Cellular and Molecular Targets of MLL-AF9 in a Novel Conditional Mouse Model

Abstract

1280 Previous studies have shown that the expression of several leukemia-associated mixed lineage leukemia (MLL) fusion genes transformed human and mouse bone marrow cells in vitro and in vivo. In order to dissect the molecular and cellular targets of the MLL-AF9 fusion, we generated a novel inducible doxycycline (DOX)-regulated transgenic mouse model. Conditional ex vivo activation of MLL-AF9 induced aberrant self-renewal and impaired differentiation of long-term or short-term hematopoietic stem (LT-HSC and ST-HSC), common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) cells in a fully reversible manner. Direct activation of the fusion in vivo or after transplantation of transgenic bone marrow cells into irradiated hosts induced an aggressive and transplantable disease after a median latency of 80days characterized as acute myelo-monocytic leukemia closely mimicking the human disease. Fusion gene expression and leukemia induction was DOX dosage dependent and reversible upon DOX removal. Activation of MLL-AF9 in isolated LT-HSC or GMP cells in vitro or in vivo resulted in the accumulation of immature blast-like cells with similar immunophenotypes. However, MLL-AF9-expressing stem and progenitor cells displayed distinct properties such as colony formation, differentiation and resistance to chemotherapeutic drugs. Turning-off the fusion resulted in multi-lineage differentiation of LT-HSC-derived cells, whereas GMP-derived cells were limited to mature macrophages and granulocytes suggesting partial maintenance of their original identity. In line with these in vitro observations, lower cell numbers of transplanted LT-HSCs induced a more aggressive leukemia with a significantly shorter latency as compared to ST-HSC, CMP or GMPs. Immunophenotypically 15% of the LT-HSC derived leukemias displayed a CMP–like phenotype and had a median latency of 37d (“early”) whereas the rest of the cases displayed a GMP-like phenotype with a median latency of 73d (“late”). In contrast, only GMP-like phenotypes and longer latencies were observed upon transplanting ST-HSCs (75d), CMPs (72d) or GMPs (100d). Transplantation of blasts from “early” LT-HSC- and GMP-derived leukemias into secondary recipients induced the disease after similar latency, however, cytarabine (Ara-C) treatment significantly delayed only the disease induced by GMP- but not by LT-HSC-derived blasts. Gene expression profiling in immortalized pre-leukemic cells revealed down-regulation of over 300 genes, including several well-known MLL targets such as Meis1, HoxA5, HoxA9 and HoxA10 upon reducing the levels of MLL-AF9 expression. Likewise, we observed a global decrease in histone H3 lysine 79 dimethylation consistent with a Dot1l function in MLL-AF9 driven leukemia. LT-HSC-derived (“early”) blasts displayed distinct genetic signatures with > 400 genes highly and > 1300 genes lowly expressed (p001 fc1.5), clearly separating them from the GMP-derived blasts. Evi-1 and Erg, two prognostic markers in patient-derived gene signatures, stood out among these genes. The aggressive “early” LT-derived murine leukemias showed high Evi-1 and Erg expression levels (Evi-1 high, Erg high) as compared to the “late” LT-derived (Evi-1 low, Erg high) or the GMP-derived leukemias (Evi-1 low, Erg low). These observations suggest that the previously reported poor prognosis associated with elevated EVI-1 and/or ERG expression might directly reflect the cell of origin of the disease. We are currently exploiting our highly informative MLL-AF9 disease model to evaluate the functional relevance of novel origin-dependent MLL-AF9 target genes and to identify novel prognostic markers and therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Glycoproteome remodelling in MLL-rearranged B-cell precursor acute lymphoblastic leukemia

“…Increased FUT4 RNA also is included in the signature that clusters MLL-r leukemias together from other subclasses [3] and was suggested to regulate migration and adhesion of B-ALL cells by integrin α5β1 [76]. Thus, we believe that increased Lewis X synthesis could contribute to MLL-AF4-driven migration and invasion [77].…”

GATA2 as an outcome signature: promoting bladder cancer migration and inhibiting PD-L1 expression

“…The present study found that embryonic development-related genes were prognostic risk factors for BLCA. Invasion and metastasis of BLCA are thought to be associated with EMT [30,31].Tumor cell stemness regulates tumor cell differentiation, proliferation, invasion and migration [4,[32][33][34].Numerous studies showed that cancer stem cells regulate EMT [35][36][37].Embryonic development is often closely related to stem cells and EMT [5,38,39]. Therefore, we established a micro-landscape of embryonic development-related genes for predicting prognostic risk in BLCA patients.…”

Inflammatory Recruitment of Healthy Hematopoietic Stem and Progenitor Cells in the Acute Myeloid Leukemia Niche

“…To conceptually validate the presence of inflammation in HSPCs beyond the C1498 model system, we utilized a more commonly used and translationally relevant MLL-AF9 AML mouse model (27,28). Typically reliant of retrovirus overexpression, the model system we describe here mimics human disease instead through doxycycline (DOX) mediated regulation of human MLL-AF9 fusion oncogene expression to (29,30). To evaluate the inflammatory state in HSPCs in the iMLL-AF9 model in vivo, we generated chimeric recipients (CD45.2) bearing both iMLL-AF9 BM (CD45.1; iMLL-AF9 leukemic BM) and wildtype BM (CD45.1/2; resembling healthy BM) (Fig.…”