Kevin A. Link

AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3

AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3

Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells

RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.

p53 Independent epigenetic-differentiation treatment in xenotransplant models of acute myeloid leukemia

Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo regimens of the deoxycytidine analogue decitabine that deplete the chromatin-modifying enzyme DNA methyl-transferase 1 without phosphorylating p53 or inducing early apoptosis were determined. These decitabine regimens but not equimolar DNA-damaging cytarabine upregulated the key late differentiation factors CCAAT enhancer-binding protein ɛ and p27/cyclin dependent kinase inhibitor 1B (CDKN1B), induced cellular differentiation and terminated AML cell cycle, even in cytarabine-resistant p53- and p16/CDKN2A-null AML cells. Leukemia initiation by xenotransplanted AML cells was abrogated but normal hematopoietic stem cell engraftment was preserved. In vivo, the low toxicity allowed frequent drug administration to increase exposure, an important consideration for S phase specific decitabine therapy. In xenotransplant models of p53-null and relapsed/refractory AML, the non-cytotoxic regimen significantly extended survival compared with conventional cytotoxic cytarabine. Modifying in vivo dose and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to standard therapy.

Core binding factor at the crossroads: determining the fate of the HSC.

Hematopoietic development requires coordinated actions from a variety of transcription factors. The core binding factor (CBF), consisting of a Runx protein and the CBFβ protein, is a transcription factor complex that is essential for emergence of the hematopoietic stem cell (HSC) from an endothelial cell stage. The hematopoietic defects observed in either Runx1 or CBFβ knockout mice underscore the necessity of this complex for definitive hematopoiesis. Despite the requirement for CBF in establishing definitive hematopoiesis, Runx1 loss has minimal impact on maintaining the HSC state postnatally, while CBFβ may continue to be essential. Lineage commitment, on the other hand, is significantly affected upon CBF loss in the adult, indicating a primary role for this complex in modulating differentiation. Given the impact of normal CBF function in the hematopoietic system, the severe consequences of disrupting CBF activity, either through point mutations or generation of fusion genes, are obvious. The physiologic role of CBF in differentiation is subverted to an active process of self‐renewal maintenance by the genetic aberrations, through several possible mechanisms, contributing to the development of hematopoietic malignancies including myelodysplastic syndrome and leukemia. The major impact of CBF on the hematopoietic system in both development and disease highlights the need for understanding the intricate functions of this complex and reiterate the necessity of continued efforts to identify potential points of therapeutic intervention for CBF‐related diseases. J. Cell. Physiol. 222:50–56, 2010.

The thrombopoietin/MPL/Bcl-xL pathway is essential for survival and self-renewal in human preleukemia induced by AML1-ETO

AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO-induced MPL.

UBASH3B/Sts-1-CBL axis regulates myeloid proliferation in human preleukemia induced by AML1-ETO

The t(8;21) rearrangement, which creates the AML1-ETO fusion protein, represents the most common chromosomal translocation in acute myeloid leukemia (AML). Clinical data suggest that CBL mutations are a frequent event in t(8;21) AML, but the role of CBL in AML1-ETO-induced leukemia has not been investigated. In this study, we demonstrate that CBL mutations collaborate with AML1-ETO to expand human CD34+ cells both in vitro and in a xenograft model. CBL depletion by shRNA also promotes the growth of AML1-ETO cells, demonstrating the inhibitory function of endogenous CBL in t(8;21) AML. Mechanistically, loss of CBL function confers hyper-responsiveness to thrombopoietin and enhances STAT5/AKT/ERK/Src signaling in AML1-ETO cells. Interestingly, we found the protein tyrosine phosphatase UBASH3B/Sts-1, which is known to inhibit CBL function, is upregulated by AML1-ETO through transcriptional and miR-9-mediated regulation. UBASH3B/Sts-1 depletion induces an aberrant pattern of CBL phosphorylation and impairs proliferation in AML1-ETO cells. The growth inhibition caused by UBASH3B/Sts-1 depletion can be rescued by ectopic expression of CBL mutants, suggesting that UBASH3B/Sts-1 supports the growth of AML1-ETO cells partly through modulation of CBL function. Our study reveals a role of CBL in restricting myeloid proliferation of human AML1-ETO-induced leukemia, and identifies UBASH3B/Sts-1 as a potential target for pharmaceutical intervention.

MEIS1 regulates an HLF–oxidative stress axis in MLL-fusion gene leukemia

Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment.

Supraphysiologic levels of the AML1-ETO isoform AE9a are essential for transformation.

Significance The AE9a protein (alternative splicing at exon 9) is often used to model t(8;21) leukemia. Our study demonstrates that increased oncogene dosage is a critical parameter of AE9a transformation, likely as a result of impaired transcriptional regulation of AML1-ETO target genes. This insight could assist in identifying those downstream genes most critical for t(8;21)-associated transformation. Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34+ cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.

Production and purification of high-titer foamy virus vector for the treatment of leukocyte adhesion deficiency.

Compared to other integrating viral vectors, foamy virus (FV) vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34+ cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy. However, infectious titers of FV-human(h)CD18 were low and not suitable for manufacturing of clinical-grade product. Herein, we developed a scalable production and purification process that resulted in 60-fold higher FV-hCD18 titers from ~1.7 × 104 to 1.0 × 106 infectious units (IU)/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimized gag, heparin affinity chromatography, tangential flow filtration, and ultracentrifugation, which reproducibly resulted in 5,000-fold concentrated and purified virus, an overall yield of 19 ± 3%, and final titers of 1–2 × 109 IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO) concentration, thereby avoiding DMSO-induced toxicity to CD34+ cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production.

461. Scale-Up and Manufacturing of High-Titer Foamy Virus Vector Containing Human CD18 for the Treatment of Leukocyte Adhesion Deficiency

Compared to other integrating viral vectors, foamy virus (FV) vectors have several distinct advantages as a gene transfer tool, including: the ability to carry larger expression cassettes, the increased stability due to a DNA genome formed in developing vector particles, and the random integration pattern without preference for promoter regions or active genes as is the case with gamma-retroviral and lentiviral vectors. In addition, the FV envelope has tropism for most cell types and is not associated with disease in humans. Combined, these properties make FV vectors the ideal candidate for gene therapy application. Proof of principle was provided by Bauer et al. (Nat. Med. 2008) showing cure of canine Leukocyte Adhesion Deficiency (LAD) using autologous CD34+ cells transduced with FV vector carrying the CD18 gene, without short or long-term adverse effects. Here we demonstrate the optimization of large scale manufacturing of FV vector carrying the human CD18 gene for the treatment of human LAD. As compared to the methods used in the canine LAD study, a 60-fold increase in FV-CD18 titer was observed using a combination of polyethylenimine (PEI) transfection and use of a codon-optimized gag, from approximately 1.70E+04 to 1.00E+06 Infectious Units/mL (IU/mL) on Raw 264.7 cells. An evaluation of purification strategies showed efficient vector purification and concentration of FV vector through a combination of heparin-affinity chromatography on the AKTA ready, a disposable fluid path chromatography unit, tangential flow filtration and ultracentrifugation, with an overall 5000-fold concentration and 20-30% recovery. This approach was compatible with large scale manufacturing in compliance with Good Manufacturing Practices (GMP). Although 5% DMSO is needed for optimal recovery of infectious FV vector from -80°C storage, highly concentrated vector can be diluted to reduce the DMSO concentration and toxicity to CD34+ cells, while maintaining effective transduction rates of up to 50%. This process is currently being finalized for clinical manufacturing in support of a Phase I trial for the treatment of LAD.Note: MN and DL equally contributed to this study.