Abstract 3153: Imatinib mesylate treatment improves reprogramming efficiency, and morphology of chronic myeloid leukemia derived induced pluripotent stem cells
Authors: Benjamin Esther , Joshi Gaurav , Rajamani Bharathi , Nandy Krittika , Velayudhan Shaji , Balasubramanian Poonkuzhali 
Published: June 2022

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL1 fusion gene with constitutively active tyrosine kinase activity. Although the tyrosine kinase inhibitors (TKI) have revolutionized the treatment for CML, withdrawal of TKI therapy in patients on deep molecular response causes disease relapse, primarily due to the persistence of leukemic stem cells (LSCs) that are insensitive to TKI. As LSCs are a rare population of cells, it is challenging to understand the molecular basis of the disease relapse to tailor strategies to eliminate them selectively. Induced pluripotent stem cells (iPSCs) derived from primary CML are increasingly used for disease modeling and high through drug screening. Cryopreserved CD34+ from three chronic phase CML patients (n=3) and CD34+ cells from a normal donor after mobilization were expanded in SFEM II supplemented with CD34+ Expansion Supplement (10X) including UM729 for 3-days and were nucleofected with episomal reprogramming plasmids as described previously (Manian et al., 2018). The nucleofection efficiency was 15% in the expanded CD34+ CML and normal cells. While the normal CD34+ cells formed iPSCs with the characteristic flat morphology between 20-24 days, the CML CD34+ cells formed several dome-shaped colonies after 30 days. Two normal CD34+ iPSC colonies continued to maintain their morphology for 12 passages. All the CML iPSC colonies (n=11) expressed the same type of BCR-ABL1 transcript as the CD34+ cells and did not carry any mutations in the BCR-ABL1 kinase domain. However, only three of the 11 CML-iPSC colonies could be maintained without significant differentiation after five passages. They lacked typical iPSC morphology and appeared as small cell aggregates or dome-shaped colonies. To understand whether this atypical morphology of the CML iPSC colonies was due to the expression of the BCR-ABL fusion protein within the cells, the medium was supplemented with 10μM imatinib. There was an increase in cell proliferation rate and gain of typical iPSCs morphology similar to normal iPSCs within two days after adding IM. Withdrawal of IM from the IM-treated CML iPSC resulted in a dome-shaped morphology, reduced proliferation rate, and reduction in TRA1-60 expression after 96 hrs. The CML iPSC cultured in the presence of IM showed significant downregulation of phospho-CRKL, a BCR-ABL1 downstream signaling protein, compared to the colonies cultured in the absence of IM. These results suggest that suppression of BCR-ABL1 by treatment with IM produced stable CML iPSC-like phenotype while IM withdrawal resulted in dome-shaped colonies and pushed the cells towards differentiation. This proof-of-concept study identified a unique CML LSCs mimetic model. It would serve as an excellent platform for screening small molecules to eliminate LSCs as it thrives on TKI therapy.

Citation Format: Esther Benjamin, Gaurav Joshi, Bharathi Rajamani, Krittika Nandy, Shaji Velayudhan, Poonkuzhali Balasubramanian. Imatinib mesylate treatment improves reprogramming efficiency, and morphology of chronic myeloid leukemia derived induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3153.