Establishing human leukemia xenograft mouse models by implanting human bone marrow-like scaffold-based niches

To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches

Oncogene EVI1 Drives Acute Myeloid Leukemia Via a Targetable Interaction with CTBP2

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Since transcription factors like EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies we demonstrate interaction with CTBP1 and CTBP2 via a single PLDLS motif in EVI1 is indispensable for leukemic transformation. A 4x PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. Our findings may also have important implications for other tumour types with aberrant expression of EVI1 or for cancers transformed by other CTBP1/2 dependent oncogenic transcription factors

Optimal selection of natural killer cells to kill myeloma: the role of HLA-E and NKG2A

Immunotherapy with allogeneic natural killer (NK) cells offers therapeutic perspectives for multiple myeloma patients. Here, we aimed to refine NK cell therapy by evaluation of the relevance of HLA-class I and HLA-E for NK anti-myeloma reactivity. We show that HLA-class I was strongly expressed on the surface of patient-derived myeloma cells and on myeloma cell lines. HLA-E was highly expressed by primary myeloma cells but only marginally by cell lines. HLA-E(low) expression on U266 cells observed in vitro was strongly upregulated after in vivo (bone marrow) growth in RAG-2(-/-) γc(-/-) mice, suggesting that in vitro HLA-E levels poorly predict the in vivo situation. Concurrent analysis of inhibitory receptors (KIR2DL1, KIR2DL2/3, KIR3DL1 and NKG2A) and NK cell degranulation upon co-culture with myeloma cells revealed that KIR-ligand-mismatched NK cells degranulate more than matched subsets and that HLA-E abrogates degranulation of NKG2A+ subsets. Inhibition by HLA-class I and HLA-E was also observed with IL-2-activated NK cells and at low oxygen levels (0.6 %) mimicking hypoxic bone marrow niches where myeloma cells preferentially reside. Our study demonstrates that NKG2A-negative, KIR-ligand-mismatched NK cells are the most potent subset for clinical application. We envision that infusion of high numbers of this subclass will enhance clinical efficacy

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies

e-Blood Reconstructing the human hematopoietic niche in immunodeficient mice:opportunities for studying primary multiple myeloma

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies