Identifying the Impact of ADAR1 Inhibition on LSC Niche Remodeling in 3D Nanobioreactors

Background In acute myeloid leukemia (AML), adenosine deaminase associated with RNA (ADAR1) p150 splice isoform overexpressing leukemia stem cells (LSCs) contribute to relapse and an overall survival rate of only 20% at 5 years. Recently, we reported the successful completion of pre-IND studies with a small molecule ADAR1p150 antagonist, Rebecsinib, which significantly reduces LSC self-renewal in stromal co-cultures and humanized LSC mouse models while sparing normal hematopoietic stem and progenitor cells (HSPCs) (Crews, Balaian...Burkart, Jamieson. Cell Stem Cell 2023, March cover). However, the capacity of Rebecsinib to modify LSC-supportive niches that promote LSC persistence had not been examined. Thus, we adapted our 3D niche nanobioreactor system, which was developed with NASA to study normal CD34+ HSPC maintenance in response to macroenvironmental and microenvironmental stressors (Pham, Balaian...Jamieson. BioRxiv 2024), to study the impact of ADAR1 inhibition on LSC niche remodeling. Methods Human CD34+ cells were immunomagnetic bead-selected from AML or normal aged bone marrow (ABM) samples that were collected and stored according to IRB approved protocols. In 7.5 ml gas permeable surgifoam-sponge containing 3D nanobioreactors, AML CD34+ cells were treated with varying doses of Rebecsinib compared with DMSO as a control. After 2 weeks of treatment in the nanobioreactor, cells were plated on stroma established from the CD34 negative fraction of AML bone marrow, ABM or a young stromal cell line, HS-27A cell line. After 2 weeks, CD34+ cells were transferred to new stromal co-cultures that had been pre-treated with Rebecsinib or DMSO and co-cultured for an additional 2 weeks followed by clonogenic survival and replating assays as well as RNA sequencing (RNA-seq) analyses to detect ADAR1 expression and adenosine to inosine (A-to-I) editing activity. Results Rebecsinib (1 to 5 uM) treatment significantly reduced primary AML (n=3) LSC survival following co-culturing for 2 weeks with autologous CD34 negative AML bone marrow derived stromal cells in the nanobioreactor while normal HSPCs (n=3) cultured in 3D nanobioreactors with autologous ABM stroma were spared at the same doses. Co-culture of surviving LSCs after Rebecsinib treatment on primary autologous AML or HS-27A young stroma resulted in increased LSC colony survival and replating. However, pre-treatment of the AML stroma with Rebecsinib for 1 week prior- to co-culture resulted in loss of LSC maintenance as typified by reduced survival and self-renewal compared to untreated stroma. Moreover, Rebecsinib pre-treated AML stroma enabled normal HSC survival and self-renewal in contrast to DMSO-treated control stroma. Furthermore, RNA-seq analyses revealed that Rebecsinib treatment of AML stroma significantly decreased DROSHA ,CRAT and TP53BP1 and altered A-to-I editing of MDM2 and other tumor suppressor regulatory transcripts. In contrast, Rebecsinib treatment of ABM stroma resulted in increased expression of genes associated with adhesion, such as CXCL3, CXCL8 ,CXCR5. Conclusions Malignant niche remodeling with Rebecsinib reverses LSC persistence while sparing normal HSC maintenance and may represent a novel avenue for preventing AML relapse.