Amendment #1: Dose escalation scheme modified to a 3+3 study design. Starting dose level revised to 0.125 mg/kg and subsequent cohort(s) maximum dose level will not exceed 1.0 mg/kg. Duration of patient participation updated to 28 weeks, with an increase in the follow-up period from 8 to 12 weeks. Inclusion criteria modified to clarify inclusion of patients who have no alternative treatment options. Inclusion criteria modified to clarify patient understands and is able to provide written informed consent. Replace Exclusion Criteria specifying exclusionary disorders with an Exclusion Criteria giving judgement to investigator.

Changed definition of erythroid response in transfusion dependent patients to include a reduction of ≥ 4 units/8 weeks, which is consistent with the IWG guidelines for HI-E. Addition of FACT-An assessment as exploratory objective, exploratory endpoint. Increased number of cohorts from 5 to 7 and patient total from 60 to 72 to accomodate higher dose levels. Increased max dose from 1.0 mg/kg to 1.75 mg/kg. Inclusion criteria 3 modified to change definition of TD patients to include patients who recieve transfusions within 8 weeks prior to C1D1 for any reason. Addition of exclusion criteria to prevent enrollment of subjects previously treated with ACE-536 or sotatercept or transfusions within 7 days of C1D1. New post treatment follow up visit added. Dose-limiting toxicity was changed to treatment-emergent SAE of grade 3 or higher. ITT Population redefined to be consistent with safety population. EE population redefined to include maximum number of patients with evaluable data. Prolonged collection of transfusion history added. Toxicology section updated with information from ACE-536 IB Ver 004.

Updated dose modification and titration rules for expansion cohort. Clarification added to birth control inclusion criteria. Additional pregnancy tests added to SOE. Clarification added that post treatment follow up visit to be performed +/- 7 days. Added 2 day window for Day 85 visit. Updated language to respresent currently ongoing toxicity studies in rabbits. Updated language implemented to provide objective measures for determining RBC transfusion requirements during ACE-536 treatment period. Dose modification rules updated to restrict dose reductions to dose levels tested in the escalation cohorts. Updated dose modification rules to replace 25% dose reductions with defined dose levels that were reviewed by the SRT in the escalation phase. Clarified that molecular testing of bone marrow aspirate may be performed. Updated to clarify that any available data prior to enrollment may be used to confirm eligibility. C1D1 results are not required to confirm eligibility.

Addition of recently approved INN for investigational product. Addition of ability to include other QOL tools to evaluate patient outcomes. Approximately 50 additional patients split into two groups (2A and 2B) are to be enrolled, in order to gain experience in additional patient populations that have not been well represented by patients enrolled under the current protocol. Updated dose escalation table to reflect the addition of expansion cohort 2 and the planned total number of patients from ‘up to 72’ to ‘up to 128’. Inclusion criteria updated to add enrollment criteria for expansion cohort 2 patients. Updated Study Drug Packaging, Labeling and Storage to include information on the lyophilized powder formulation which may be used in this study. Statistical language added to justify sample size of 50 patients in expansion cohort 2. Removed QoL Questionnaire at C5D1 for expansion cohort 2 patients only. Added transfusion frequency evaluation for the Post-Treatment Follow up visit. Clarification of the bone marrow aspirate/biopsy requirements for dose escalation in expansion cohort 1 and 2. Clarification that bone biomarkers are not required for expansion cohort 2 patients. Clarification of visit schedule for patients with dose delay. Updated to allow patients that have initiated iron chelation after C1D1 to continue on study. Provide further clarification on the use of “pre-transfusion hemoglobin threshold” during study. Updated Patient Dose with the starting dose of 1.0 mg/kg and the maximum dose level of 1.75 mg/kg. Revised dose administration limit to four injections and increased the volume per injection to accommodate doses with a total volume > 4 mL.

Number of study centres increased from 15 to 20. Cohort definitions expanded to increase feasibility of enrollment of patients in expansion cohorts. Additional reason added for lack of effect for patients who have not clearly progressed, however are not receiving benefit from treatment. Removed restriction of requiring 24 hours between a RBC transfusion and dosing because no longer considered a risk. Language added to include option to delay patient dose. Language added to better define reporting of adverse events of special interest based on investigator’s brochure.

Medical monitor updated. Expansion cohort 3 added to enroll RS-, EPO < 500 U/L, transfused ≤ 6 RBC units, and ESA-naïve patients to further understand response to ACE-536 in this population. Total number of patients increased from ‘up to 128’ to ‘up to 153’ throughout to reflect addition of expansion cohort 3. Clarification added to Inclusion Criteria(s) to incorporate cohort specific requirements. Peripheral blasts added to hematology. Additional criteria added to comply with new additions to dose modification rules. WBC dose modification rule edited to detail actions to be taken to ensure patients are not treated if showing signs of disease progression. Additional dose modification rule added to ensure patients are not treated if showing signs of disease progression. Language and corresponding reference added to better clarify use of IPSS-R for assessment of adverse events of special interest. Providing details of IPSS-R scoring system due to addition of assessment of adverse events of special interest by IPSS-R.