Luspatercept Reduces Clonal Hematopoiesis-Associated Cardiac Stress Via Modulation of mTORC1 and Inflammatory Signaling Pathways

Introduction: Clonal hematopoiesis (CH) is driven by somatic mutations in hematopoietic stem cells that yield clonal progeny of mutant cells in blood (variant allele frequency > 2%) and found in 10-30% of individuals aged > 65 years. CH-related mutations (eg, DNMT3A, TET2, ASXL1, and SF3B1) were shown to be associated with increased mTORC1 (master regulator of growth, metabolism, and stress signaling) and inflammatory pathway activation that may drive both hematologic malignancies and cardiovascular complications. Elevated mTORC1 activation due to pressure overload stimulates pathologic cardiac hypertrophy, impairs autophagy, and worsens cardiovascular disease. Recently, patients with myelodysplastic syndrome (MDS) were reported to have deregulation of autophagy and high rates of cardiovascular comorbidities, including elevated levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), a biomarker for cardiovascular morbidity and mortality. Currently, no treatments are available to treat CH mutations or related complications. Luspatercept (inhibitor of activin receptor-Smad2/3 signaling) is approved as first-line treatment of anemia in adult patients with lower-risk MDS based on the COMMANDS trial (NCT03682536). Here, we explore clinical data from the COMMANDS trial and cellular and preclinical models to identify potential novel mechanisms of luspatercept that may have an impact on CH-associated consequences.

Methods: Transcriptomics, proteomics, and phosphoproteomics (bone marrow) were assessed at baseline and week 24 using patient samples from the COMMANDS trial. Genomic DNA was isolated from bone marrow and myeloid-specific 36 somatic genes mutations were identified by targeted next-generation sequencing (400X, sensitivity 3%) at baseline. Murine analog of luspatercept (RAP-536) was used for transverse aortic constriction (TAC)-induced pressure overload study in wild type (WT) and TET2 knockout (TET2-KO) C57BL/6 mice. Cardiac hypertrophy and function were assessed by echocardiography. Serum BNP level and Western blot analysis of heart tissues were done following 7-week treatment with RAP-536.

Results: In all, 85% (123/145) of patients treated with luspatercept from the COMMANDS trial had at least one of the most frequent CH-related mutations (eg, DNMT3A, TET2, ASXL1, SF3B1), with modest myeloid-based expansion at baseline (R=0.3, P ≤ 0.0002). Luspatercept treatment significantly reduced anemia from baseline in patients with CH-related mutations (primary endpoint response 62%, n/N = 76/123) and downregulated NT-proBNP levels (Δ median = 590 pg/mL in responders, P = 0.02). In responding patients with CH-related mutations, luspatercept treatment reduced mTORC1 pathway activation and inflammatory signaling at the transcriptomic, proteomic, and phosphoproteomic levels. Consistent with this, we also observed inhibition of GDF11 (one of the Smad2/3 ligands inhibited by luspatercept) mediated mTORC1 activity and activation of autophagy/lysosome markers following luspatercept treatment in human cardiomyocyte cell line. Using a murine model of pressure overload (TAC)-induced heart failure, which has been shown to have elevated mTORC1 and inflammatory signaling, we showed that RAP-536 significantly reduced TAC-induced cardiac hypertrophy (left ventricular [LV] mass reduction by echocardiography) in both WT (21%, P = 0.001) and TET2-KO (16%, P = 0.01) mice. Accordingly, we observed reduction of LV end diastolic volume in both in WT (18%, P = 0.018) and TET2-KO (24%, P = 0.006) mice. Moreover, we also observed reduction of serum BNP levels (WT: 59%, P < 0.0001; TET2-KO: 69%, P = 0.0002) and downregulation of IL-6 (gene and protein level) in heart tissues following RAP-536 treatment, suggesting potential CH-mutation agnostic cardiovascular benefits.

Conclusions: Our reverse and forward translational approach identified novel mechanisms of luspatercept that may have potential implications in patients with CH-related mutations and associated consequences (hematologic and cardiovascular). Moreover, these findings offer insights into how luspatercept treatment can modify cellular and tissue microenvironments, which could be leveraged in tailored therapeutic approaches, either alone or in conjunction.