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Results: 155 patients underwent allogeneic HCT for AML at our institution during the study period. Disease status was defined as CR in 80 (52%), CRi in 55 (35%) and AD in 20 (13%). There were no significant demographic or transplant characteristics between the groups. The 5-year probability of survival for CR patients was 51.3% (95%CI 40.2-65.4), compared to 24.4% (95%CI 14.1-42.4) for CRi and 12.7% (95%CI 3.6-45.2) for AD (p<0.001) (adjusted HR 1.92 (95% CI 1.20-3.06, p = 0.006) for CRi and 2.82 (95% CI 1.39-5.72, p < 0.001) for AD). 100 day NRM was 6.3%, 23.6% and 35.0% for CR, CRi and AD groups respectively (p < 0.001), with 5-year with cumulative incidence of NRM of 26.8%, 46.8% and 48.1% (p < 0.001). The cumulative incidence of relapse at two years was higher for the AD group (36.9%), but did not differ significantly between the CR and CRi groups (19.3% vs 20.6%, p = 0.86). These finding persistent in MRD negative patients (n = 107), with 5 year OS 37.5% vs 56.7% (p = 0.006) and NRM of 46.4% vs 30.4% (p = 0.003) for CR and CRi groups respectively, with no difference in the probability of relapse. Summary/Conclusion: Our data shows that patients undergoing HCT in CRi have a poorer survival and higher NRM without increased relapse risk compared to those transplanted in CR. The persistently increased NRM in CRi patients without MRD supports the notion that the poorer survival may result from poor fitness for transplant and increased susceptibility to transplant related complications rather than an imminent relapse. With the advent of increasingly sensitive MRD technologies, these data may suggest that further pre-transplant optimization to promote marrow recovery may be permissible in order to minimize NRM without risking increased relapses in CRi patients. The high NRM may also caution against pursuing MRD negativity at all cost, particularly if that risks incomplete count recovery.

The BMP-SMAD pathway is activated when a dimeric ligand (BMP) interacts with a dimeric serine threonine kinase receptor (BMPRII) and triggers the activation of a dimeric BMP type I receptor (BMPRI). Catalytically active BMPRIs phosphorylate SMAD1/5/8 that, upon SMAD4 binding, translocate to the nucleus to regulate the expression of BMP target genes, including hepcidin. Hepcidin is the main regulator of iron homeostasis that controls body iron levels by binding and blocking the sole iron exporter ferroportin. In agreement, hepcidin expression is homeostatically activated by serum and liver iron, and its deficiency is a common hallmark of Hereditary Hemochromatosis (HH) and the major cause of iron overload in beta thalassemia. The components of the BMP-SMAD pathway relevant for hepcidin regulation are ALK2 and ALK3 (BMPRI); BMPR2 and ACVR2A (BMPRII), BMP2 and BMP6 (BMP ligands). Recently, we have identified the immunophilin FKBP12 as an inhibitor of hepcidin and demonstrated that FKBP12 binds ALK2 to avoid ligand-independent activation of the BMP-SMAD pathway. To investigate the mechanism of BMP-SMAD pathway and hepcidin regulation by FKBP12, we performed in vitro, ex vivo and in vivo studies. We found that FKBP12 sequestration by the immunosuppressive drug Tacrolimus (TAC) stabilizes ALK2-ALK2 homodimers and ALK2-ALK3 heterodimers in a transfected human hepatoma cell line. In addition, it increases the interaction of ALK2 with ACVR2A and BMPR2. To investigate the role of FKBP12 on BMP-SMAD signaling, BMPRI and II were silenced in murine primary hepatocytes. Despite FKBP12 co-immunoprecipitates only with ALK2, silencing of Alk2 and Alk3 completely blunts TAC-mediated BMP-SMAD pathway activation, suggesting that FKBP12 functionally interacts also with ALK3. Acvr2a silencing impairs TAC-dependent hepcidin upregulation, whereas Bmpr2 silencing does not. As expected, Fkbp12 silencing abrogates hepcidin upregulation by TAC, confirming the main role of this immunophilin in hepcidin regulation. In vivo, TAC treatment upregulates hepcidin in wild type and HH mouse models, but surprisingly, Fkbp12 mRNA downregulation by ASOs does not. Interestingly, Fkbp 2, 4 and 8 are highly expressed in murine hepatocytes and, according to literature data, are able to bind to TAC. Of note, Fkbp12 is the least expressed immunophilin in murine primary hepatocytes. To further investigate the FKBPs involved in TAC-dependent hepcidin regulation, Fkbp2, 4 and 8 were knockdown in murine primary HCs that were then treated with TAC. The TAC effect is preserved in siFkbp2- and siFkbp4-derived HCs, but abolished when Fkbp8 was downregulated. Overall these data suggest that: 1) FKBP12 regulates BMP-SMAD signaling by favoring ALK2-ALK3 homo and heterodimerization, and interaction with BMPRII in the absence of ligands; 2) TAC-mediated hepcidin upregulation is dependent upon ALK2, ALK3, ACVR2A, FKBP12 and FKBP8. 3) In vivo, TAC treatment upregulates hepcidin whereas Fkbp12 silencing does not, suggesting the existence of redundancy between the different FKBPs. Further studies are needed to dissect the role of FKBP8 in BMP-SMAD pathway and hepcidin regulation. Disclosures Aghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Muckenthaler: Silence Therapeutics: Research Funding. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.