AML Derived Induced Pluripotent Stem Cells Reveal a Genotype-Independent Effect of Monocytic Differentiation in Mediating Venetoclax Resistance
Authors: Jones Matthew F , Peretz Cheryl A C , Kennedy Vanessa E. , Tran Elaine , Koh Andrew , Kotini Andriana , Olszewska Malgorzata , Papapetrou Eirini , Smith Catherine C. 
Published: December 2024

Background
Combination regimens incorporating the BCL2 inhibitor venetoclax are increasingly ubiquitous in the treatment of AML. Resistance to venetoclax remains a major clinical problem. Several mechanisms of resistance to venetoclax have been described, including selection for RAS-mutant clones (DiNardo et al. Blood 2020) and in vitro resistance of AML blasts with monocytic immunophenotypes (Kuusanmäki et al., Haematologica 2020). We have performed single-cell DNA sequencing with immunophenotyping of patients treated with the combination of gilteritinib with venetoclax and observed simultaneous expansion of RAS mutant clones and increased expression of monocytic markers at relapse (Kennedy et al, Blood 2022). Whether monocytic differentiation is a bystander consequence of selection for RAS mutations or an independent driver of resistance remains unknown. Lack of a model system in which the differentiation state of AML cells can be modified in an isogenic background hampers efforts to study monocytic differentiation as an independent mechanism of resistance to venetoclax.
Methods
We used a panel of 4 previously characterized (Kotini et al., Blood Cancer Disc 2023) AML-derived induced pluripotent stem cell (AML-iPSC) lines to test the effect of differentiation state on venetoclax sensitivity. The cell lines AML-9.9 and AML-9.9-FLT3 are derived from the same patient and identical except that AML-9.9-FLT3 line has a FLT3-ITD mutation. AML-4.10 and AML-4.24 are derived from the same patient and are genetically identical except that AML-4.10 has a KRAS G12D mutation. These lines were subjected to hematopoietic differentiation first to immature progenitors (“primitive”) and then further differentiated down the monocytic lineage, after which differentiation state was characterized by flow cytometry and morphology. The presence of a functional leukemia stem cell (LSC) subpopulation was assayed by colony formation in cytokine-enriched methylcellulose. Venetoclax sensitivity in vitro was tested by staining with a caspase-cleavable dye at different points along the spectrum of myeloid maturity.
Results
We found that this panel of AML-iPSCs can be robustly induced to form monocytic cells in vitro. In conditions favoring primitive differentiation state, there is a detectable population that expresses a previously described monocytic leukemia stem cell immunophenotype (Pei et al., Cancer Disc 2023). In early stages of monocytic differentiation, these lines upregulate markers of mature monocytes including CD11b, CD64 and CD68 and retain colony forming capacity in enriched methylcellulose, which indicates persistence of an LSC subpopulation after differentiation. Treatment of AML-iPSCs with venetoclax at primitive differentiation states reveals no significant difference in sensitivity as assessed by Caspase 3/7+ staining in AML-4.10 (KRAS G12D) vs AML 4.24 (KRAS WT). On the other hand, treatment of these cell lines after inducing monocytic differentiation results in a marked and significant reduction in venetoclax sensitivity in all 4 cell lines, regardless of genotype.
Conclusions
In our AML-iPSC model system, the presence of KRAS-G12D mutation has no significant effect on venetoclax sensitivity compared to an isogenic control while monocytic differentiation confers significant venetoclax resistance to all AML-iPSC lines regardless of genotype. These results suggest that monocytic differentiation is an independent determinant of venetoclax resistance rather than a passenger effect of genetic mutation. Future directions will include in vivo testing of differentiated AML-iPSC sensitivity to venetoclax and clinically relevant combination therapies. Single cell transcriptomic profiling will be performed to thoroughly characterize the evolution of heterogenous cell states in AML-iPSCs during treatment. This model system will be a useful tool to investigate the mechanisms of cell-state dependent mechanisms of treatment resistance in AML.