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Diamond-Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is associated with anemia, congenital anomalies, and cancer predisposition. It is categorized as a ribosomopathy, because more than 80% or patients have haploinsufficiency of either a small or large subunit-associated ribosomal protein (RP). The erythroid pathology is due predominantly to a block and delay in early committed erythropoiesis with reduced megakaryocyte/erythroid progenitors (MEPs). To understand the molecular pathways leading to pathogenesis of DBA, we performed RNA sequencing on mRNA and miRNA from RPS19-deficient human hematopoietic stem and progenitor cells (HSPCs) and compared existing database documenting transcript fluctuations across stages of early normal erythropoiesis. We determined the chromatin regulator, SATB1 was prematurely downregulated through the coordinated action of upregulated miR-34 and miR-30 during differentiation in ribosomal insufficiency. Restoration of SATB1 rescued MEP expansion, leading to a modest improvement in erythroid and megakaryocyte expansion in RPS19 insufficiency. However, SATB1 expression did not affect expansion of committed erythroid progenitors, indicating ribosomal insufficiency affects multiple stages during erythroid differentiation.

Supplementary Figures 1-2 from ERBB Receptor Activation Is Required for Profibrotic Responses to Transforming Growth Factor β

Supplementary Results from ERBB Receptor Activation Is Required for Profibrotic Responses to Transforming Growth Factor β

Supplementary Table 1 from ERBB Receptor Activation Is Required for Profibrotic Responses to Transforming Growth Factor β

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with severe anemia, congenital malformations, and an increased risk of developing cancer. The chromatin-binding special AT-rich sequence-binding protein-1 (SATB1) is downregulated in megakaryocyte/erythroid progenitors (MEPs) in patients and cell models of DBA, leading to a reduction in MEP expansion. Here we demonstrate that SATB1 expression is required for the upregulation of the critical erythroid factors heat shock protein 70 (HSP70) and GATA1 which accompanies MEP differentiation. SATB1 binding to specific sites surrounding the HSP70 genes promotes chromatin loops that are required for the induction of HSP70, which, in turn, promotes GATA1 induction. This demonstrates that SATB1, although gradually downregulated during myelopoiesis, maintains a biological function in early myeloid progenitors.

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 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.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is typically associated with mutations in one of at least 20 ribosomal genes and is associated with anemia, congenital abnormalities, and cancer predisposition. The current treatment for DBA is associated with toxicities including iron overload from repeated transfusions, immune suppression from chronic corticosteroid therapy or consequences from stem cell transplantation. Developing new therapies for DBA remains a challenge, since it is a rare disease and the connection between faulty ribosomes and defects in erythropoiesis is still being explored. As such, repurposing existing, approved drugs is one approach to find new ways to treat this disease. We recently identified that Nemo-like Kinase (NLK) is hyper-activated and contributes to disease pathogenesis in the erythroid progenitors of patients with DBA, irrespective of the genetic mutation. NLK is an atypical member of the MAPK family of kinases. Due to a high degree of conservation, kinase inhibitors that specifically target NLK are not currently available, although several small molecules inhibit NLK as an off-target. We are actively pursuing a number of these compounds as potential therapies for DBA. In addition to known kinase inhibitors, we have examined the active components of widely available nutritional supplements. Although rigorous clinical evaluation is required, these supplements are well tolerated and offer an alternative or a complement to conventional drug therapy for DBA. Ginsenoside Rb1, an active component of ginseng, increases erythropoiesis in in vitro models of DBA (2.6-fold) at 50mM (p=0.048) with an EC 50 of 2.3mM. Importantly, ginsenoside Rb1 does not impact healthy erythropoiesis or other hematopoietic lineages and is nontoxic to normal cells. Our results demonstrate that ginsenoside Rb1 does not inhibit NLK kinase activity directly, but rather induces a microRNA (miR-208) that binds to NLK mRNA leading to degradation of transcript by 35.9% (p=0.007) before the protein can be translated. Another nutritional supplement that has been indicated to improve erythropoiesis (in DBA and non-diseased models) and is currently in clinical trials is the amino acid leucine. In DBA progenitors, 5mM leucine increases erythropoiesis from 8.8 to 16.3% of healthy controls. Leucine acts by stimulating the activity of mTORC1 but similar to erythropoiesis, mTORC1 activity is only stimulated from 26.4 to 57.2% of controls in DBA progenitors. Our results demonstrate that leucine does not impact NLK expression or activity directly. Aberrantly activated NLK phosphorylates and inhibits the activation of mTORC1 (target of leucine). The suppression of NLK by 50mM ginsenoside Rb1 restored mTORC1 activity to basal (94.7% of non-diseased control) but also restored leucine sensitivity (from 57.2 to 88.1% of non-diseased controls). Importantly, combining 5mM leucine and 50mM ginsenoside Rb1 increased erythropoiesis from 1.9-fold and 2.6-fold when used alone, to 8.9-fold (or 78.3% of healthy controls) together in our in vitro models of DBA (CI=0.31). Ginseng and leucine offer promising alternatives to steroids and other immunosuppressive drugs for DBA patients. The goal of these studies is to raise the hemoglobin in DBA patients to avoid the need for red cell transfusions. As nutritional supplements are widely available and well tolerated, this class of compounds provides alternatives to currently approved drugs to treat DBA. Disclosures Glader: Agios: Consultancy.

Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

Blood cell development is regulated through intrinsic gene regulation and local factors including the microenvironment and cytokines. The differentiation of hematopoietic stem and progenitor cells (HSPCs) into mature erythrocytes is dependent on these cytokines binding to and stimulating their cognate receptors and the signaling cascades they initiate. Many of these pathways include kinases that can diversify signals by phosphorylating multiple substrates and amplify signals by phosphorylating multiple copies of each substrate. Indeed, synthesis of many of these cytokines is regulated by a number of signaling pathways including phosphoinositide 3-kinase (PI3K)-, extracellular signal related kinases (ERK)-, and p38 kinase-dependent pathways. Therefore, kinases act both upstream and downstream of the erythropoiesis-regulating cytokines. While many of the cytokines are well characterized, the nuanced members of the network of kinases responsible for appropriate induction of, and response to, these cytokines remains poorly defined. Here, we will examine the kinase signaling cascades required for erythropoiesis and emphasize the importance, complexity, enormous amount remaining to be characterized, and therapeutic potential that will accompany our comprehensive understanding of the erythroid kinome in both healthy and diseased states.

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.

The del(5q) myelodysplastic syndrome (MDS) is a distinct subtype of MDS, associated with deletion of the ribosomal protein S14 (RPS14) gene that results in macrocytic anemia. This study sought to identify novel targets for the treatment of patients with del(5q) MDS by performing an in vivo drug screen using an rps14-deficient zebrafish model. From this, we identified the secreted gelatinase matrix metalloproteinase 9 (MMP9). MMP9 inhibitors significantly improved the erythroid defect in rps14-deficient zebrafish. Similarly, treatment with MMP9 inhibitors increased the number ofcolony forming unit-erythroid colonies and the CD71+erythroid population from RPS14 knockdown human BMCD34+cells. Importantly, we found that MMP9 expression is upregulated in RPS14-deficient cells by monocyte chemoattractant protein 1. Double knockdown of MMP9 and RPS14 increased the CD71+population compared with RPS14 single knockdown, suggesting that increased expression of MMP9 contributes to the erythroid defect observed in RPS14-deficient cells. In addition, transforming growth factorβ(TGF-β) signaling is activated in RPS14 knockdown cells, and treatment with SB431542, a TGF-βinhibitor, improved the defective erythroid development of RPS14-deficient models. We found that recombinant MMP9 treatment decreases the CD71+population through increased SMAD2/3 phosphorylation, suggesting that MMP9 directly activates TGF-βsignaling in RPS14-deficient cells. Finally, we confirmed that MMP9 inhibitors reduce SMAD2/3 phosphorylation in RPS14-deficient cells to rescue the erythroid defect. In summary, these study results support a novel role for MMP9 in the pathogenesis of del(5q) MDS and the potential for the clinical use of MMP9 inhibitors in the treatment of patients with del(5q) MDS.

The induction of the master erythroid transcription factor, GATA1 during early erythropoiesis is critical for efficient red blood cell production. However, GATA1 is expressed at low levels in hematopoietic stem cells (HSCs) and is moderately induced at both the common myeloid progenitor (CMP) and megakaryocyte/erythroid progenitor (MEP) stages prior to lineage commitment. Diamond Blackfan Anemia is a rare disease, usually associated with ribosomal gene mutations, leading to significant decrease in GATA1 expression and block in early committed erythroid differentiation. Mild defects in other myeloid lineages are also observed, with limited clinical relevance. The importance of GATA1 downregulation in disease pathogenesis is manifested by rare patients with DBA carrying GATA1 mutations. To understand signaling pathways that contribute to the pathogenesis DBA, we perform RNA-seq with mRNA from human CD34+ fetal liver cells and found that the chromatin organizer, Special AT-rich sequence binding protein 1 (SATB1) was prematurely downregulated. Our results further demonstrated that sustained SATB1 expression is critical to maintain required levels of GATA1 protein at both the CMP and MEP stages of differentiation, but not in committed erythroid progenitors. In mice, SATB1 is modestly expressed in HSCs and upregulated during lymphopoiesis. SATB1 is downregulated during myeloid and erythroid differentiation and antagonizes myeloid and erythroid expansion. However, in human hematopoietic stem and progenitor cells (HSPCs), SATB1 is required for efficient expansion of these lineages. SATB1 maintains 78% expression in human MEPs, but is undetectable in early committed erythroid progenitors. In RPS19-insufficient human HSPCs, SATB1 was downregulated to 22% in MEPs (p=0.02). Re-expression of SATB1 corrected a significant subset of deregulated mRNAs, including GATA1 regulators. In the absence of SATB1, one such GATA1 regulator, heat shock protein 70 (HSP70), failed to be induced in ribosome-competent human MEPs, reducing GATA1 protein expression by 35.7% (p= 0.026). Concurrently, MEP expansion was inhibited by 64.5% (p=0.023), reducing erythroid and megakaryocyte expansion by 18.2% (p=0.024) and 20.4% (p=0.183) respectively. SATB1 facilitated the formation of chromatin loops linking together an enhancer element with HSP70 promoters required for HSP70 induction in early differentiation. Although GATA1 is significantly upregulated in committed erythroid progenitors, RPS19-insufficient human CD235+ erythrocytes express GATA1 28.4% of controls (p= 0.011). SATB1 re-expression increased GATA1 expression to 31.4% (p=0.089). Similarly, SATB1 re-expression increased CD235+ expansion from 13.9% to 39.5% (p=0.02) compared to controls. Our data indicate that premature SATB1 downregulation contributes to erythroid failure in DBA by reducing MEP expansion, but aberrant GATA1 expression observed in more mature erythrocytes is predominantly SATB1-independent. However, SATB1-re-expression improved CD11b+ myeloid expansion from 81.2% to 90.4% (p=0.045) and CD41a+ megakaryocyte expansion from 76.7% to 214.7% (p=0.038) respectively. Our results demonstrate that SATB1 plays an important role in human hematopoiesis and is an important regulator of GATA1. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

Introduction Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that leads to the accumulation of immature blasts in the blood and bone marrow. While the 5-year relative survival rate has increased from 6.4% to 28.7% in the last 40 years, death rates have remained steady at 3-4%. Less toxic, more effective targeted treatments are needed to improve outcomes for patients with AML. Previous studies identified cAMP response element-binding protein (CREB) as a potential therapeutic target for AML therapy. CREB is overexpressed in AML and is associated with poor prognosis. We identified a small molecule, XX-650-23, that inhibits interaction between CREB and its co-activator, CREB Binding Protein. However, XX-650-23 does not have optimal physical chemical properties for clinical application in humans. Niclosamide, an FDA approved anthelmintic drug, shares structural similarity with XX-650-23, and suppresses the proliferation of AML cells in vitro and in vivo by inhibiting CREB-dependent signaling. The aim of this study is to define the molecular pathways that mediate the effects of niclosamide in AML cells. This will allow for more precise selection of patient populations for treatment, identify potential sources of niclosamide resistance and reveal combination therapies that make use of the distinct pathways targeted. Methods To identify genetic factors modulating cellular sensitivity to niclosamide treatment, we performed a CRISPR/Cas9 library screen in the presence or absence of niclosamide. HL60 cells expressing Cas9 were infected with a custom sgRNA lentivirus library of 220 genes from a previous genome-wide shRNA screen. After puromycin selection, HL60 cells expressing pooled sgRNAs were grown in either niclosamide or 0.1% DMSO for 20 days. Genomic DNA was extracted from control and niclosamide-treated cells. Frequency of sgRNA were measured by next generation sequencing and analyzed using the Cas9 high-throughput maximum likelihood estimator (casTLE) algorithm. Results Sixty-five gene hits were found to be significant (p≤0.05). Of the top 220 hits from the previous genome-wide shRNA screen, 26 were identified as positive hits (p ≤0.05) in the CRISPR/Cas9 knock out screen. The CRISPR screen identified genes enriched in a number of pathways including programmed cell death, nucleotide biosynthesis, mitochondrial processes and canonical glycolysis, as measured by gene ontology term analysis. Numerous gene deletions conferred resistance to niclosamide treatment. Genes DHODH (score= 123; effect= 2.9) and HSPA9 (score= 144; effect = 2.5), involved in nucleotide biosynthesis and mitochondrial processes, were significantly enriched in the surviving population. Perturbation of known metabolic pathways sensitized AML cells to niclosamide, including ATP synthase F1 subunit beta (ATP5B) (score= 316; effect= -5.1), hexokinase 2 (HK2) (score= 312; effect= -5.4), phosphofructokinase (PFKP) (score= 195; effect= -1.9) and mitochondrial phosphate carrier protein (SLC25A3) (score= 271; effect= -7.8). SLC25A3 and ATP5B play a role in oxidative phosphorylation and mitochondrial proton transmembrane transport, whereas HK2 and PFKP are key to glycolysis, suggesting that niclosamide's mechanism of action is dependent on energy metabolism. Furthermore, we found that disruption of Bcl-2 (BCL2) was sensitizing to niclosamide treatment (score= 129; effect= -4.7). To confirm the sensitizing effect of Bcl-2 inhibition on niclosamide treatment, HL60 cells were treated with niclosamide and Bc1-2 inhibitor venetoclax. Pretreatment of HL60 cells with niclosamide led to significantly decreased cell viability upon venetoclax treatment (p Overall, our study identified genes with strong signatures for cell death, nucleotide biosynthesis, mitochondrial function and metabolic processes. Compounds inhibiting genes identified in this screen could be combined with niclosamide for synergistic effect. Targets overrepresented in the surviving population represent sources of resistance to niclosamide therapy. This study provides insight into mechanisms of action and resistance in AML cells treated with niclosamide and novel targets for combination therapy. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Niclosamide is an anthelmintic, FDA approved to treat tapeworm infections.

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.

Deficiency of ribosomal proteins (RPs) leads to Diamond Blackfan Anemia (DBA) associated with anemia, congenital defects, and cancer. While p53 activation is responsible for many features of DBA, the role of immune system is less defined. The Innate immune system can be activated by endogenous nucleic acids from non-processed pre-rRNAs, DNA damage, and apoptosis that occurs in DBA. Recognition by toll like receptors (TLRs) and Mda5-like sensors induces interferons (IFNs) and inflammation. Dying cells can also activate complement system. Therefore we analyzed the status of these pathways in RP-deficient zebrafish and found upregulation of interferon, inflammatory cytokines and mediators, and complement. We also found upregulation of receptors signaling to IFNs including Mda5, Tlr3, and Tlr9. TGFb family member activin was also upregulated in RP-deficient zebrafish and in RPS19-deficient human cells, which include a lymphoid cell line from a DBA patient, and fetal liver cells and K562 cells transduced with RPS19 shRNA. Treatment of RP-deficient zebrafish with a TLR3 inhibitor decreased IFNs activation, acute phase response, and apoptosis and improved their hematopoiesis and morphology. Inhibitors of complement and activin also had beneficial effects. Our studies suggest that innate immune system contributes to the phenotype of RPS19-deficient zebrafish and human cells.

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.

Sorting nexin 9 (SNX9) is shown to differentiate Smad3 from Smad2 nuclear delivery by mediating the association of phosphorylated Smad3 with importin 8 and the nuclear membrane. While the absence of SNX9 had negligible effects on transforming growth factor β receptor activity or Smad2 signaling, Smad3-dependent targets and phenotypes were inhibited., Transforming growth factor β (TGFβ) is a pleiotropic protein secreted from essentially all cell types and primary tissues. While TGFβ’s actions reflect the activity of a number of signaling networks, the primary mediator of TGFβ responses are the Smad proteins. Following receptor activation, these cytoplasmic proteins form hetero-oligomeric complexes that translocate to the nucleus and affect gene transcription. Here, through biological, biochemical, and immunofluorescence approaches, sorting nexin 9 (SNX9) is identified as being required for Smad3-dependent responses. SNX9 interacts with phosphorylated (p) Smad3 independent of Smad2 or Smad4 and promotes more rapid nuclear delivery than that observed independent of ligand. Although SNX9 does not bind nucleoporins Nup153 or Nup214 or some β importins (Imp7 or Impβ), it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates nuclear translocation of pSmad3 but not SNX9.

The role of non-coding Ribonucleic Acids (ncRNAs) in biology is currently an area of intense focus. Hematopoiesis requires rapidly changing regulatory molecules to guide appropriate differentiation and ncRNA are well suited for this. It is not surprising that virtually all aspects of hematopoiesis have roles for ncRNAs assigned to them and doubtlessly much more await characterization. Stem cell maintenance, lymphoid, myeloid and erythroid differentiation are all regulated by various ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and various transposable elements within the genome. As our understanding of the many and complex ncRNA roles continues to grow, new discoveries are challenging the existing classification schemes. In this review we briefly overview the broad categories of ncRNAs and discuss a few examples regulating normal and aberrant hematopoiesis.

After basolateral (BL) cell surface delivery, retromer promotes type II TGF-β receptor exit and recycling to the BL plasma membrane. In the absence of retromer, however, type II receptors aberrantly sort and are mislocalized such that both BL and apical expression is observed independent of the Rab11-positive apical recycling endosome., Transforming growth factor β (TGF-β) is critical for the development and maintenance of epithelial structures. Because receptor localization and trafficking affect the cellular and organismal response to TGF-β, the present study was designed to address how such homeostatic control is regulated. To that end, we identify a new role for the mammalian retromer complex in maintaining basolateral plasma membrane expression of the type II TGF-β receptor (TβRII). Retromer and TβRII associate in the presence or absence of TGF-β ligand. After retromer knockdown, although TβRII internalization and trafficking to a Rab5-positive compartment occur as in wild-type cells, receptor recycling is inhibited. This results in TβRII mislocalization from the basolateral to both the basolateral and apical plasma membranes independent of Golgi transit and the Rab11-positive apical recycling endosome. The data support a model in which, after initial basolateral TβRII delivery, steady-state polarized TβRII expression is maintained by retromer/TβRII binding and delivery to the common recycling endosome.

TGF-β modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that non-Smad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-β. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-β treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-β-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-β and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases.

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome. The disease usually presents within the first year of life and is associated with anemia, congenital abnormalities, and an increased risk of developing cancer. The most prevalent mutations are found in the ribosomal protein RPS19, accounting for over 25% of cases but approximately 70% of DBA patients carry mutations in ribosomal genes. Downregulation of GATA1 has been attributed to the disease, but the upstream mechanisms leading to aberrant erythropoiesis is only beginning to be elucidated. SATB1 is highly upregulated in lymphocytes (especially thymocytes) and steadily downregulated in all myeloid ineages during differentiation. However the more modest expression of SATB1 in early progenitors is required for self-renewal of HSCs and globin switching in early erythropoiesis. Using transcriptomics, we identified SATB1 expression is prematurely lost in RPS19-insufficient erythropoiesis and that over a third (16/42) of RPS19-sensitive early erythroid genes were rescued upon SATB1 re-expression. One of the most deregulated transcripts encode the chaperone HSP70. GATA1 is an essential master regulator in erythroid differentiation and high GATA1 protein levels are required for efficient erythropoiesis. In DBA, GATA1 protein expression is drastically diminished. Two mechanisms contribute to this. At the translational level, GATA1 transcripts fail to be translated adequately due to ribosomal inefficiency at binding short, unstructured 5'UTRs. At the protein level,HSP70 loss leads to reduced GATA1 protein stability. During healthy erythropoiesis HSP70 genes are drastically upregulated (3.21, 4.18 and 1.37- fold) and this is lost in RPS19 insufficiency. Here we report that upregulation of HSP70 in healthy erythropoiesis requires SATB1. SATB1 binds to 3 sites within a 40kb region surrounding the HSP70 gene loci and recruits a distal predicted enhancer element to the proximal promoters via formation of two chromatin loops. During RPS19-insufficiency this chromatin organization is lost, but SATB1 re-expression restores chromatin looping and HSP70 expression, stabilizing GATA1 and promoting efficient erythropoiesis. As with HSP70, it is likely that permissive chromatin organization is essential for other RPS19-sensitive erythroid genes rescued by SATB1 re-expression. Disclosures No relevant conflicts of interest to declare.

The del(5q) myelodysplastic syndrome (MDS) is a unique subtype of MDS characterized by an interstitial deletion of the long arm of chromosome 5 with macrocytic anemia, normal to increased platelet count, and hypoloblated megakaryocytes. Although haploinsufficiency of several genes encoded within the commonly deleted region have been implicated in the hematologic phenotype of del (5q) MDS, the deletion of the RPS14 gene, which encodes a component of the 40S ribosomal subunit, is a key determinant of the severe macrocytic anemia seen in patients. Lenalidomide is FDA-approved and highly effective for the treatment for del(5q) MDS. However, there are rare resistant cases resulting from p53 mutations in del(5q) MDS patients and 50% of patients acquire resistance within 2 to 3 years. Given these limitations of lenalidomide treatment, as well as frequent side effects, we sought to identify novel targets for the treatment of del(5q) MDS patients by performing an in vivo drug screening assay using a zebrafish model of MDS. We generated stable genetic mutation of zebrafish RPS14 using CRISPR-Cas9 gene targeting technique. This zebrafish model mirrors the anemic phenotype seen in the 5q-syndrome. We found that mmp9 gene expression was abnormally induced by RPS14 deficiency in zebrafish and MMP9 inhibitors partially rescued erythroid defects in RPS14-deficient zebrafish. To study the role of MMP in human hematopoietic stem cells, we treated MMP9 inhibitors in RPS14 or luciferase control knockdown human bone marrow stem and progenitor (BMCD34+) cells for 6 days and then evaluated erythroid development. Our results showed that treatment with MMP9 inhibitors in RPS14 knockdown CD34+ cells significantly increased erythroid colony numbers compared with DMSO control (1.5 to 2.5-fold increase). Similarly, FACS analysis demonstrated increased the CD71+ erythroid population in RPS14 downregulated cells by MMP9 inhibitor treatment (4-fold increase compared to control), but no difference in the CD11b+ myeloid population. This data suggests that MMP9 inhibitors have specific roles in the erythroid development in RPS14 deficient cells. To understand the mechanism of MMP9 inhibitors, we first examined MMP9 expression in RPS14-deficient cells. After transduction of human BMCD34+ cells with lentivirus expressing RPS14 shRNAs, we sorted GFP+ cells and observed MMP9 levels by RT-qPCR and Western blot analysis. We found a 4-fold increase in the expression levels of MMP9 in the RPS14 knockdown cells, supporting the hypothesis that MMP9 is increased in RPS14-deficient cells ultimately leading to decreased erythropoiesis. To determine the effects of MMP9 treatment during erythropoiesis, we treated human BMCD34+ cells with recombinant MMP9 protein (rMMP9). Cells were treated with activated rMMP9 for 2 days, and then examined by FACS analysis. Interestingly, we found that rMMP9 treatment decreased the CD71+ erythroid population by 40% compared to vehicle control. We confirmed that enhanced expression of MMP9 is critical to the negative regulation of erythroid development in RPS14 knockdown cells by a performing double knockdown experiment. We co-transduced lentivirus expressing shMMP9-mCherry and shRPS14-GFP shRNAs and examined erythropoiesis by FACS analysis. Our results demonstrated that MMP9 and RPS14 double knockdown partially rescued the decreased CD71+ population in RPS14 knockdown cells. However, MMP9 knockdown alone did not affect CD71+ erythroid development. Taken together, our data suggest that increased MMP9 contributes to the erythroid defect observed in RPS14-deficient cells. In this study, our results showed that MMP9 inhibitors rescued the erythroid defects in RPS14 downregulated del(5q) MDS models. This suggests a novel role for MMP9 in the pathogenesis of del(5q) MDS and MMP9 inhibitors in the treatment of patients with del(5q) MDS. Disclosures No relevant conflicts of interest to declare.

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.

Transforming growth factor-β receptor recycling is regulated by the clathrin adaptor Dab2 protein. In the absence of Dab2, receptors localize in a perinuclear locale because they are unable to transit from the early endosomal antigen 1-positive early endosome to the Rab11-positive endosomal recycling compartment., Transforming growth factor (TGF)-β family proteins form heteromeric complexes with transmembrane serine/threonine kinases referred to as type I and type II receptors. Ligand binding initiates a signaling cascade that generates a variety of cell type-specific phenotypes. Whereas numerous studies have investigated the regulatory activities controlling TGF-β signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-β receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-β receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-β, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-β receptor recycling was abrogated. Loss of Dab2 resulted in enlarged early endosomal antigen 1-positive endosomes, reflecting the inability of cargo to traffic from the early endosome to the endosomal recycling compartment and, as documented previously, diminished Smad2 phosphorylation. The results support a model whereby Dab2 acts as a multifunctional adaptor in mesenchymal cells required for TGF-β receptor recycling as well as Smad2 phosphorylation.

Engagement of the transforming growth factor-β (TGF-β) receptor complex activates multiple signaling pathways that play crucial roles in both health and disease. TGF-β is a key regulator of fibrogenesis and cancer-associated desmoplasia; however, its exact mode of action in these pathologic processes has remained poorly defined. Here, we report a novel mechanism whereby signaling via members of the ERBB or epidermal growth factor family of receptors serves as a central requirement for the biological responses of fibroblasts to TGF-β. We show that TGF-β triggers upregulation of ERBB ligands and activation of cognate receptors via the canonical SMAD pathway in fibroblasts. Interestingly, activation of ERBB is commonly observed in a subset of fibroblast but not epithelial cells from different species, indicating cell type specificity. Moreover, using genetic and pharmacologic approaches, we show that ERBB activation by TGF-β is essential for the induction of fibroblast cell morphologic transformation and anchorage-independent growth. Together, these results uncover important aspects of TGF-β signaling that highlight the role of ERBB ligands/receptors as critical mediators in fibroblast responses to this pleiotropic cytokine. Cancer Res; 70(19); 7421–30. ©2010 AACR.

Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-beta targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-beta in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-beta including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-beta pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-beta pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-beta pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.

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 mutation in one of at least 12 ribosomal proteins, with RPS19 being the most prevalent, accounting for over 25% of cases. In mice, complete loss of RPS19 is not viable, yet mutations that result in haploinsufficiency result in failure of erythropoiesis due to a blockage during differentiation of early erythroblast progenitors. An increase in p53 stability has been attributed to the disease, but the downstream signaling molecules mediating aberrant erythropoiesis have not been completely characterized. Here, we report that Nemo-like Kinase (NLK) is chronically hyper-activated in RPS19 insufficient CD34+ HSPCs derived from fetal liver, cord blood and mobilized peripheral blood differentiating toward the erythroblast lineage, as well as DBA-patient derived EBV-transformed Human B lymphoblastic cell lines In in vitro kinase assays, significant increases in the extent of phosphorylation of three (3) NLK kinase substrates - NLK (3.86 fold), c-Myb (5.22 fold) and Raptor (9.82 fold) was observed in the presence of immunopurified NLK during RPS19 insufficiency. This activation is due to p53-mediated upregulation of TNFα that activates NLK in neighboring cells in a paracrine fashion. In differentiating HSPCs, RPS19 insufficiency results in increased phosphorylation of the critical erythroid transcription factor c-Myb (9.6 fold) and autophagy-regulating Raptor (5.3 fold), both of which are restored to basal levels upon expression of siRNA against NLK. Phosphorylation of c-Myb by NLK results in increased ubiquitination and proteasomal degradation with subsequent loss of c-Myb-induced genes critical for transcriptional regulation of erythropoiesis (including LMO2 and KLF1). Raptor phosphorylation by NLK inhibits mTOR activation, resulting in premature autophagy in erythroblast progenitors. Knockdown of NLK using siRNAs significantly increased c-Myb downstream gene expression (p Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise as potential druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

The nonreceptor protein tyrosine kinase c-Abl regulates cell proliferation and survival. Recent studies provide evidence that implicate c-Abl as a mediator for fibrotic responses induced by transforming growth factor-β (TGF-β), but the precise mechanisms underlying this novel oncogene function are unknown. Here, we report that when expressed in normal fibroblasts, a constitutively active mutant of Abl that causes chronic myelogenous leukemia (CML) stimulated the expression and transcriptional activity of the early growth response factor 1 (Egr-1). Mouse embryonic fibroblasts (MEFs), lacking c-Abl, were resistant to TGF-β stimulation. Responsiveness of these MEFs to TGF-β could be rescued by wild-type c-Abl, but not by a kinase-deficient mutant form of c-Abl. Furthermore, Abl kinase activity was necessary for the induction of Egr-1 by TGF-β in normal fibroblasts, and Egr-1 was required for stimulation of collagen by Bcr-Abl. Lesional skin fibroblasts in mice with bleomycin-induced fibrosis of skin displayed evidence of c-Abl activation in situ, and elevated phospho-c-Abl correlated with increased local expression of Egr-1. Collectively, these results position Egr-1 downstream of c-Abl in the fibrotic response, delineate a novel Egr-1-dependent intracellular signaling mechanism that underlies the involvement of c-Abl in certain TGF-β responses, and identify Egr-1 as a target of inhibition by imatinib. Furthermore, the findings show in situ activation of c-Abl paralleling the upregulated tissue expression of Egr-1 that accompanies fibrosis. Pharmacological targeting of c-Abl and its downstream effector pathways may, therefore, represent a novel therapeutic approach to blocking TGF-β-dependent fibrotic processes.

Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. p21-activated kinase-2 (PAK2) and activation of abelson nonreceptor tyrosine kinase (c-abl) have been shown recently to be smad-independent, fibroblast-specific targets downstream of the activated TGF-beta receptor. In the current study we show that in cultured NRK49F-renal fibroblasts (but not in tubular or mesangial cells) TGF-beta similarly activates PAK2 as well as c-abl and induces cell proliferation. Inhibition of the c-abl kinase with imatinib mesylate prevents increased proliferation after TGF-beta addition without affecting PAK2. These in vitro findings were extended to rats with unilateral obstructive nephropathy, a disease model of TGF-beta-driven renal fibrogenesis. In obstructed kidneys, PAK2 and c-abl activity were increased but only c-abl activation was blocked by imatinib. Treatment with imatinib did not prevent renal interstitial infiltration of macrophages or phosphorylation and nuclear translocation of smad2/3 in obstructed kidneys. In contrast, imatinib substantially inhibited an increase in the number of interstitial fibroblasts and myofibroblasts and reduced the expression and interstitial accumulation of collagen type III, collagen type IV and fibronectin. These findings indicate that TGF-beta-induced activation of the nonreceptor c-abl tyrosine kinase regulates fibroblast proliferation and, by this means, is a costimulatory signal in TGF-beta-dependent renal fibrogenesis. Inhibition of c-abl activity with imatinib mesylate ameliorates experimental renal fibrosis in rats.

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.

Transforming growth factor beta (TGF-beta) causes growth arrest in epithelial cells and proliferation and morphological transformation in fibroblasts. Despite the ability of TGF-beta to induce various cellular phenotypes, few discernible differences in TGF-beta signaling between cell types have been reported, with the only well-characterized pathway (the Smad cascade) seemingly under identical control. We determined that TGF-beta receptor signaling activates the STE20 homolog PAK2 in mammalian cells. PAK2 activation occurs in fibroblast but not epithelial cell cultures and is independent of Smad2 and/or Smad3. Furthermore, we show that TGF-beta-stimulated PAK2 activity is regulated by Rac1 and Cdc42 and dominant negative PAK2 or morpholino antisense oligonucleotides to PAK2 prevent the morphological alteration observed following TGF-beta addition. Thus, PAK2 represents a novel Smad-independent pathway that differentiates TGF-beta signaling in fibroblast (growth-stimulated) and epithelial cell (growth-inhibited) cultures.

Approximately 53% of patients with Diamond-Blackfan Anemia (DBA) have mutations in one of nine ribosomal protein components (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPL5, RPL11 and RPL35A), with RPS19 being the most commonly affected (25% of all patients). Mutations found in patients result in protein haploinsufficiency and subsequent ribosomal stress, which increases the expression and activity of p53, leading to a failure of HSC differentiation during erythropoiesis. While RPS19-/- mice are not viable, we have generated a model system using different shRNA constructs to generate varying degrees of RPS19 knockdown in cultured cells and animal models. Cultured cells (including CD34+ HSCs derived from fetal liver or cord blood) recapitulate features of DBA erythropoietic failure in vitro and in vivo, including p53 upregulation and defects in erythrocyte differentiation, while anemias and skeletal defects are observed in zebrafish models. Using next-generation miRNA sequencing on the Illumina HiSeq2000 platform, we identified a number of miRNAs differentially expressed between human fetal liver CD34+ HSCs expressing normal or reduced RPS19. Preprocessing, alignment, and miRNA quantification were carried out using miRDeep2 [1] software. Differential expression analysis was performed with DESeq2 [2], which tests for differential expression based on a model using negative binomial distribution for count data from high-throughput sequencing assays. Candidates underwent a second round of screening based on robustness of expression differential (fold increase/decrease, variation of fold increase/decrease across replicates, and increase/decrease relative to RPS19 expression) and possible links to hematopoiesis and erythropoiesis, as reported in the scientific record. All three miR-34 isoforms (a, b and c) are robustly upregulated upon RPS19 reduction, with the extent of miR-34 upregulation being inversely proportional to RPS19 expression. When RPS19 protein expression is suppressed by approximately 50% (as observed in DBA patients), miR-34a, b and c isoforms are upregulated by 3.2, 2.2 and 2.6 fold respectively. In other systems, increased miR-34 results in modulation of a number of factors linked to erythropoiesis, including downregulation of c-Myc, Myb and NOTCH signaling. Genome-wide transcriptional analysis using next-generation sequencing on the Illumina HiSeq platform in CD34+ HSCs indicates c-Myc (4.2 fold), c-Myb (2.8 fold) and a number of NOTCH effectors [SIRT1 (1.8 fold) andHES1 (1.9 fold)] are indeed downregulated at the transcriptional level. Results were validated with qRT-PCR and western blot analysis, however, while transcriptional modulation fully accounts for c-Myc downregulation at the mRNA and protein level, we propose that c-Myb protein levels are modulated both transcriptionally and post-translationally, as protein expression is significantly further downregulated than mRNA message (6.3 fold). We propose that miR-34 upregulation in RPS19-depleted cells occurs through p53 and initiates genomic changes incompatible with erythropoiesis through downregulation of transcription factors c-Myc, c-Myb and the NOTCH signaling pathway. Disclosures No relevant conflicts of interest to declare.

Transforming growth factor β (TGFβ) has significant profibrotic activity both in vitro and in vivo. This reflects its capacity to stimulate fibrogenic mediators and induce the expression of other profibrotic cytokines such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF/ErbB) ligands. Here we address both the mechanisms by which TGFβ induced ErbB ligands and the physiological significance of inhibiting multiple TGFβ-regulated processes. The data document that ErbB ligand induction requires PDGF receptor (PDGFR) mediation and engages a positive autocrine/paracrine feedback loop via ErbB receptors. Whereas PDGFRs are essential for TGFβ-stimulated ErbB ligand up-regulation, TGFβ-specific signals are also required for ErbB receptor activation. Subsequent profibrotic responses are shown to involve the cooperative action of PDGF and ErbB signaling. Moreover, using a murine treatment model of bleomycin-induced pulmonary fibrosis we found that inhibition of TGFβ/PDGF and ErbB pathways with imatinib plus lapatinib, respectively, not only prevented myofibroblast gene expression to a greater extent than either drug alone, but also essentially stabilized gas exchange (oxygen saturation) as an overall measure of lung function. These observations provide important mechanistic insights into profibrotic TGFβ signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFβ.—Andrianifahanana, M., Wilkes, M. C., Gupta, S. K., Rahimi, R. R., Repellin, C. E., Edens, M., Wittenberger, J., Yin, X., Maidl, E., Becker, J., Leof, E. B. Profibrotic TGFβ responses require the cooperative action of PDGF and ErbB receptor tyrosine kinases.

Transforming growth factor-β (TGF-β) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-β signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-β has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-β signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-β. We show that TGF-β activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2–dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-β–mediated anchorage-independent growth without affecting TGF-β transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-β action. mTORC2 promotes TGF-β–induced morphologic transformation and is required for TGF-β–induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-β–mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-β and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia. [Cancer Res 2009;69(1):84–93]

Transforming growth factor β (TGF-β) signaling via Smad proteins occurs in various cell types. However, whereas the biological response to TGF-β can be as distinct as growth promoting (i.e., mesenchymal cells) versus growth inhibiting (i.e., epithelial cells), few discernible differences in TGF-β signaling have been reported. In the current study, we examined the role of Ras in the proliferative response to TGF-β and how it might interface with Smad-dependent and Smad-independent TGF-β signaling targets. TGF-β stimulated Ras activity in a subset of mesenchymal, but not epithelial, cultures and was required for extracellular signal-regulated kinase (ERK)–dependent transcriptional responses. Although dominant negative Ras had no effect on TGF-β internalization or Smad-dependent signaling (i.e., phosphorylation, nuclear translocation, or SBE-luciferase activity), it did prevent the hyperphosphorylation of the Smad transcriptional corepressor TG-interacting factor (TGIF). This was not sufficient, however, to overcome the mitogenic response stimulated by TGF-β, which was dependent on signals downstream of p21-activated kinase 2 (PAK2). Moreover, although the initial activation of Ras and PAK2 are distinctly regulated, TGF-β–stimulated PAK2 activity is required for Ras-dependent ERK phosphorylation and Elk-1 transcription. These findings show the requirement for crosstalk between two Smad-independent pathways in regulating TGF-β proliferation and indicate that the mechanism(s) by which TGF-β stimulates growth is not simply the opposite of its growth inhibitory actions. [Cancer Res 2007;67(8):3673–82]

Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.

Transforming growth factor-β (TGF-β) stimulates cellular proliferation and transformation to a myofibroblast phenotype in vivo and in a subset of fibroblast cell lines. As the Smad pathway is activated by TGF-β in essentially all cell types, it is unlikely to be the sole mediator of cell type–specific outcomes to TGF-β stimulation. In the current study, we determined that TGF-β receptor signaling activates phosphatidylinositol 3-kinase (PI3K) in several fibroblast but not epithelial cultures independently of Smad2 and Smad3. PI3K activation occurs in the presence of dominant-negative dynamin and is required for p21-activated kinase-2 kinase activity and the increased proliferation and morphologic change induced by TGF-β in vitro.

Idiopathic pulmonary fibrosis is a progressive and fatal fibrotic disease of the lungs with unclear etiology. Prior efforts to treat idiopathic pulmonary fibrosis that focused on anti-inflammatory therapy have not proven to be effective. Recent insight suggests that the pathogenesis is mediated through foci of dysregulated fibroblasts driven by profibrotic cytokine signaling. TGF-beta and PDGF are 2 of the most potent of these cytokines. In the current study, we investigated the role of TGF-beta-induced fibrosis mediated by activation of the Abelson (Abl) tyrosine kinase. Our data indicate that fibroblasts respond to TGF-beta by stimulating c-Abl kinase activity independently of Smad2/3 phosphorylation or PDGFR activation. Moreover, inhibition of c-Abl by imatinib prevented TGF-beta-induced ECM gene expression, morphologic transformation, and cell proliferation independently of any effect on Smad signaling. Further, using a mouse model of bleomycin-induced pulmonary fibrosis, we found a significant inhibition of lung fibrosis by imatinib. Thus, Abl family members represent common targets for the modulation of profibrotic cytokine signaling.

Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. We therefore investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding.