FLT3-ITD impedes retinoic acid, but not arsenic, responses in murine acute promyelocytic leukemias.

Acute promyelocytic leukemia (APL) is often associated with activating FLT3 signaling mutations. These are highly related to hyperleukocytosis, a major adverse risk factor with chemotherapy-based regimens. APL is a model for oncogene-targeted therapies: all-trans retinoic acid (ATRA) and arsenic both target and degrade its ProMyelocytic Leukemia/Retinoic Acid Receptor α (PML/RARA) driver. The combined ATRA/arsenic regimen now cures virtually all patients with standard-risk APL. Although FLT3-internal tandem duplication (ITD) was an adverse risk factor for historical ATRA/chemotherapy regimens, the molecular bases for this effect remain unknown. Using mouse APL models, we unexpectedly demonstrate that FLT3-ITD severely blunts ATRA response. Remarkably, although the transcriptional output of initial ATRA response is unaffected, ATRA-induced PML/RARA degradation is blunted, as is PML nuclear body reformation and activation of P53 signaling. Critically, the combination of ATRA and arsenic fully rescues therapeutic response in FLT3-ITD APLs, restoring PML/RARA degradation, PML nuclear body reformation, P53 activation, and APL eradication. Moreover, arsenic targeting of normal PML also contributes to APL response in vivo. These unexpected results explain the less favorable outcome of FLT3-ITD APLs with ATRA-based regimens, and stress the key role of PML nuclear bodies in APL eradication by the ATRA/arsenic combination.

PLZF is a regulator of homeostatic and cytokine-induced myeloid development.

A major question in hematopoiesis is how the system maintains long-term homeostasis whereby the generation of large numbers of differentiated cells is balanced with the requirement for maintenance of progenitor pools, while remaining sufficiently flexible to respond to periods of perturbed cellular output during infection or stress. We focused on the development of the myeloid lineage and present evidence that promyelocytic leukemia zinc finger (PLZF) provides a novel function that is critical for both normal and stress-induced myelopoiesis. During homeostasis, PLZF restricts proliferation and differentiation of human cord blood-derived myeloid progenitors to maintain a balance between the progenitor and mature cell compartments. Analysis of PLZF promoter-binding sites revealed that it represses transcription factors involved in normal myeloid differentiation, including GFI-1, C/EBPalpha, and LEF-1, and induces negative regulators DUSP6 and ID2. Loss of ID2 relieves PLZF-mediated repression of differentiation identifying it as a functional target of PLZF in myelopoiesis. Furthermore, induction of ERK1/2 by myeloid cytokines, reflective of a stress response, leads to nuclear export and inactivation of PLZF, which augments mature cell production. Thus, negative regulators of differentiation can serve to maintain developmental systems in a primed state, so that their inactivation by extrinsic signals can induce proliferation and differentiation to rapidly satisfy increased demand for mature cells.

Comprehensive genomic screens identify a role for PLZF-RARalpha as a positive regulator of cell proliferation via direct regulation of c-MYC.

The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RARalpha) and RARalpha-PLZF. Using a combination of chromatin immunoprecipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RARalpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RARalpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RARalpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RARalpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RARalpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RARalpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RARalpha.

HOX deregulation in acute myeloid leukemia.

The deregulation of homeobox (HOX) genes in acute myeloid leukemia (AML) and the potential for these master regulators to perturb normal hematopoiesis is well established. To date, overexpression of HOX genes in AML has been attributed to specific chromosomal aberrations and abnormalities involving mixed-lineage leukemia (MLL), an upstream regulator of HOX genes. The finding reported in this issue of the JCI by Scholl et al. that caudal-type homeobox transcription factor 2 (CDX2), which is capable of affecting HOX gene expression during embryogenesis, is overexpressed in 90% of patients with AML and induces a transplantable AML in murine models provides an alternative mechanism for HOX-induced leukemogenesis and yields important insights into the hierarchy of HOX gene regulation in AML (see the related article beginning on page 1037).

Transcriptional regulation of FHL1 by TLX1/HOX11 is dosage, cell-type and promoter context-dependent.

TLX1/HOX11 encodes an NK-like homeodomain transcription factor that is both normally required for embryonic development and aberrantly expressed in T-cell acute lymphoblastic leukemia. Previous studies have shown that TLX1 can regulate target genes including ALDH1A1 and FHL1. However, whereas ALDH1A1 is consistently regulated by TLX1, endogenous FHL1 is only induced in a proportion of fibroblast or T-cell clones stably expressing TLX1. Here, we provide an explanation for these findings by demonstrating that the induction of FHL1, but not ALDH1A1, requires a high level of TLX1 expression in NIH 3T3 cells. In luciferase reporter assays, TLX1-mediated repression rather than activation of the FHL1 gene promoter and the magnitude of this effect was strongly influenced by the cellular background. Together, these results characterize TLX1 as a dual function regulator whose activity in respect to FHL1 is critically dependent upon its cellular concentration, as well as cell type and promoter context.

Overexpression of stem cell associated ALDH1A1, a target of the leukemogenic transcription factor TLX1/HOX11, inhibits lymphopoiesis and promotes myelopoiesis in murine hematopoietic progenitors.

TLX1/HOX11 is an oncogenic transcription factor in human T-cell leukemia, however, the molecular basis for its transforming activity has remained elusive. The ALDH1A1 gene, whose product participates in retinoic acid synthesis, was previously identified as a TLX1-responsive gene. Here, we confirm regulation of ALDH1A1 transcription by TLX1 and show that ALDH1A1 can profoundly perturb murine hematopoiesis by promoting myeloid differentiation at the expense of lymphopoiesis. Together, these data demonstrate that ALDH1A1 plays a key role in normal hematopoiesis, and confirm ALDH1A1 as a TLX1 transcriptional target that may contribute to the ability of this homeoprotein to alter cell fate and induce tumor growth.

Identification of genes associated with blood feeding in the cat flea, Ctenocephalides felis.

BACKGROUND: The cat flea (Ctenocephalides felis) is a blood-feeding ectoparasitic insect and particular nuisance pest of companion animals worldwide. Identification of genes that are differentially expressed in response to feeding is important for understanding flea biology and discovering targets for their control. METHODS: C. felis fleas were maintained and fed for 24 h using an artificial rearing system. The technique of suppression subtractive hybridization was employed to screen for mRNAs specifically expressed in fed fleas. RESULTS: We characterized nine distinct full-length flea transcripts that exhibited modulated or de novo expression during feeding. Among the predicted protein sequences were two serine proteases, a serine protease inhibitor, two mucin-like molecules, a DNA topoisomerase, an enzyme associated with GPI-mediated cell membrane attachment of proteins and a component of the insect innate immune response. CONCLUSIONS: Our results provide a molecular insight into the physiology of flea feeding. The protein products of the genes identified may play important roles during flea feeding in terms of blood meal digestion, cellular growth/repair and protection from feeding-associated stresses.

Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia.

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.

Expression and purification of the human homeodomain oncoprotein HOX11.

HOX11 is a transcription factor belonging to the homeodomain family that is essential for spleen development during embryogenesis. It is also tumorigenic, being associated with T-cell acute lymphoblastic leukemia in children. In order to understand the functional role of HOX11 in both normal development and malignancy, protein-DNA and protein-protein interaction studies involving this factor are required. Such investigations would be facilitated by the availability of significant amounts of purified HOX11 protein. However, expression of full-length HOX11 in bacteria has been reported to be problematic owing to fusion protein instability. Here, we report the purification of human HOX11 expressed in Escherichia coli as a soluble and functional glutathione S-transferase (GST) fusion protein. In addition, a mutant version of HOX11 was produced (HOX11 Delta H3) which lacked the DNA-recognition helix (helix 3) of the homeodomain. Through a single purification procedure using glutathione-Sepharose, 2mg of the recombinant proteins were obtained per liter of bacterial culture. Notably, recombinant GST-HOX11 fusion proteins had a markedly higher stability when purified at low temperature (4 degrees C). Purification to near-homogeneity was achieved as judged by SDS-PAGE and the purified proteins were recognized by anti-HOX11 antibodies. The biological activity of the recombinant protein was verified by the specific binding of GST-HOX11, but not GST-HOX11 Delta H3, to DNA containing consensus HOX11 recognition sites.