A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia.

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

Keratinocyte growth factor and stem cell factor to improve thymopoiesis after autologous CD34+ cell transplantation in rhesus macaques.

Deficient thymopoiesis and retarded recovery of naive CD4(+) T cells are important determinants of insufficient immune-competence following hematopoietic stem cell transplantation (HSCT). Although keratinocyte growth factor (KGF) may protect the thymic epithelium, stem cell factor (SCF) is involved in early thymopoiesis. We evaluated whether KGF alone or combined with SCF would affect thymopoiesis and hematologic recovery following myeloablative autologous HSCT into rhesus macaques. Purpose-bred adult rhesus macaques received 10(6) autologous CD34(+)-selected mononuclear bone marrow cells (BMC)/kg after 9 Gy myeloablative conditioning. Animals were treated with phosphate-buffered saline (PBS) (n = 2), KGF alone (n = 2), or KGF combined with SCF (n = 2). KGF-treated animals showed accelerated hematologic recovery, improved thymopoiesis, and enhanced naive T-cell recovery following transplantation. Improved T cell recovery was not associated with protection against cytomegalovirus reactivation nor with improved antibody response to tetanus toxoid vaccination. Animals treated with KGF and SCF experienced severe adverse events that precluded evaluation of thymopoiesis and T cell recovery. Collectively, our data confirm that KGF may enhance thymopoiesis.

Stem cell factor consistently improves thymopoiesis after experimental transplantation of murine or human hematopoietic stem cells in immunodeficient mice.

Deficient thymopoiesis is a pivotal determinant of impaired immune competence following hematopoietic stem cell transplantation (HSCT). Stem cell factor (SCF) is essentially involved in early thymopoiesis. We evaluated whether SCF administration would improve recovery of thymopoiesis following HSCT in immunodeficient mice receiving: 1) bone marrow (BM) transplantation of congenic mice; or 2) human fetal liver HSCT in the human immune system mouse model. Following murine BM transplantation, SCF significantly enhanced thymopoiesis and peripheral T cell recovery in lymph nodes and spleen. SCF did not affect BM lymphoid progenitor recovery and/or expansion. Median thymic cellularity increased from 0.9 in PBS- to 266 × 10(4)/thymus in SCF-treated mice (p = 0.05). Following human HSCT in human immune system mice, higher thymic cellularity was observed in SCF-treated mice. Double-negative and early double-positive thymocyte subsets increased, but especially late double-positive, CD4 single-positive, and CD8 single-positive thymocyte subsets were significantly enhanced (p < 0.05). These results show that exogenous supply of SCF may significantly improve murine and human posttransplant thymopoiesis, for which the effect is probably exerted by directly promoting T cell development intrathymically rather than by enhanced entry of prethymically expanded lymphoid progenitors.

Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells.

The human body contains over 500 individual lymph nodes, yet the biology of their formation is poorly understood. Here we identify human lymphoid tissue-inducer cells (LTi cells) as lineage-negative RORC+ CD127+ cells with the functional ability to interact with mesenchymal cells through lymphotoxin and tumor necrosis factor. Human LTi cells were committed natural killer (NK) cell precursors that produced interleukin 17 (IL-17) and IL-22. In vitro, LTi cells gave rise to RORC+ CD127+ NK cells that retained the ability to produce IL-17 and IL-22. Postnatally, similar populations of LTi cell-like cells and RORC+ CD127+ NK cells were present in tonsils, and both secreted IL-17 and IL-22 but no interferon-gamma. Our data indicate that lymph node organogenesis is controlled by an NK cell precursor population with adaptive immune features and demonstrate a previously unappreciated link between the innate and adaptive immune systems.

The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.

Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation.

Deficient thymopoiesis and retarded recovery of newly developed CD4(+) T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage(-)sca-1(+)c-kit(+)flt3(+) lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.

Addition of treosulfan to a nonmyeloablative conditioning regimen results in enhanced chimerism and immunologic tolerance in an experimental allogeneic bone marrow transplant model.

Treosulfan (L-threitol-1,4-bismethanesulfonate) is an alkylating agent with routine clinical application in the treatment of ovarian cancer. In this murine study we show that this drug also has the ability to deplete primitive hematopoietic stem cells in a dose-dependent manner as determined by the cobblestone area-forming cell assay and is similar to its parent compound busulfan. Because busulfan is frequently used as part of the conditioning regimen before stem cell transplantation, we investigated an alternative nonmyeloablative protocol in an allogeneic bone marrow transplantation model in which low-dose treosulfan was added to an immune-suppressive regimen consisting of T cell-depleting antibodies, fludarabine, and thymic irradiation. Although this treatment protocol produced minimal myelosuppression, the addition of treosulfan proved to be important for allowing stable multilineage and mixed chimerism in C57BL/6 recipients of major histocompatibility complex-mismatched B10.A bone marrow without evidence of graft-versus-host disease. Donor lymphocyte infusion performed at 10 weeks after bone marrow transplantation had the effect of transforming the state of mixed chimerism to full donor-type cells, again without evidence of graft-versus-host disease. Donor-specific immunologic tolerance in the mixed chimeric animals was indicated by the acceptance of donor-type and rejection of third-party skin grafts. Thus, low-dose treosulfan may be considered as a useful component of a truly nonmyeloablative conditioning protocol in providing for mixed hematopoietic chimerism and, consequently, in establishing a platform for adoptive immunotherapy.

Relation between CXCR-4 expression, Flt3 mutations, and unfavorable prognosis of adult acute myeloid leukemia.

Recently it was shown that, analogous to normal hematopoietic cells, the level of CXC chemokine receptor 4 (CXCR-4) expression on acute myeloid leukemia (AML) cells correlates with stromal cell derived factor-1 alpha (SDF-1)-induced chemotaxis. As we speculated that an anomalous organ distribution of AML cells could affect cell survival and thus result in an altered fraction surviving chemotherapy, we examined a possible correlation between patient prognosis and CXCR-4 expression in AML patients. We found that patients with a high CXCR-4 expression in the CD34(+) subset had a significantly reduced survival and a higher probability of relapse, resulting in a median relapse-free survival (RFS) of only 8.3 months. CXCR-4 expression was significantly higher in fetal liver tyrosine kinase-3 (Flt3)/internal tandem duplication (ITD) AML than in Flt3/wild-type (wt) AML. Covariate analysis indicated that the prognostic significance of Flt3/ITDs with respect to RFS was no more apparent when analyzed in conjunction with the expression of CXCR-4 in the CD34(+) subset, suggesting that the poor prognosis of Flt3/ITD AML might be subordinate to the increased CXCR-4 expression. Using a granulocyte colony-stimulating factor receptor (G-CSF-R)-expressing 32D cell line, we observed that SDF-1/CXCR-4 interaction is required for the survival of myeloid differentiating cells, and it also induces a block in G-CSF-induced myeloid differentiation. These data suggest that the SDF-1/CXCR-4 axis may influence therapy responsiveness and defines unfavorable prognosis in AML.