Stephanie Halene

Immune response to green fluorescent protein: implications for gene therapy.

Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.

Development and function of human innate immune cells in a humanized mouse model

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition

Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2s normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.

Comorbidities and survival in a large cohort of patients with newly diagnosed myelodysplastic syndromes

Comorbid conditions have rarely been systematically studied among patients with myelodysplastic syndromes (MDS). We conducted a large population-based study to assess the role of comorbidity in the survival of newly diagnosed MDS patients. This study included 1708 MDS patients (age > or =66 years) diagnosed in the US during 2001-2002, with follow-up through the end of 2004. Hazard ratios (HRs) were estimated using multivariate Cox proportional hazard models. The median survival time was approximately 18 months. Fifty one percent of MDS patients had comorbid conditions. Patients with comorbid conditions had significantly greater risk of death than those without comorbidities. The HR was 1.19 (95% confidence interval (CI): 1.05-1.36) and 1.77 (95% CI: 1.50-2.08) for those with a Charlson index of 1-2 and > or = 3, respectively. The risk of death increases with Charlson index. MDS patients who have congestive heart failure or chronic obstructive pulmonary disease had significantly shorter survival than patients without those conditions, whereas diabetes did not appear to have an impact on survival. This study confirms comorbidity as a significant and independent determinant of MDS survival, and the findings underscore the importance to take comorbid conditions into account when assessing the prognosis of MDS.

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity

The oncometabolite 2-hydroxyglutarate renders IDH1/2 mutant cancer cells deficient in homologous recombination and confers vulnerability to synthetic lethal targeting with PARP inhibitors. Target 2HG or not 2HG, that is the question Mutations in isocitrate dehydrogenase 1 and 2, which result in overproduction of 2-hydroxyglutarate (2HG), are observed in multiple tumor types, including gliomas and acute myelogenous leukemia. An additional form of 2HG is produced under hypoxia, which is also frequent in tumors. 2HG is considered to be an oncometabolite, or a metabolite that promotes carcinogenesis, and inhibitors of mutant isocitrate dehydrogenase are in development to target this process. However, Sulkowski et al. found that it may be possible to take advantage of 2HG overproduction instead. The authors discovered that 2HG overproduction impairs homologous recombination used in DNA repair and sensitizes cancer cells to treatment with PARP inhibitors, another class of cancer drugs that are already in clinical use. 2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)–dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) inhibitors. This “BRCAness” phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1–dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Gene Therapy Using Hematopoietic Stem Cells: Sisyphus Approaches the Crest

Gene transfer targeting cells of the blood and immune system was one of the first areas of investigation in the field of gene therapy. Despite the encouraging results achieved in early studies using murine bone marrow, the task of gene transfer into human hematopoietic stem cells proved to be far more difficult. As a result, progress has been disappointingly slow and initial clinical trials generally failed to achieve significant levels of gene marking. The continued application of new advances in vectorology and hematopoietic stem cell biology has now led to improvements in preclinical models that are being translated into clinical trials. The progress and remaining problems are discussed in this review article.

Role of RhoA-Specific Guanine Exchange Factors in Regulation of Endomitosis in Megakaryocytes

Polyploidization can precede the development of aneuploidy in cancer. Polyploidization in megakaryocytes (Mks), in contrast, is a highly controlled developmental process critical for efficient platelet production via unknown mechanisms. Using primary cells, we demonstrate that the guanine exchange factors GEF-H1 and ECT2, which are often overexpressed in cancer and are essential for RhoA activation during cytokinesis, must be downregulated for Mk polyploidization. The first (2N-4N) endomitotic cycle requires GEF-H1 downregulation, whereas subsequent cycles (>4N) require ECT2 downregulation. Exogenous expression of both GEF-H1 and ECT2 prevents endomitosis, resulting in proliferation of 2N Mks. Furthermore, we have shown that the mechanism by which polyploidization is prevented in Mks lacking Mkl1, which is mutated in megakaryocytic leukemia, is via elevated GEF-H1 expression; shRNA-mediated GEF-H1 knockdown alone rescues this ploidy defect. These mechanistic insights enhance our understanding of normal versus malignant megakaryocytopoiesis, as well as aberrant mitosis in aneuploid cancers.

Gaucher disease gene GBA functions in immune regulation

Inherited deficiency of acid β-glucosidase (GCase) due to biallelic mutations in the GBA (glucosidase, β, acid) gene causes the classic manifestations of Gaucher disease (GD) involving the viscera, the skeleton, and the lungs. Clinical observations point to immune defects in GD beyond the accumulation of activated macrophages engorged with lysosomal glucosylceramide. Here, we show a plethora of immune cell aberrations in mice in which the GBA gene is deleted conditionally in hematopoietic stem cells (HSCs). The thymus exhibited the earliest and most striking alterations reminiscent of impaired T-cell maturation, aberrant B-cell recruitment, enhanced antigen presentation, and impaired egress of mature thymocytes. These changes correlated strongly with disease severity. In contrast to the profound defects in the thymus, there were only limited cellular defects in peripheral lymphoid organs, mainly restricted to mice with severe disease. The cellular changes in GCase deficiency were accompanied by elevated T-helper (Th)1 and Th2 cytokines that also tracked with disease severity. Finally, the proliferation of GCase-deficient HSCs was inhibited significantly by both GL1 and Lyso-GL1, suggesting that the “supply” of early thymic progenitors from bone marrow may, in fact, be reduced in GBA deficiency. The results not only point to a fundamental role for GBA in immune regulation but also suggest that GBA mutations in GD may cause widespread immune dysregulation through the accumulation of substrates.

Complex oncogene dependence in microRNA-125a–induced myeloproliferative neoplasms

Deregulation of microRNA (miRNA) expression can lead to cancer initiation and progression. However, limited information exists on the function of miRNAs in cancer maintenance. We examined these issues in the case of myeloproliferative diseases and neoplasms (MPN), a collection of hematopoietic neoplasms regarded as preleukemic, thereby representing early neoplastic states. We report here that microRNA-125a (miR-125a)–induced MPN display a complex manner of oncogene dependence. Following a gain-of-function genomics screen, we overexpressed candidate miR-125a in vivo, which led to phenotypes consistent with an atypical MPN characterized by leukocytosis, monocytosis, splenomegaly, and progressive anemia. The diseased MPN state could be recapitulated in a doxycycline-inducible mouse model. Upon doxycycline withdrawal, the primary MPN phenotypes rapidly resolved after the discontinuation of miR-125a overexpression. However, reinduction of miR-125a led to complex phenotypes, with some animals rapidly developing lethal anemia with extensive damages in the spleen. Forced expression of miR-125a resulted in elevated cellular tyrosine phosphorylation and hypersensitivity toward hematopoietic cytokines. Furthermore, we demonstrate that miR-125a targets multiple protein phosphatases. Our data demonstrate that miR-125a–induced MPN is addicted to its sustained overexpression, and highlight the complex nature of oncogenic miRNA dependence in an early neoplastic state.

Serum response factor is an essential transcription factor in megakaryocytic maturation

Serum response factor (Srf) is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to Srf(F/F) mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/Srf(F/F) knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41(+) megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.