Transdifferentiation Of Primary Human Philadelphia Chromosome-Positive B Cell Acute Lymphoblastic Leukemia Cells Into Non-Leukemic Macrophages
Authors: McClellan James S. , Gentles Andrew J. , Ryan Christine , Majeti Ravi 
Published: October 2019

Abstract

Background
Despite the addition of tyrosine kinase inhibitors to treatment regimens for Philadelphia chromosome-positive (Ph+) precursor B cell acute lymphoblastic leukemia (B-ALL), the prognosis for adults with this disease remains poor, highlighting the need for better treatments. Ph+ B-ALL is characterized by frequent deletions in transcription factors required for normal B cell development. While these mutations are not obviously “druggable” by conventional means, these findings indicate that B cell maturation arrest is crucial to disease pathogenesis. This suggests that strategies to induce differentiation of B-ALL blasts might provide a novel avenue for therapy, but, to date, no such therapies have been developed. Previous studies have demonstrated that normal B cell progenitors retain the potential to transdifferentiate into cells of the myeloid lineage, and we hypothesized that B-ALL cells might possess this latent myeloid potential as well.

Methods and Results
To investigate the transdifferentiation potential of B-ALL cells, we FACS-purified B-ALL blasts from 12 patients with Ph+ disease and exposed them to myeloid differentiation-promoting cytokines in vitro for 12 days. In 6 of 12 cases, culturing leukemic blasts under these conditions led to their transdifferentiation into cells that morphologically resembled normal human macrophages. These cells expressed typical monocyte/macrophage markers including CD14, CD11b, and CD11c and downregulated the B cell marker CD19. In addition, the transdifferentiated cells were able to perform phagocytosis and generate an oxidative burst, functions that are typical of macrophages. Using gene expression profiling and gene set enrichment analysis (GSEA), we found that the transdifferentiated B-ALL blasts possess a gene expression profile that most closely resembles macrophage/monocyte gene expression signatures. Furthermore, using fluorescence in situ hybridization for BCR-ABL and immunoglobulin heavy chain clonality assays, we demonstrated that these macrophages were clonally related to the B-ALL blasts initially isolated from the patients. The CCAAT/enhancer-binding protein alpha (CEBPα) has been shown to induce transdifferentiation of normal precursor B cells to the macrophage linage, and we sought to determine whether this effect would be seen in primary B-ALL blasts as well. Similar to the effect seen using myeloid cytokines, we found that ectopic expression of CEBPα in B-ALL blasts led to upregulation of CD14 and downregulation of CD19 in these primary human B-ALL cells. To determine if transdifferentiation impairs leukemogenicity, we transplanted macrophages derived from primary B-ALL samples into immunodeficient NOD/SCID/IL2R-gamma null (NSG) mice. The transdifferentiated cells failed to engraft whereas primary Ph+ B-ALL blasts showed robust engraftment, demonstrating a detrimental effect of myeloid transdifferentiation on leukemogenicity. B-ALL blasts that remained CD19hi/CD14lo after culture were still capable of engrafting NSG mice and causing disease, indicating that ex vivo culturing alone did not eliminate leukemogenicity.

Conclusions
Here, we demonstrate that primary Ph+ B-ALL blasts can transdifferentiate to the myeloid lineage by either exposure to myeloid differentiation-promoting cytokines or ectopic expression of CEBPα. The resulting cells resemble normal human macrophages in morphology, immunophenotype, gene expression, and function. Most importantly, we show that transdifferentiated B-ALL blasts failed to engraft immunocompromised mice and cause leukemia. Our findings suggest that the induction of transdifferentiation represents a novel therapeutic strategy for the treatment of Ph+ B-ALL.

Disclosures:
No relevant conflicts of interest to declare.