Impairment of the Stromal SDF-1-Mediated Hematopoietic Support By GDF-11 in MDS Is Rescued By Luspatercept

Introduction:

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis due to genetic and functional abnormalities of hematopoietic stem and progenitor cells (HSPCs). Accumulating evidence now also points to the bone marrow microenvironment (BMME) as a key mediator of MDS pathophysiology.

Increased levels of transforming growth factor beta (TGF-β) superfamily ligands, including growth differentiation factor 11 (GDF-11), in the bone marrow have been linked to ineffective erythropoiesis and activation of SMAD2/3 signalling in MDS. Luspatercept (ACE-536) is a novel recombinant fusion protein containing modified activin receptor type IIB linked to the fragment crystallisable (Fc) domain of human immunoglobulin G1 and is a first-in-class erythroid maturation agent. Luspatercept binds to selected TGF-β superfamily ligands, including GDF-11 and activin B, restoring late-stage erythropoiesis in MDS mice and patients. Whether luspatercept modulates the BMME is unknown.

Aim:

We investigated the potential impact of luspatercept on the biology of mesenchymal stromal cells (MSCs).

Methods:

MSCs from patients with either high-or low-risk MDS and age-matched healthy donors (HD) were treated with GDF-11 in the presence or absence of RAP-536, a homologue of luspatercept harboring the same activin receptor IIB domain. Subsequently, Smad signaling pathway activation was analyzed by Western blot. Moreover, MSC phenotype, stromal derived factor-1 (SDF-1/CXCL12) expression and secretion as well as the osteogenic differentiation potential were recorded. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with GDF-11 ± RAP-536 before freshly isolated CD34+ HSPCs were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly. After four weeks, the HSPCs were seeded in methylcellulose medium to perform a CFU-GEMM assay. Further, engraftment and migration potential of co-cultured HSPCs was analyzed in zebrafish embryos in vivo.

Results:

GDF-11/RAP-536 treatment did not impact viability, proliferation, and growth pattern of MSCs. The osteogenic differentiation was significantly improved by RAP-536 treatment which was shown by a 2.3-fold increase of ALP activity. GDF-11 induced Smad2/3 phosphorylation in MSCs which was inhibited by RAP-536 to about 50% of activity. The MSC phenotype characterized by the expression of CD73, CD90, CD105, CD146, CD29 and CD44 was not significantly influenced by RAP-536 treatment.

Interestingly, the chemokine SDF-1 which plays an important role for the interaction and support of HSPCs was significantly up-regulated in MDS and HD MSCs by RAP-536 at both the mRNA (1.9-fold) and the protein level (2.1-fold; 313.0±35.0 vs. 649.7±76.8 pg/ml, *p< 0.05, n=3). This restored SDF-1 secretion of MSCs resulted in functional effects of HSPCs when co-cultured with MSCs. The number of CAF-C was significantly higher after two, three, and four weeks of HSPCs cultured on pre-treated MSC layers (12.0-fold / 3.4-fold / 1.75-fold). The phenotypical analysis of HSPCs demonstrated increased expression levels of CXCR4 in the adherent fraction. Addition of the CXCR4 antagonist AMD3100 blocked enhanced colony formation by RAP-536 confirming the previous observation on SDF-1 modulation. Clonal growth of CAF-C-derived HSPCs after four weeks of co-culture on un- or pre-treated MSC monolayers was analyzed in a secondary colony-forming cell assay (CFU) for an additional 14 days. The frequency of CFUs was increased in all lineages with significant differences in total colony numbers (15±5.8 vs. 35.8±14.7, *p< 0.05, n=6).

These results were validated in vivo in a zebrafish model. HSPCs were co-cultured with GDF-11 and RAP-536 pre-treated MDS-MSCs for one week before injection into the zebrafish circulation. One and two days post-injection, a significantly higher cell proportion could be detected in the caudal hematopoietic (CHT) region as a result of RAP-536 pre-treatment (Fig.1).

Conclusion:

These data provide first evidence that RAP-536 has also the capacity to modulate MSCs which might contribute to the restoration of hematopoiesis in MDS.

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