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Engineering an inducible leukemia-associated transcription factor enables large-scale ex vivo production of functional human phagocytes

ABSTRACT: Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible transcription factor to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) transcription factor to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 years of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated towards CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established a novel transcription factor that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable transcription factors have the potential to be applied as molecular tools to produce functional immune cells for cell therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE237826 | GEO | 2024/05/22

REPOSITORIES: GEO

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Project description:Expression of meningioma 1 (MN1) has been proposed to be a negative prognostic molecular marker in adult AML with normal cytogenetics, however its role in pediatric leukemia is unknown. We found elevated MN1 expression in 53 of 88 pediatric leukemia cases: significant amounts of MN1 were found in immature B-cell ALL and most cases of infant leukemia but no MN1 expression was detected in T-cell acute lymphoblastic leukemia (T-ALL). Interestingly 17 of 19 cases harboring MLL-X fusions showed also elevated MN1 expression. Lentiviral siRNA mediated MN1 knock-down resulted in cell cycle arrest and impaired clonogenic growth of 3 MLL-X-positive human leukemia cell lines overexpressing MN1 (THP-1, RS4;11, MOLM13). In a mouse MLL/ENL-induced leukemia MN1 overexpression resulted from retroviral provirus insertion. Strikingly co-expression of MN1 with MLL/ENL resulted in significantly reduced latency for induction of an AML phenotype in mice suggesting functional cooperation. MN1 overexpression in MLL/ENL-carrying cells resulted in expansion of the L-GMP population and facilitated disease induction in secondary recipients. Gene expression profiling allowed to define a number of potential MN1 hematopoietic targets. Up-regulation of CD34, FLT3, HLF, or DLK1 was validated in bone marrow transiently overexpressing MN1, in MN1-induced mouse leukemias, as well as in some cases of pediatric leukemias overexpressing MN1. Taken together, our work suggests that MN1 overexpression is essential for growth of leukemic cells, and that MN1 can act as a cooperating oncogene with MLL-X fusion genes most probably through modification of a distinct gene expression program that leads to expansion of a leukemia initiating cell population. In three independent experiments bone marrow cells were transduced with MSCV-MN1-IRES/YFP or empty vector. 72h after transduction EYFP-positive cells were FACS-sorted and RNA isolated by ion-exchange chromatography with RNAmini (Qiagen) according to the manufacturerâs protocol

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Engineering an inducible leukemia-associated transcription factor enables large-scale ex vivo production of functional human phagocytes

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