Wendy A. Hudson

Malignant Transformation Initiated by Mll-AF9: Gene Dosage and Critical Target Cells

The pathways by which oncogenes, such as MLL-AF9, initiate transformation and leukemia in humans and mice are incompletely defined. In a study of target cells and oncogene dosage, we found that Mll-AF9, when under endogenous regulatory control, efficiently transformed LSK (Lin(-)Sca1(+)c-kit(+)) stem cells, while committed granulocyte-monocyte progenitors (GMPs) were transformation resistant and did not cause leukemia. Mll-AF9 was expressed at higher levels in hematopoietic stem (HSC) than GMP cells. Mll-AF9 gene dosage effects were directly shown in experiments where GMPs were efficiently transformed by the high dosage of Mll-AF9 resulting from retroviral transduction. Mll-AF9 upregulated expression of 192 genes in both LSK and progenitor cells, but to higher levels in LSKs than in committed myeloid progenitors.

Xenotransplantation of human lymphoid malignancies is optimized in mice with multiple immunologic defects

While it is known that mice with genetic immune defects are useful for establishing durable engraftment of human tumor xenografts, the relative role of components of host innate and adoptive immunity in engraftment has not been determined. We directly compared the ability of four strains of genetically immunodeficient mice (NOD/SCID, SCID, Nude and Rag-1-deficient) to successfully engraft and support the human cell lines Daudi, Raji, Namalwa and Molt-4 as subcutaneous tumors. We additionally examined the effect of further immunosuppression of the mice by whole body irradiation at a dose of 600 cGy for Nude and Rag-1 and 300 cGy for SCID mice and by administration of anti-natural killer (asialo-GM1) antibody on tumor growth. Mice with each of the defects supported xenografts to varying degrees. We found differences in growth characteristics in the cell lines tested, with Namalwa consistently producing the largest tumors. With all cell lines studied, optimal growth was achieved using NOD/SCID mice. Overall, tumor growth was somewhat enhanced by pretreatment with radiation with little additional benefit from the addition of anti-asialo-GM1 antibody. The importance of multiple components of the innate and adoptive immune system in xenotransplantation were best demonstrated when results in untreated NOD/SCID mice were compared to SCID, nude and RAG-1-deficient mice. The NOD/SCID mouse with or without additional immunosuppression provides the optimal model for the study of the biology and treatment of human leukemias and lymphomas.

A role for MEIS1 in MLL-fusion gene leukemia

Leukemias with MLL rearrangements are characterized by high expression of the homeobox gene MEIS1. In these studies, we knocked down Meis1 expression by shRNA lentivirus transduction in murine Mll-AF9 leukemia cells. Meis1 knockdown resulted in decreased proliferation and survival of murine Mll-AF9 leukemia cells. We also observed reduced clonogenic capacity and increased monocytic differentiation. The establishment of leukemia in transplantation recipients was significantly delayed by Meis1 knockdown. Gene expression profiling of cells transduced with Meis1 shRNA showed reduced expression of genes associated with cell cycle entry and progression. shRNA-mediated knockdown of MEIS1 in human MLL-fusion gene leukemia cell lines resulted in reduced cell growth. These results show that MEIS1 expression is important for MLL-rearranged leukemias and suggest that MEIS1 promotes cell-cycle entry. Targeting MEIS1 may have therapeutic potential for treating leukemias expressing this transcription factor.

EVI1 is critical for the pathogenesis of a subset of MLL-AF9–rearranged AMLs

The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)-rearranged leukemias are EVI1(pos). High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1(pos) MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1(neg) MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1(pos) HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9-transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1(neg). Moreover, shRNA-mediated knockdown of Evi1 in an Evi1(pos) MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1(pos) MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.

Pharmacokinetics and biodistribution of radioimmunoconjugates of anti-CD19 antibody and single-chain Fv for treatment of human B-cell Malignancy

Abstract The comparative advantages and disadvantages of intact antibodies and single-chain Fv as immunotoxins and radioimmunoconjugates have been widely discussed but not directly compared. In this study, the in vivo properties of anti-CD19 B43 monoclonal antibody and its derived single-chain Fv (FVS191) were studied in athymic nude mice bearing CD19-positive human lymphomas. B43 mab and FVS191 were labeled with iodine-125 using iodine-beads, and immunoreactivities were determined to be 57% and 72%, respectively. Scatchard analysis showed a similar high affinity for both. The results of pharmacokinetic studies revealed that FVS191 had a rapid biphasic clearance from the circulation (T1/2α = 2.5 min, T1/2β = 3.7 h); The T1/2α and T1/2β phases of B43 mab were determined to be 0.72 h and 57 h respectively. Biodistribution studies compared the uptake of labeled antibodies by CD19-positive and by CD19-negative tumors. The peak percentages of injected dose were 5.7% at 12 h for B43 and 2.45% at 1 h for FVS191. Radiolocalization indices (RI) demonstrated tumor-specific uptake for both, but higher uptake for B43. The optimal RI was seen at 15 min for FVS191 and 6 h for B43. FVS191 was unstable in vivo, approximately 50% of the injected dose being degraded in blood in 100 min. Radioactivity detected in the urine was present mainly as the deiodinized form of FVS191. The results suggest that B43 mab is favored over FVS191 in biodistribution properties and in vivo stability. Because B43 Mab showed early tumor-specific uptake, high RI values, and favorable tissue-to-blood ratios, it is a potential candidate for radioimmunotherapy and immunotoxin therapy of B-cell leukemia and lymphoma.

Modeling human infant MLL leukemia in mice: leukemia from fetal liver differs from that originating in postnatal marrow

To the editor: Leukemia in human infants often begins with an in utero chromosomal translocation resulting in an MLL fusion oncogene that is most frequently MLL-AF4 or MLL-AF9.1 Recent gene expression studies suggest that human infant MLL fusion gene leukemia originates in cells that differ from both infant leukemia without the fusion gene and non-infant childhood leukemia. However, the cell of origin remains undefined.2,3 Mouse models of human MLL leukemia have proven to be informative. An example is the MLL-AF9 knock-in model, in which the fusion gene is expressed at physiologic levels.4–6 In this model, hematopoietic stem cells (HSCs) from postnatal murine marrow are readily transformed and leukemias develop that resemble human myeloid post natal MLL leukemias.6 The current study was designed to model human fetal–originated infant MLL leukemia. We compared the leukemia that developed from MLL-AF9 fetal liver cells with that arising from adult marrow cells. Mice were transplanted with fetal MLL-AF9 liver cells (sorted HSCs or unsorted cells) or HSC cells from adult MLL-AF9 bone marrow. We found that the time to development of leukemia was significantly longer after transplantation of fetal liver HSCs than that from marrow HSCs (Figure 1A). Extensive analysis of the leukemias by histopathology, immunohistochemistry, and flow cytometry showed important differences in recipients of adult marrow HSCs (Figure 1B) compared with recipients of fetal liver HSCs (Figure 1C) and unsorted fetal liver cells (Figure 1D). When donor cells were from adult marrow, the leukemia was always myeloid in character with myeloperoxidase (MPO) positivity but negative for CD45R/B220, a phosphatase that appears early in hematopoietic cellular differentiation. However, when fetal liver cells (either sorted HSCs in Figure 1C or unsorted in Figure 1D) were used as donor cells, the leukemia often showed more undifferentiated cells. In some cases fetal liver–derived leukemia cells were positive for CD45R/ B220 (Figure 1C-D) and either positive or negative for MPO. Figure 1 MLL-AF9 leukemia from fetal liver differs from that originating in postnatal marrow. (A) The latency of leukemia in recipient mice that received 100 hematopoietic MLL-AF9 stem cells (HSCs) from either embryonic day (E) 14.5 fetal liver or adult bone marrow. ... The delay in overt murine MLL leukemia from both fetal liver and bone marrow compared with many human infants with MLL fusion gene leukemia suggests that secondary cooperating events (genetic or epigenetic) may differ between species. The shorter latency of onset of leukemia in bone marrow compared with fetal liver cells suggests that by the time the targeted cells have progressed in maturity to the marrow stem/progenitor stage secondary changes have already developed. Our previous observation that MLL-AF9–transformed fetal liver had limited replating capacity compared with MLL-AF9 marrow is consistent with this hypothesis.5 The MLL fusion partner, AF9, may also be important in the timing of the onset of leukemia. Human infant MLL fusion gene leukemia involving an AF9 partner occurs with greater delay than with other partners as another group7 has observed. Overall, these results provide further impetus for the study of the cellular origins of human and murine MLL leukemia.

Sleeping Beauty Insertional Mutagenesis in Mice Identifies Drivers of Steatosis-Associated Hepatic Tumors

Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis-associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Cancer Res; 77(23); 6576-88. ©2017 AACR.