Base-Editing Human Hematopoietic Stem Cells to Enable Venetoclax Post-Transplant Maintenance Therapy for AML
Year: 2025
Type: article
Abstract: For many patients with acute myeloid leukemia (AML), hematopoietic stem cell transplant (HSCT) remains the only therapy with curative potential. Unfortunately, relapse after HSCT is the most common cause of treatment failure. Venetoclax , a selective inhibitor of BCL2, has emerged as a promising therapy in both up-front and relapsed AML, and there is interest in post-HSCT venetoclax to prevent relapse. However, post-HSCT venetoclax administration causes dose-limiting cytopenias in approximately 50% of patients due to its activity on donor hematopoietic stem cells (HSCs). We hypothesized that introducing a venetoclax resistance mutation into human HSCs would increase the therapeutic window of BCL2 inhibition post-HSCT, thereby serving as a platform to reduce relapse risk without hematopoietic toxicity. We prioritized mutations that are likely to impair venetoclax binding to BCL2, preserve BCL2 function, and are amenable to CRISPR adenine base editing (ABE). Based on these considerations, we generated human HSCs harboring the BCL2 F104L mutation with high efficiency via CRISPR-Cas9 ABE technology (median = 75%, n = 10). BCL2 F104L HSCs were significantly more resistant to venetoclax in comparison to control HSCs ( Figure 1A , IC 50 not reached vs. IC 50 97.76 nM). BCL2 F104L HSCs retained sensitivity to cytarabine , indicating selective resistance to venetoclax. BCL2 F104L HSCs retained stem cell properties, with equivalent colony formation between BCL2 F104L and control HSCs. Intracellular BH3 profiling revealed that BCL2 function and overall apoptotic priming is preserved in BCL2 F104L HSCs in the absence of venetoclax. To evaluate the long-term impact of the BCL2 F104L mutation on hematopoietic engraftment and development, we transplanted human BCL2 F104L or control HSCs in immunocompromised NSG mice . DNA sequencing of human CD45+ cells isolated from the bone marrow of engrafted mice showed stable allele frequency of the BCL2 F104L mutation in comparison to pre-injection sequencing, suggesting the mutation confers neither a growth advantage nor disadvantage to HSCs ( Figure 1B ). Flow cytometry analysis of peripheral blood samples show long-term multi-lineage repopulating potential is preserved in BCL2 F104L HSCs despite lower initial engraftment efficiency ( Figure 1C ). Future work will investigate our hypothesis that mice with BCL2 F104L HSCs will tolerate venetoclax without hematopoietic aplasia . In this proof-of-concept study, we have leveraged a naturally-occurring resistance mutation to generate a base-edited human stem cell product. This strategy could increase the therapeutic index of BCL2 inhibition at early timepoints post-HSCT to prevent relapse and improve overall survival in patients with AML.
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $3,650
OpenAlex ID: W4407976596