Anemia as associated with end-stage renal disease, chemo- and radiation therapies, inflammation, myelodysplasia, iron dysregulation and hemoglobinopathies leads to chronic bouts of accelerated, yet stressed and often compromised erythropoiesis. A specific understanding of key regulators of such stress erythropoiesis is of basic importance for insight into new anemia therapies. rhEPO per se can be therapeutic, but has substantial hypertensive side-effects, evokes thrombolytic events, is a high-cost recombinant glycoprotein, and may worsen certain cancers. Via investigations of EPO's effects on primary erythroid progenitors, we have uncovered and initially characterized two important new factors (and associated pathways) that selectively regulate stress erythropoiesis. First, our studies of a novel EPO-induced intracellular Spi2A serpin (via a new KO model) reveal that under select stress conditions, erythroblasts become highly sensitive to lysosome damage to the extent of leaching lysosomal executioner cathepsins- which are inhibited by Spi2A. If unchecked, cathepsin-associated damage and erythroblast death escalate, with ROS and iron as cytotoxic co-factors. We also demonstrate that a Cathepsin B/L inhibitor can phenocopy Spi2A's cytoprotective effects. A second EPO co-induced erythropoietic regulator we have discovered (and are beginning to understand) is the molecular adaptor and pseudokinase, Trib3. By developing a Trib3-KO mouse model, we reveal non-redundant novel stress erythropoiesis specific roles for Trib3 during both early, and late- stage erythroid cell formation We will now advance insight into these two new stress erythropoiesis factors, and coupled pathways, via the following SPECIFIC AIMS: SA#1 will first define how Spi2A and linked pathways impact on stress erythropoiesis due to ?-thalassemia and iron imbalance (#1.1). At a mechanistic level, consequences of Spi2A- deficiency on erythroblast autophagy will be determined and molecular damage incurred by erythroblast lysosomes during oxidative stress plus Spi2A-deficiency will be defined (#1.2). Via LOF studies, SA#2 first will define effects exerted by Trib3 pseudokinase on early- and late-stage erythropoiesis during ?-thalassemia, and iron imbalance (#2.1). Aim 2.2 will determine Trib3's roles in unfolded protein response pathways together with Trib3's regulatory molecular action mechanisms in early- and late- stage erythroid cells. SA#3 will advance studies to primary human erythroid progenitors (including thalassemia), and will first determine effects of lysosomal compromise, and cathepsin inhibition on erythroblast cytoprotection (#3.1). Aim 3.2 will define effects of Trib3 LOF and GOF during erythropoietic stress. Substantial insight will be gained concerning new regulators of red cell formation and novel target pathways within compromised erythroid progenitors for anti- anemia agents.