Developing a Pediatric-Relevant Murine MLL-r B-ALL Model to Study Factors Involved in Lineage Switching
Authors: Liao Wenjuan , Liu Tian , Grandvallet Contreras Julian , Harris Lynn Heltemes , Phang Tzu , Farrar Michael A. , Kohler M. Eric , Fry Terry J , Ernst Patricia 
Published: December 2024

Relapse following anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in childhood relapsed/refractory B-acute lymphoblastic leukemia (r/r B-ALL) poses a formidable challenge, with a particularly concerning manifestation known as Lineage Switch (LS) characterized by a transition to an acute myeloid leukemia (AML) phenotype. Lineage switching is particularly associated with certain cytogenetic subtypes including KMT2A/MLL rearranged (MLL-r) B-ALL. However, murine models of MLL-r leukemia predominate in AML rather than B-ALL. Human xenografts do enable MLL-r B-ALL engraftment but lack the complete niche to faithfully replicate the evolution from B-ALL to AML.
We present here an iterative approach to developing an accurate syngeneic MLL-r B-ALL model using retroviral delivery of fusion oncoproteins. To study the lineage-unstable nature of MLL-r B-ALL, MLL-r CD19-positive B-ALL cells were first generated in vitro in an IL-7-dependent, stroma-free condition via retroviral delivery of MLL-r alleles. During transformation, we show ongoing Ig heavy chain rearrangements and acquisition of a stable pro B cell phenotype similar to that of human MLL-r B-ALL. RNA sequencing revealed a particular suite of myeloid gene expression despite immunophenotyping consistent with a pro B cell identity. Despite this stable phenotype in vitro, engraftment into murine newborn recipients was accompanied by a rapid lineage switch from CD19-positive B-ALL to CD19-negative AML. Immunoglobulin heavy chain D-J rearrangements were present in AML cells from relapsed recipients, suggesting that the B-ALL and AML were progeny of B-ALL. We show that blocking myeloid lineage access by generating MLL-r Cebpa-/- B-ALL is not sufficient to support B-ALL in vivo. When wild-type (WT) and RAGγC-/- newborn recipients were compared, the B-ALL switch to AML occurred with delayed kinetics in RAGγC-/- recipients, suggesting that elevated levels of free IL-7 could be a factor in supporting B-ALL in vivo. To test this hypothesis, we employed IL7R blocking antibodies and IL-7 injections. Strikingly, manipulating IL7 signaling influenced the kinetics of lineage switch. Further, constitutive activation of Stat5 (Stat5CA) significantly delayed the AML LS, leading to a biphenotypic leukemia in both WT and RAGγC-/- recipients. CD19 CAR T infusion accelerated lineage switching in our MLL-r B-ALL models with and without Stat5CA.
In summary, we developed a universally applicable, novel syngeneic murine B-ALL model to study factors involved in lineage switching to AML in vivo. Using this model, we made three key discoveries: 1) lack of access to the myeloid lineage is insufficient to maintain B-ALL in vivo in mice, 2) the IL7-Stat5 axis can be enhanced to maintain B-ALL stability and more accurately model human MLL-r B-ALL, and 3) there are distinct transcriptional differences between de novo AML and lineage-switched AML, even when driven by the same MLL fusion oncoprotein.