 119 and 143 nM, respectively, at 7 days of culture), associated with increased apoptosis and reduced cycling of the cells. To address the in vivo therapeutic efficacy of SEL120-34A, mice injected with AML cells 10 days earlier were treated orally for 12 consecutive days using doses of 20 and 40 mg/kg before sacrificed. No tolerability issues were observed with mice maintaining a stable weight through the treatment. Whereas the control group had 87% leukemia cells in the peripheral blood at the end-point analysis, SEL120-34A treated animals showed a dose-dependent selective anti-leukemic activity with a corresponding 78% (20 mg/kg) and 67% (40 mg/kg, p=0.011) of leukemia cells. Similarly, a significant selective dose-dependent anti-leukemic activity of SEL120-34A was observed also in the bone marrow. In addition, a dose-dependent reduced white blood cell count and smaller spleen size upon SEL120-34A treatment was observed, demonstrating that CDK8 inhibition has selective anti-leukemic activity in vivo. Notably, SEL120-34A treatment resulted in granulocytic (Gr1+CD11b-) differentiation of the AML cells (5.8% of the AML cells in the control group; 21.9% at 20 mg/kg, p=0.03; and 22.3%, p=0.0037 at 40 mg/kg). Moreover, SEL120-34A treatment resulted in strong inhibition of Stat5 S726 and Stat1 S727 phosphorylation in AML bone marrow cells harvested from the mice. To test the efficacy of CDK8 inhibition on AML patient cells, four AML patient derived xenografts were treated with SEL120-34A ex vivo. In all four samples, two of which contained activating mutations in FLT3, SEL120-34A treatment resulted in a significant antileukemic activity with decreased number of AML cells and an increase in apoptotic cells. Taken together, our data from murine and human AML models indicate that CDK8 inhibition has therapeutic efficacy in primitive AML cells. SEL120-34A treatment in vivo resulted in reduced leukemia cell burden in both blood and bone marrow accompanied by granulocytic differentiation. Treatment of AML cells in cultures consistently resulted in a reduction in AML cell number and increased apoptosis. Ongoing and future experiments will address whether SEL120-34A treatment also extends survival of mice with AML in syngeneic and patient-derived xenograft models. These data highlight CDK8 as a promising therapeutic target in AML and provides preclinical proof of concept for anti-leukemic efficacy of the clinical candidate SEL120-34A in relevant AML models. Disclosures Mazan: Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Wiklik:Selvita S.A.: Employment. Combik:Selvita S.A.: Employment. Masiejczyk:Selvita S.A.: Employment. Fiedor:Selvita S.A.: Employment. Obacz:Selvita S.A.: Employment. Bialas:Selvita S.A.: Employment. Chesy:Selvita S.A.: Employment. Gabor-Worwa:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rzymski:Selvita S.A.: Employment, Equity Ownership. Fioretos:Cantargia: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life. Approximately 70% of DBA patients possess a mutations in one of at least 12 ribosomal proteins, with RPS19 and RPL11 being the most prevalent, accounting for over 25% and 5% of cases respectively. Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Nemo-like Kinase (NLK) is chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as erythroid progenitors from DBA patients. In an RPS19-insufficient human model, genetic silencing of NLK (shRNA) increased erythroid expansion by 2.2 fold, indicating aberrant NLK activation contributes to the pathogenesis of the disease. In an independent, high-throughput kinase inhibitor screen examining progenitor expansion in RPS19-insufficient Kit+ murine cells, a number of compounds were identified that increased progenitor expansion. SB431542 and SD208 are recognized TGFβ inhibitor compounds, but were the only TGFβ inhibitors of a panel of 11 that increased progenitor expansion. Both active compounds robustly inhibited NLK activity in vitro and in vivo while the remaining 9 inhibitors had no significant impact on NLK. Both compounds increased erythroid expansion in murine (3.1 and 5.4 fold) and human (3.2 and 6.3 fold) models of DBA with no effect on wild type erythropoiesis (EC50 5 µM and 0.7 µM). No further increase in erythroid expansion was observed when NLK expression was silenced by shRNA. Virtually identical results were observed in CD34+ progenitors from 3 DBA patient bone marrow aspirates with 2.3, 1.9 and 2.1 fold increases in CD235+ erythroblast generation compared to untreated. NLK hyperactivation was limited to differentiating committed erythroid progenitors and was not detected in megakaryocytic, other myeloid progenitors or lymphoblastoid cells lines from DBA patients. During differentiation, non-erythroid lineages upregulate miR181, which results in NLK transcript degradation and loss of NLK expression. The absence of NLK in non-erythroid progenitors prevents NLK activation during ribosomal insufficiency. CRISPR/Cas9 mutation of the miR181 binding site in the NLK 3'UTR in RPS19-insufficient CD34+ HSPCs prevented NLK downregulation, increased NLK activity, and sensitized megakaryocyte and other myeloid lineages (80.5% and 76% reduction relative to controls). This is comparable to the erythroid expansion defect in RPS19-insufficiency (80.7% reduction). In differentiating erythroid progenitors, RPS19 insufficiency increased phosphorylation of the mTORC1 component Raptor (5.3 fold), reducing mTOR activity by 82%. This was restored to basal levels upon pharmacological or genetic inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTORC1 activity with leucine has been proposed to increase translational efficiency in DBA patients. Probably due to NLK phosphorylation of raptor, DBA patients did not respond as anticipated. While leucine treatment did increase mTOR activity in both control (100% to 188%) and RPS19-insufficiency (27 % to 42% of control), the combined treatment of leucine with NLK inhibition resulted in increased mTOR activity to 142% of control and significantly improved erythroid expansion. Identification of aberrantly activated enzymes, such as NLK, that are specifically expressed in erythroid progenitors, offer therapeutic promise as potential druggable targets in the clinical management of DBA that can be used in combination with existing therapies. Disclosures Glader: Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Porteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

Diamond-Blackfan Anaemia (DBA) is a rare blood cell aplasia that presents clinically at approximately 2-3 months of age and its main characteristic is reduced erythroid precursors in the bone marrow, i.e. anemia. Mutations in different ribosomal protein (RP) genes have been associated with DBA, with mutations in RPS19 accounting for 20-25% of all cases. It has been proposed that RPS19 deficiency causes perturbations in ribosome biogenesis, thus activation of the p53-dependent Nucleolar Surveillance Pathway (NSP). In this context free RPs (predominantly L5 and L11) in a complex with 5S rRNA sequester the E3 ubiquitin ligase murine double minute 2 (MDM2), leading to the accumulation of p53 and subsequent activation of its transcriptional targets mediating cell cycle arrest or apoptosis. In DBA, one of the molecular mechanisms impairing the proliferation and thus reducing the number of erythroid progenitors that can progress to mature red blood cells is an elevation of p53 protein mediating activation of the NSP. In order to identify potential therapeutics that could be repurposed to prevent the activation of NSP in DBA patients, we have screened compound libraries of clinically approved therapeutics to identify pathways implicated in the p53-dependent NSP due to RPS19 deficiency. We quantitated both cell number and the level of p53 expression, identifying compounds that can result in low and high expression of p53, the latter for potential use in cancer therapy. Using an RPS19 depleted A549 cell line as a model system, the screen successfully identified different therapeutic groups. In the DBA context, we were most interested in the compounds that reduced p53 and had no negative effect on cell number. A selection of 22 molecules were re-evaluated in vitro, again using RPS19 depleted A549 cells, through the quantification of p53 protein expression and densitometry analysis. From this, 10candidates were evaluated ex vivofor their effects on proliferation using bone marrow obtained from an inducible Rps19 knockdown (DBA) mouse model. While we are currently testing a number of compounds in vivo using the Rps19 DBA mouse model (as described), one of the compounds tested thus far has demonstrated a partial rescue of the cKit+ population, no changes in erythroid precursors but interestingly a reversal of the defect in the Granulocyte-Monocyte Progenitor (GMP) population. Impairment in lineage progression in the GMP compartment has also been reported to present in bone marrow failure Shwachman-Diamond Syndrome. We are currently evaluating the mechanism by which this drug is rescuing the c-Kit+ and GMP populations in these mice. In summary, our high-throughput screening approach and follow up studies have identified a suite of novel therapies that may be beneficial for repurposing for the treatment of bone marrow failure by increasing hematopoietic progenitor cells. We plan to evaluate this, and potentially other therapies, in a clinical trial with DBA patients. Disclosures Flygare: LU Holding: Patents & Royalties: Patent.

A safe and efficient therapy for patients with the bone marrow failure syndrome Diamond-Blackfan Anemia (DBA) is urgently needed. To identify novel drug candidates for DBA, a small molecule screen was performed using c-Kit positive E14.5 fetal liver cells from a DBA mouse model in which the DBA phenotype is induced upon doxycycline (DOX)-inducible silencing of Rps19, the most frequently mutated gene in DBA (Jaako et al. PMID: 21989989). Test compounds were added 24 hours after cell seeding and DOX addition. After four days the number of live metabolically active cells in each well was estimated based on quantitation of intracellular ATP. Fifteen commercial annotated small-molecule libraries (3 800 molecules) and 10 500 selected compounds from a diverse compound library were screened. Between the screens we identified 20 molecules that reproducibly increased proliferation of rps19-deficient erythroid progenitors 4-8 fold in a concentration-dependent manner. Hits from annotated libraries included inhibitors of TGFb receptor, DYRK and Casein kinases. The most potent hits however were compounds in a series of thienopyridines, with an unknown target profile, but with a core structure suggesting kinase inhibition activity. Database searches revealed that the structure-activity relationships (SAR) of 12 active and 10 inactive analogues were similar to that of a series of thienopyridines previously reported as bone anabolic agents by unknown mechanism (Saito et al. PMID: 23453217). The most potent analogues described by Saito et al. as bone anabolic agents were synthesized, and the compounds rescue also RPS19-deficient erythroid cell proliferation in a potent manner (EC50= 20-50 nM). In an attempt to identify the molecular target of these compounds, six actives including thienopyridines 15k and 15w were subjected to kinase profiling against 468 kinases (DiscoverX). Three of the compounds targeted cyclin- dependent kinases CDK8 and its paralog CDK19, and in particular the most potent molecule (15w) is a highly selective CDK8 inhibitor. To confirm whether CDK8 inhibition underlies the rescue of the DBA phenotype, structurally unrelated and potent CDK8 inhibitors including CCT-251545, Senexin A, Senexin B, and Sel120-34A were also evaluated. All tested CDK8-inhibitors rescue proliferation and erythroid maturation of c-kit+ cells from the DBA mouse in a concentration-dependent manner. To further investigate the potential of CDK8 as a therapeutic target in DBA several CDK8 inhibitors were evaluated in erythroid cultures of primary DBA patient cells. CDK8 inhibitors 15w, Senexin B and Sel120-34A increase erythroid progenitor proliferation 5-10 fold of CD34+ peripheral blood cells from three DBA patients (RPS19, RPS26, RPL35a mutations), and increase the fraction of transferrin receptor (CD71) and glycophorin A positive cells in culture. Healthy CD34+ peripheral blood treated with CDK8 inhibitors show no increase in proliferation. Finally, we show that bone marrow failure and anemia in the DBA mouse model is partially rescued (RBC and Hemoglobin levels, p Disclosures Rzymski: Selvita S.A.: Employment, Equity Ownership. Johansson:LU Holding: Patents & Royalties. Lundbäck:LU Holding: Patents & Royalties. Mazan:Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

This article reviews the regulation of production of red blood cells at several levels: (1) the ability of erythropoietin and adhesion to a fibronectin matrix to stimulate the rapid production of red cells by inducing terminal proliferation and differentiation of committed erythroid CFU-E progenitors; (2) the regulated expansion of the pool of earlier BFU-E erythroid progenitors by glucocorticoids and other factors that occurs during chronic anemia or inflammation; and (3) the expansion of thehematopoietic cell pool to produce more progenitors of all hematopoietic lineages., National Institutes of Health (U.S.) (grant P01 HL 32262), National Institutes of Health (U.S.) (grant DK06735), Amgen Inc., Swedish Research Council, Maja och Hjalmar Leanders Stiftelse, National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award, Sweden-America Foundation

Pellet Cladding Interaction (PCI) during power changes can be a limiting factor for nuclear reactors operation as it can cause fuel cladding failure by Iodine-induced Stress Corrosion Cracking (ISCC) [1]. In the as-fabricated condition, there is a gap between the fuel cladding and the pellets. During reactor operation when fast power transient changes occur, the fuel experiences power ramp and the pellets expand more than the cladding, such that the gap is reduced and eventually closed. When the fuel and the cladding are in direct contact, further fuel swelling and thermal expansion produces tensile stresses in the cladding. Moreover, pellet cracking occurs causing the stress to increase locally in the cladding at the location of the cracks. ISCC initiates at locations where both the stress and the iodine content locally reach critical levels. The cracking initiates at the inner surface of the cladding and propagates towards the outer surface [2, 3].ISCC can be investigated out of pile by means of an expanding mandrel test technique [4, 5]. In this design of the mandrel test equipment, the fuel pellet is simulated using a ceramic sheath with 4 longitudinal slits to facilitate the transport of iodine inside the cladding, which contains a Zr slug (mandrel) inserted in the center. The Zr slug is axially compressed and expands radially forcing the surrounding ceramic sheath to crack. The ceramic fragments can strain the cladding with further compression of the Zr slug.Different versions of the mandrel technique have previously been used to test unirradiated cladding. However, previous studies show that ISCC is sensitive to the condition of the cladding inner surface as well as the material strength, thus the irradiated cladding is expected to behave differently with respect to ISCC than unirradiated cladding.To evaluate the susceptibility to PCI failure of a given fuel rod, power ramp tests are performed in experimental reactors [2]. However, a good out of pile test to complement single ramp tests is crucial to improve the understanding of the mechanisms behind the PCI failures as well as the effect of different parameters.Within the SCIP program3 , in addition to ramp testing, a mandrel testing technique that enables out-of-pile testing of irradiated cladding has therefore been developed. The equipment is designed to investigate critical parameters that are known to affect PCI failures such as: irradiation, the strain rate, pellet performance (expansion and cracking), manufacturing (missing pellet surface) and cladding properties (texture, hardness, liner composition etc) [2].The tests are conducted under carefully controlled environmental conditions. Ar gas (99,99%) is passed through a temperature-controlled bath with iodine crystals (reagent grade 99,8%).The effect of the following parameters can be investigated:- Strain rate,- Iodine concentration,- Oxygen potential,- Temperature,- Different crack pattern of the pellet or missing chip,- Cladding properties.The mandrel equipment at Studsvik is specially designed and installed in a hot cell to enable testing on irradiated cladding. The mandrel test technique has been qualified within SCIP using unirradiated cladding. The effects of stress/strain rate, iodine concentration and oxygen potential, on the ISCC behavior were investigated by mandrel testing coldworked non-liner Zircaloy-2 cladding materials.After qualification of the test equipment, several tests have been performed on irradiated non-liner recrystallized Zircaloy-2 claddings. The tests were performed at a temperature of 320°C and iodine partial pressure in the range of 0 to 200 Pa. The tests were run by controlling the strain rate with an initial fast strain rate (21.6%/h) up to 0.6% strain from the initial diameter followed by a slow strain rate (0.36%/h) up to a strain of 2% and with a holding time of 20 hours or until failure. After the mandrel tests, the samples have been characterized by visual inspection, profilometry, LOM and SEM4 .The results of the irradiated recrystallized Zircaloy-2 claddings showed that in the presence of iodine partial pressures in the range of 60 to 200 Pa, ISCC occurred at 0.7-0.78% total (elastic + plastic) diametral strain. The low strain to failure as well as typical ISCC features like fluting, observed at the fractured surfaces, are consistent with previous hot cell examinations of PCI failures, and thus the new expanding mandrel technique seems promising.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of Mdm2, the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.Leukemia accepted article preview online, 19 May 2015. doi:10.1038/leu.2015.128.