Lack of Gdf11 does not improve anemia or prevent the activity of RAP-536 in a mouse model of β-thalassemia

TO THE EDITOR:

β-thalassemia (BT) is a global health problem affecting millions of patients, and current therapies to treat BT present major clinical challenges.^1,2  Luspatercept (ACE-536), an erythroid maturation agent that functions independently of the erythropoietin (EPO) pathway,³  has been shown to improve anemia in BT.^4,5  In clinical trials, treatment with ACE-536 increased hemoglobin (Hb) levels and significantly reduced red blood cell (RBC) transfusions in adults with BT, with little to no adverse effects.^4,5  ACE-536 is a fusion protein composed of a modified extracellular domain of activin receptor IIB (ACVR2B) and the Fc part of human immunoglobulin G1. This fusion protein competes with ACVR2B to bind members of the transforming growth factor-β (TGF-β) superfamily.⁶  Studies in murine models of BT using the murine analog of ACE-536 (RAP-536) show that RAP-536 targets the protein growth differentiation factor 11 (GDF11).^3,7,8  These studies have proposed that overexpression of GDF11 blocks terminal erythroid differentiation by increasing oxidative stress and that treatment with the ligand trap ACE/RAP-536 sequesters GDF11, unblocking terminal erythroid differentiation and, thereby, ameliorating ineffective erythropoiesis. However, ACE-536 and RAP-536 have been shown to stimulate RBC synthesis in healthy humans and mice, where GDF11 overexpression has not been reported.^5,9  Because of the incongruency of the proposed model, our study resorted to genetic tools to reduce GDF11. We investigated whether deletion of the Gdf11 gene improved anemia in a BT mouse model (Hbb^th3/+).¹⁰  We also examined whether RAP-536 was efficacious in the absence of Gdf11.

Gdf11 was functionally inactivated in the entire hematopoietic compartment of mice (Gdf11^Δ2-3/Δ2-3) by crossing Vav^Cre-transgenic animals¹¹  with Hbb^+/+ and Hbb^th3/+ LoxP flanked Gdf11 (Gdf11^flox/flox) mice¹²  to produce Hbb^+/+Vav^CreGdf11^Δ2-3/Δ2-3 and Hbb^th3/+Vav^CreGdf11^Δ2-3/Δ2-3 mice (supplemental Figure 1, available on the Blood Web site). The resulting progeny were viable, and complete blood count (CBC) results did not show changes in RBC, Hb, hematocrit (Hct), or reticulocyte levels (Figure 1A-H). Gdf11 recombination in the spleens of Hbb^th3/+Vav^CreGdf11^Δ2-3/Δ2-3 mice was confirmed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) (Figure 1Q). Similarly, crossing EpoR^Cre-transgenic animals¹³  with Gdf11^flox/flox mice, in which Gdf11 was reduced in the early erythroid progenitor, did not result in changes to the hematological profile (supplemental Figure 2).

We then tested whether GDF11 produced by nonhematopoietic tissues indirectly influenced erythropoiesis. Because Gdf11-null mice (Gdf11^−/−) are embryonically lethal,¹²  we generated mice with a pancellular deletion of Gdf11 using the tamoxifen (TAM)-inducible Rosa^Cre strain¹⁴  and generating Hbb^+/+Rosa^CreGdf11^Δ2-3/Δ2-3 and Hbb^th3/+Rosa^CreGdf11^Δ2-3/Δ2-3 mice (supplemental Figure 3). Neither Hbb^+/+Rosa^CreGdf11^Δ2-3/Δ2-3 mice nor Hbb^th3/+Rosa^CreGdf11^Δ2-3/Δ2-3 mice showed any alterations in RBC, Hb, Hct, or reticulocyte levels 2 weeks (Figure 1I-P), 5 weeks (supplemental Figure 5A-H), or 5 to 6 months (supplemental Figure 5I-P) post-TAM treatment. We confirmed Gdf11 recombination by qRT-PCR in the spleens of Hbb^th3/+Rosa^CreGdf11^Δ2-3/Δ2-3 mice (Figure 1Q) and by polymerase chain reaction in the spleen, liver, heart, duodenum, kidney, and bone marrow of Hbb^+/+Rosa^CreGdf11^Δ2-3/Δ2-3 and Hbb^th3/+Rosa^CreGdf11^Δ2-3/Δ2-3 animals (supplemental Figure 4). These findings were consistent in females (supplemental Figure 6) and males (supplemental Figure 7). No differences were detected in the spleen-to-body weight ratios of Vav^CreGdf11^Δ2-3/Δ2-3 or Rosa^CreGdf11^Δ2-3/Δ2-3 mice compared with controls (supplemental Figure 8). GDF11 has also been proposed to exert a negative effect on late-stage erythropoiesis in myelodysplastic syndromes, a heterogeneous group of clonal hematopoietic disorders.^3,15  To test this, we crossed NUP98-HOXD13¹⁶  (NHD13), a mouse model that recapitulates all key features of myelodysplastic syndromes, with Vav^CreGdf11^Δ2-3/Δ2-3 mice. Results show that genetic removal of Gdf11 from all hematopoietic lineages in NHD13 mice did not confer a therapeutic benefit (supplemental Figure 9).

We then evaluated whether the effect of RAP-536 is mediated by the synthesis of Gdf11 from erythroid or nonerythroid cells. We administered 12 doses of RAP-536 to Hbb^+/+ and Hbb^th3/+ mice of the Vav^CreGdf11^Δ2-3/Δ2-3- and Rosa^CreGdf11^Δ2-3/Δ2-3-derived lines. In all mice treated, RAP-536 significantly increased RBC, Hb, and Hct parameters; additionally, reticulocyte counts were normalized in Hbb^th3/+ mice (Figure 2A-P). Thus, lack of Gdf11 did not prevent responsiveness to RAP-536. Because activin receptor ligand traps have been shown to stimulate RBC synthesis and Hb increases in healthy humans,^5,17  we investigated whether CD34⁺ cells respond to RAP-536 treatment in vitro. CD34⁺ were differentiated and expanded in a 3-phase liquid erythroid-specific differentiation medium containing RAP-536.¹⁸  Cell counts were conducted at the end of an 8-day differentiation assay to determine whether cells cultured in RAP-536 produced more mature cells. No differences were observed in the number of hemoglobinized cells after treatment with 5 µg/mL or 150 µg/mL RAP-536 (Figure 2Q).

Next, we investigated messenger RNA (mRNA) expression levels of GDF11 and ACVR2B in early erythroid progenitors derived from healthy and BT donor CD34⁺ cells. Because GDF11 has been proposed to inhibit early erythroid progenitors from differentiating, cells were cultured as previously mentioned¹⁸  and collected for mRNA early in the differentiation phases: 24 hours later and 4 days later. qRT-PCR results showed low levels of GDF11 and ACVR2B in CD34⁺ cells from BT (Figure 2R) and healthy (Figure 2S) donors compared with TFRC and GYPA controls (supplemental Figure 10B for probes used). Similarly, Ter119⁺ erythroid progenitors isolated from Hbb^th3/+ spleens (Figure 2T; supplemental Figure 10A) showed low expression of Gdf11 and Acvr2b in phosphate-buffered saline– and RAP-536–treated mice (Figure 2U). However, in RAP-536–treated mice, Tfrc and Gypa levels were significantly different compared with phosphate-buffered saline controls, which is consistent with a reduction in the erythroid progenitor pool and an increase in RBC differentiation.

The EPO-independent ability of RAP-536 to increase RBC number and Hb content offers the potential for understanding an undiscovered pathway in erythropoiesis. Studies have identified GDF11 as the primary target of RAP-536.^3,7,8  However, evidence for overexpression of GDF11 was obtained using nonspecific antibodies for GDF11¹⁹  in BT erythroid cells⁷  and does not explain how RAP-536 is also effective in Hbb^+/+ mice, in which GDF11 overexpression in erythroid cells has not been observed. RAP-536 has also been reported to bind other members of the TGF-β family, including GDF8 and activin B.^3,20  Based on these observations, analyses were performed in Hbb^+/+ mice using antibodies against activin B, GDF8, or GDF8/11.²⁰  These studies indicate that the antibodies had modest effects on RBC synthesis, and none could recapitulate the effect of RAP-536.²⁰  Here, we show that lack of Gdf11 does not improve anemia in Hbb^th3/+ mice or increase hemoglobin in Hbb^+/+ mice. In accordance with these findings, Hbb^th3/+ and Hbb^+/+ animals, with a deletion in the hematopoietic compartments and a pancellular deletion of Gdf11, respond to RAP-536. In addition, we show that Gdf11 and Acvr2b expression is low in a BT mouse model and in human erythroid cells. Altogether, our studies strongly indicate that decreasing GDF11 in erythroid cells has no effect on anemia. Lastly, GDF11 has been reported to have protective properties on cardiovascular health; patients with decreased levels of GDF11 had increased incidences of heart failure, stroke, and death.²¹  Animal studies have also implicated GDF11 as important in neuronal vascularization and skeletal muscle development.^22-24  Targeting GDF11 as an agent to increase hematological parameters could compromise cardiovascular protection or affect other important pathways in development. Future work will focus on the role of the TGF-β superfamily in erythropoiesis and on identifying the targets responsible for the therapeutic effects produced by RAP-536, because the mechanism for RAP-536 remains to be elucidated.