Splicing of erythroid transcription factor is associated with therapeutic response in myelodysplastic syndromes
Srinivas Aluri, Te Ling, Ellen Fraint, Samarpana Chakraborty, Kevin Zhang, Aarif Ahsan, Leah Kravets, Gowri Poigaialwar, Rongbao Zhao, Kith Pradhan, Anitria Cotton, Kimo Bachiashvili, Jung-In Yang, Anjali Budhathoki, Beamon Agarwal, Shanisha Gordon Mitchell, Milagros Carbajal, Srabani Sahu, Jacqueline Boultwood, Andrea Pellagatti, Ulrich Steidl, Amittha Wickrema, Satish Nandakumar, Aditi Shastri, Rajasekhar N.V.S. Suragani, Teresa V. Bowman, John D. Crispino, Sadanand Vodala, Amit Verma
Srinivas Aluri, Te Ling, Ellen Fraint, Samarpana Chakraborty, Kevin Zhang, Aarif Ahsan, Leah Kravets, Gowri Poigaialwar, Rongbao Zhao, Kith Pradhan, Anitria Cotton, Kimo Bachiashvili, Jung-In Yang, Anjali Budhathoki, Beamon Agarwal, Shanisha Gordon Mitchell, Milagros Carbajal, Srabani Sahu, Jacqueline Boultwood, Andrea Pellagatti, Ulrich Steidl, Amittha Wickrema, Satish Nandakumar, Aditi Shastri, Rajasekhar N.V.S. Suragani, Teresa V. Bowman, John D. Crispino, Sadanand Vodala, Amit Verma
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Research Article Cell biology Hematology

Splicing of erythroid transcription factor is associated with therapeutic response in myelodysplastic syndromes

Abstract

Anemia is the primary clinical manifestation of myelodysplastic syndromes (MDSs), but the molecular pathogenesis of ineffective erythropoiesis remains incompletely understood. Luspatercept, an activin receptor 2B (ACVRIIB-Fc) ligand trap, has been approved to treat anemia; however, its molecular mechanism of action is unclear. We found that activin receptor 2B (ACVR2B), its ligand growth and differentiation factor 11 (GDF11), and an effector, SMAD2, are upregulated in samples of patients with MDS. GDF11 inhibited human erythropoiesis in vitro and caused anemia in zebrafish, effects that were abrogated by luspatercept. Upon GDF11 stimulation of human erythroid progenitors, SMAD2 binding occurred in the erythroid regulatory regions, including at the GATA1 intron. Intronic SMAD2-binding led to skipping of exon 2 of GATA1, resulting in a shorter, hypomorphic isoform (GATA1s). CRISPR deletion of the SMAD2-binding intronic region decreased GATA1s production upon GDF11 stimulation. Expression of GATA1s in a mouse model led to anemia, rescued by a murine ActRIIB-Fc (RAP-536). Finally, RNA-Seq analysis of samples from the phase 3 MEDALIST trial revealed that responders to luspatercept had a higher proportion of GATA1s compared with nonresponders. Moreover, the increase in RBCs after treatment was linked to a relative decrease in GATA1s isoforms. Our study indicates that GDF11-mediated SMAD2 activation results in an increase in functionally impaired GATA1 isoforms, consequently contributing to anemia and influencing responses to luspatercept in MDS.

Authors

Srinivas Aluri, Te Ling, Ellen Fraint, Samarpana Chakraborty, Kevin Zhang, Aarif Ahsan, Leah Kravets, Gowri Poigaialwar, Rongbao Zhao, Kith Pradhan, Anitria Cotton, Kimo Bachiashvili, Jung-In Yang, Anjali Budhathoki, Beamon Agarwal, Shanisha Gordon Mitchell, Milagros Carbajal, Srabani Sahu, Jacqueline Boultwood, Andrea Pellagatti, Ulrich Steidl, Amittha Wickrema, Satish Nandakumar, Aditi Shastri, Rajasekhar N.V.S. Suragani, Teresa V. Bowman, John D. Crispino, Sadanand Vodala, Amit Verma

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Figure 3

GDF11-driven pSMAD2 binds to the intronic region of GATA1.

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GDF11-driven pSMAD2 binds to the intronic region of GATA1. (A) Schematic...
(A) Schematic showing HSC differentiation and cell stages across different days. On day 5, approximately 25 million erythroid progenitors were treated with GDF11; chromatin immunoprecipitation was carried out, followed by the sequencing of pSMAD2 pull-down peaks. (B) Venn diagram showing the number of peaks that are found in both control (Ctrl) and GDF11 and in individual samples. (C) List of genes that are top hits in the pSMAD2 ChIP. (D) Integrative genomics viewer (IGV) tracks showing pSMAD2 binding in the GATA1 intron in both control and GDF11-treated samples. The arrow indicates the pSMAD2 binding peak. Both tracks are normalized for input. (E) Schematic of RNA preparation from erythroid progenitors upon GDF11 stimulation. (F) Pathway analysis for erythroid-related pathways was conducted through gene set enrichment analysis and highlights the downregulated pathways. NES, normalized enrichment score. (G and H) A schematic representation of the alternative splicing of GATA1 into GATA1 and GATA1-short. (I) The Western blot image demonstrates that GDF11 treatment reduces the overall expression of both GATA1 full-length and GATA1s, resulting in an increased short to long isoform ratio. The effects of GDF11 can be attenuated by the addition of luspatercept (left). A bar plot displays the GATA1s to GATA1 long isoform ratio from 3 independent experiments conducted on erythroid progenitors (right). *P < 0.05, ANOVA. (J) The schematic illustrates the strategy for deleting GATA1 exon 2 using CRISPR/Cas9. The top image depicts the WT GATA1, and the bottom sashimi plot shows the GATA1 with exon 2 deleted (del). RNA-Seq verified the reduction in GATA1 exon 2. (K) A flow plot demonstrates the emergence of glycophorin A (GlyA)-positive cells after GATA1 exon 2 deletion, with indicated population percentages (left). A bar plot showing the average of 3 independent experiments (right). (J). *P < 0.05, Student’s t test.