Lueder H. Meyer

Diversity of Human Leukemia Xenograft Mouse Models: Implications for Disease Biology

Over the past decade, xenografting human leukemia cells into mice with different levels of immunodeficiency, with or without preconditioning, has provided an important tool to study various aspects of leukemia biology and to identify distinct clinical risk groups for evaluation of novel therapeutic strategies, as well as the possibility of amplifying human leukemia cells in vivo. Interestingly, these models using human acute lymphoblastic leukemia and acute myeloid leukemia cells as xenografts recapitulate many clinical features of the disease. Similar to the human environment (for example, in the bone marrow), transplanted leukemia cells in the murine setting are exposed to both favorable and unfavorable conditions for engraftment that may exert a distinct pressure for selection of subclones. Thus, results obtained in these models may vary depending on the experimental setup. The impact of in vivo growth of human leukemia cells on the background of a more or less hostile murine environment for leukemia biology and the course of the disease in patients are discussed in the context of the diversity of xenograft models.

Central nervous system acute lymphoblastic leukemia: role of natural killer cells

Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2 -rearranged BCP-ALL

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult–to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Mutations of SETBP1 and JAK3 in juvenile myelomonocytic leukemia: a report from the Italian AIEOP study group

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive disease of early childhood. Driver mutations in the Ras signaling pathways are a key feature of JMML patients. Mutations in SETBP1 and JAK3 were recently identified in a subset of JMML patients characterized by poor prognosis and progression of disease. In this study, we report the results of a screening for mutations in SETBP1 and JAK3 of a cohort of seventy Italian patients with JMML, identifying 11.4% of them harboring secondary mutations in these two genes and discovering two new mutations in the SKI domain of SETBP1. JMML xenotransplantation and colony assay provide an initial understanding of the secondary nature of these events occurring in early precursor cells and suggest a different propagating capacity of clones harboring particular mutations.

Characterization and identification of leukemia initiating cells in pediatric acute lymphoblastic leukemia.

In acute lymphoblastic leukemia (ALL) leukemia initiating cells (LICs) have been considered to be organized in an hierarchical fashion, however recent findings demonstrated LIC-activity also in more committed cells supporting a stochastic stem cell concept. Thus, the nature of leukemia initiating cells in ALL still remains elusive. As an alternative approach to define LICs by expression of cellular markers as commonly employed, we addressed LIC activities in ALL by functional investigation of cellular subfractions of distinct cell cycle phases. Patient-derived xenograft BCP-ALL cells were sorted according to cell cycle stages (i.e. G0/G1 and G2/M) and subsequently transplanted onto NOD/SCID mice. All cell fractions led to engraftment indicating LIC activity of all leukemia cells. However, cells isolated from G0/G1 cell cycle phases led to early leukemia onset in contrast to cells from late cell cycle (G2/M) constantly showing lowest LIC activity. Strikingly, this difference in LIC activity was maintained upon secondary transplantation. In an alternative approach, we investigated metabolic activities in cellular leukemia subfractions and identified low metabolic activity in cells of early G0/G1 cell cycle phases compared to increased cellular metabolism in cells of late G2/M. To address LIC-capacities of ALL cells with distinct metabolic activities on a functional level, cellular fractions were sorted according to low or high ROS levels and subsequently transplanted onto NOD/SCID mice. Interestingly, a prolonged engraftment was observed upon transplantation of ROShigh cells in contrast to faster leukemia repopulation in recipients transplanted with ROSlow cells, showing that the metabolic activity is indicative for its leukemia initiating activity. In summary, we identified LIC-activity in all leukemia subpopulations. Importantly, our findings indicate that leukemia initiating cells in ALL are enriched in early cell cycle and characterized by low metabolic activity.

High fidelity of phenotype and genotype in serial NOD/SCID pediatric acute lymphoblastic leukemia

Primary ALL cells are very difficult to culture in vitro and currently available cell lines only poorly reflect the heterogeneous nature of the disease. Many groups therefore use murine xenotransplantation models for primary pediatric ALL. But, to date it remains unclear as to what extent the characteristics of the original patient leukemia are retained over consecutive xenograft passages or whether there is selection for a clone with characteristics distinct from the bulk leukemia at diagnosis. We here address this question for 7 consecutive leukemias diagnosed in our clinic and directly transplanted onto unconditioned NOD/SCID-mice. After primary passage, clinical presentation, immunophenotype and gene expression profiles were highly similar to the diagnostic leukemia, with minor differences arising from the presence of normal human hematopoietic cells in primary patient material. Moreover, all characteristics analyzed remained stable after secondary and tertiary passages. The model precisely recapitulates the primary human leukemia in the mouse and thereby reflects the heterogeneity and inherent variability of different primary patient leukemia cells.