A critical role for CRM1 in regulating HOXA gene transcription in CALM-AF10 leukemias.

The leukemogenic CALM-AF10 fusion protein is found in patients with immature acute myeloid and T-lymphoid malignancies. CALM-AF10 leukemias display abnormal H3K79 methylation and increased HOXA cluster gene transcription. Elevated expression of HOXA genes is critical for leukemia maintenance and progression; however, the precise mechanism by which CALM-AF10 alters HOXA gene expression is unclear. We previously determined that CALM contains a CRM1-dependent nuclear export signal (NES), which is both necessary and sufficient for CALM-AF10-mediated leukemogenesis. Here, we find that interaction of CALM-AF10 with the nuclear export receptor CRM1 is necessary for activating HOXA gene expression. We show that CRM1 localizes to HOXA loci where it recruits CALM-AF10, leading to transcriptional and epigenetic activation of HOXA genes. Genetic and pharmacological inhibition of the CALM-CRM1 interaction prevents CALM-AF10 enrichment at HOXA chromatin, resulting in immediate loss of transcription. These results provide a comprehensive mechanism by which the CALM-AF10 translocation activates the critical HOXA cluster genes. Furthermore, this report identifies a novel function of CRM1: the ability to bind chromatin and recruit the NES-containing CALM-AF10 transcription factor.

Inhibitors of histone deacetylases promote hematopoietic stem cell self-renewal.

BACKGROUND: Histone deacetylases (HDAC) are associated with a variety of transcriptional repressors that control cellular differentiation and proliferation. HDAC inhibitors such as trichostatin A, trapoxin and chlamydocin could be useful tools to modulate these cellular processes. We investigated their effect on the self-renewal of hematopoietic stem cells (HSC) during ex vivo culture. METHODS: Purified murine HSC with the phenotype c-Kit+,Thy-1.1(lo), Lin(-/lo), Sca-1+ were cultured for 4 days with IL-3, IL-6 and c-Kit ligand without or with HDAC inhibitors, after which their degree of phenotypic differentiation in culture was assessed by flow cytometric analysis. To explore whether HDAC inhibitors could have a beneficial role in human HSC transplantation, mobilized peripheral blood CD34+ cells were cultured with thrombopoietin mimetic peptide, flt3 ligand, and c-Kit ligand, without or with various HDAC inhibitors. The fluorescent dye, carboxyfluorescein-diacetate succinimidylester (CFSE), was used to track division of cell subsets, and engrafting ability was evaluated in a non-obese diabetic (NOD) -SCID xenotransplantation model. RESULTS: Murine HSC cultured with HDAC inhibitors maintained a more primitive phenotype than control cultures. The number of human HSC expressing Thy-1 increased up to seven-fold during a 5-day culture with HDAC inhibitors compared with control cultures. Chlamydocin was the most effective of the HDAC inhibitors tested at promoting Thy-1 expression on human cells. CFSE tracking showed that the increase in Thy-1+ cells resulted from cell division. In a NOD-SCID repopulation assay, cells exposed to chlamydocin for 24 h displayed an average four-fold higher engrafting ability over control cells. DISCUSSION: Our studies suggest that HDAC inhibitors can induce ex vivo expansion of human HSC, and may improve engraftment in hematopoietic transplant patients when cell dose is limiting.

The elongation domain of ELL is dispensable but its ELL-associated factor 1 interaction domain is essential for MLL-ELL-induced leukemogenesis.

The MLL-ELL chimeric gene is the product of the (11;19)(q23p13.1) translocation associated with de novo and therapy-related acute myeloid leukemias (AML). ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 (ELL associated factor 1) protein. EAF1 contains a limited region of homology with the transcriptional activation domains of three other genes fused to MLL in leukemias, AF4, LAF4, and AF5q31. Using an in vitro transformation assay of retrovirally transduced myeloid progenitors, we conducted a structure-function analysis of MLL-ELL. Whereas the elongation domain of ELL was dispensable, the EAF1 interaction domain of ELL was critical to the immortalizing properties of MLL-ELL in vitro. To confirm these results in vivo, we transplanted mice with bone marrow transduced with MLL fused to the minimal EAF1 interaction domain of ELL. These mice all developed AML, with a longer latency than mice transplanted with the wild-type MLL-ELL fusion. Based on these results, we generated a heterologous MLL-EAF1 fusion gene and analyzed its transforming potential. Strikingly, we found that MLL-EAF1 immortalized myeloid progenitors in the same manner as that of MLL-ELL. Furthermore, transplantation of bone marrow transduced with MLL-EAF1 induced AML with a shorter latency than mice transplanted with the MLL-ELL fusion. Taken together, these results indicate that the leukemic activity of MLL-ELL requires the EAF1 interaction domain of ELL, suggesting that the recruitment by MLL of a transactivation domain similar to that in EAF1 or the AF4/LAF4/AF5q31 family may be a critical common feature of multiple 11q23 translocations. In addition, these studies support a critical role for MLL partner genes and their protein-protein interactions in 11q23 leukemogenesis.

Retrovirus-mediated gene transfer of MLL-ELL transforms primary myeloid progenitors and causes acute myeloid leukemias in mice.

The MLL-ELL fusion gene results from the translocation t(11;19)(q23;p13.1) that is associated with de novo and therapy-related acute myeloid leukemia. To study its transforming properties, we retrovirally transduced primary murine hematopoietic progenitors and assessed their growth properties both in vitro and in vivo. MLL-ELL increased the proliferation of myeloid colony-forming cells in methylcellulose cultures upon serial replating, whereas overexpression of ELL alone had no effect. We reconstituted lethally irradiated congenic mice with bone marrow progenitors transduced with MLL-ELL or the control MIE vector encoding the enhanced green fluorescent protein. When the peripheral blood of the mice was analyzed 11-13 weeks postreconstitution, we found that the engraftment of the MLL-ELL-transduced cells was superior to that of the MIE controls. At this time point, the contribution of the donor cells was normally distributed among the myeloid and nonmyeloid compartments. Although all of the MIE animals (n = 10) remained healthy for more than a year, all of the MLL-ELL mice (n = 20) succumbed to monoclonal or pauciclonal acute myeloid leukemias within 100-200 days. The leukemic cells were readily transplantable to secondary recipients and could be established as immortalized cell lines in liquid cultures. These studies demonstrate the enhancing effect of MLL-ELL on the proliferative potential of myeloid progenitors as well as its causal role in the genesis of acute myeloid leukemias.

Chromatin-related properties of CBP fused to MLL generate a myelodysplastic-like syndrome that evolves into myeloid leukemia.

As a result of the recurring translocation t(11;16) (q23;p13.3), MLL (mixed-lineage leukemia) is fused in frame to CBP (CREB binding protein). This translocation has been documented almost exclusively in cases of acute leukemia or myelodysplasia secondary to therapy with drugs that target DNA topo isomerase II. The minimal chimeric protein that is produced fuses MLL to the bromodomain, histone acetyltransferase (HAT) domain, EIA-binding domain and steroid-receptor coactivator binding domains of CBP. We show that transplantation of bone marrow retrovirally transduced with MLL-CBP induces myeloid leukemias in mice that are preceded by a long preleukemic phase similar to the myelodysplastic syndrome (MDS) seen in many t(11;16) patients but unusual for other MLL translocations. Structure-function analysis demonstrated that fusion of both the bromodomain and HAT domain of CBP to the amino portion of MLL is required for full in vitro transformation and is sufficient to induce the leukemic phenotype in vivo. This suggests that the leukemic effect of MLL-CBP results from the fusion of the chromatin association and modifying activities of CBP with the DNA binding activities of MLL.

Overexpression of wild-type retinoic acid receptor alpha (RARalpha) recapitulates retinoic acid-sensitive transformation of primary myeloid progenitors by acute promyelocytic leukemia RARalpha-fusion genes.

Retinoic acid receptor alpha (RARalpha) is the target of several chromosomal translocations associated with acute promyelocytic leukemias (APLs). These rearrangements fuse RARalpha to different partner genes creating the chimeric proteins: PML-RARalpha, PLZF-RARalpha, and NPM-RARalpha. Although the vast majority of APLs respond to retinoic acid therapy, those associated with PLZF-RARalpha are resistant. We have used retroviruses to express PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, RARalpha403 (a dominant negative mutant of RARalpha), and wild-type RARalpha in murine bone marrow progenitors and found that all of these constructs blocked differentiation and led to the immortalization of myeloid progenitors. This cellular transformation is specific to an alteration of the RARalpha pathway because overexpression of RARbeta, RARgamma, or RXRalpha did not result in similar growth perturbations. Pharmacological doses of RA induced differentiation and inhibited proliferation of cells transformed with either of the APL fusion genes, including PLZF-RARalpha, whereas physiological retinoic acid concentrations were sufficient to reverse the phenotype of cells transformed with wild-type RARalpha. The cellular responses to retinoic acid were accompanied by a sharp decrease in the amount of the RARalpha-fusion proteins expressed in the cells. Our findings suggest that the oncogenicity of RARalpha-fusion proteins results from their nature to behave as unliganded RARalpha in the presence of physiological concentrations of retinoic acid.

Sustained gene expression in retrovirally transduced, engrafting human hematopoietic stem cells and their lympho-myeloid progeny.

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34(+) cells lacking lineage differentiation markers (CD34(+)Lin-) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34(+) cell population retained the Lin- phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34(+)Lin-NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34(+)Lin-NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% +/- 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.

The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX.

The HRX gene (also called MLL, ALL-1, and Htrx) at chromosome band 11q23 is associated with specific subsets of acute leukemias through translocations that result in its fusion with a variety of heterologous partners. Two of these partners, ENL and AF9, code for proteins that are highly similar to each other and as fusions with HRX induce myeloid leukemias in mice as demonstrated by retroviral gene transfer and knock-in experiments, respectively. In the present study, a structure-function analysis was performed to determine the molecular requirements for in vitro immortalization of murine myeloid cells by HRX-ENL. Deletions of either the AT hook motifs or the methyltransferase homology domain of HRX substantially impaired the transforming effects of HRX-ENL. The methyltransferase homology domain was shown to bind non-sequence specifically to DNA in vitro, providing evidence that the full transforming activity of HRX-ENL requires multiple DNA binding structures in HRX. The carboxy-terminal 84 amino acids of ENL, which encode two predicted helical structures highly conserved in AF9, were necessary and sufficient for transformation when they were fused to HRX. Similarly, mutations that deleted one or both of these conserved helices completely abrogated the transcriptional activation properties of ENL. This finding correlates, for the first time, a biological function of an HRX fusion partner with the transforming activity of the chimeric proteins. Our studies support a model in which HRX-ENL induces myeloid transformation by deregulating subordinate genes through a gain of function contributed by the transcriptional effector properties of ENL.

Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL.

A subset of chromosomal translocations in acute leukemias results in the fusion of the trithorax-related protein HRX with a variety of heterologous proteins. In particular, leukemias with the t(11;19)(q23;p13.3) translocation express HRX-ENL fusion proteins and display features which suggest the malignant transformation of myeloid and/or lymphoid progenitor(s). To characterize directly the potential transforming effects of HRX-ENL on primitive hematopoietic precursors, the fusion cDNA was transduced by retroviral gene transfer into cell populations enriched in hematopoietic stem cells. The infected cells had a dramatically enhanced potential to generate myeloid colonies with primitive morphology in vitro. Primary colonies could be replated for at least three generations in vitro and established primitive myelomonocytic cell lines upon transfer into suspension cultures supplemented with interleukin-3 and stem cell factor. Immortalized cells contained structurally intact HRX-ENL proviral DNA and expressed a low-level of HRX-ENL mRNA. In contrast, wild-type ENL or a deletion mutant of HRX-ENL lacking the ENL component did not demonstrate in vitro transforming capabilities. Immortalized cells or enriched primary hematopoietic stem cells transduced with HRX-ENL induced myeloid leukemias in syngeneic and SCID recipients. These studies demonstrate a direct role for HRX-ENL in the immortalization and leukemic transformation of a myeloid progenitor and support a gain-of-function mechanism for HRX-ENL-mediated leukemogenesis.

The acute promyelocytic leukemia PML-RAR alpha protein induces hepatic preneoplastic and neoplastic lesions in transgenic mice.

The PML-RAR alpha hybrid protein generated by the t(15;17) translocation in acute promyelocytic leukemia (APL) is thought to play a central role in the oncogenic process. However, analysis of the oncogenic activity of the fusion protein in tissue culture assays or in mice has been hampered by its apparent toxicity in multiple murine cells. To circumvent this problem, we generated an inducible line of transgenic mice, MT135, in which the expression of PML-RAR alpha is driven by the metallothionein promoter. After 5 days zinc stimulation, 27 out of 54 mice developed hepatic preneoplasia and neoplasia including foci of basophilic hepatocytes, dysplasia and carcinoma with a significantly higher incidence of lesions in females than in males. The rapid onset of liver pathologies was dependent on overexpression of the transgene since it was not detected in noninduced transgenic animals of the same age. The PML-RAR alpha protein was always present in altered tissues at much higher levels than in the surrounding normal liver tissues. In addition, overexpression of PML-RAR alpha resulted in a strong proliferative response in the hepatocytes. We conclude that overexpression of PML-RAR alpha deregulates cell proliferation and can induce tumorigenic changes in vivo.

A retrovirus carrying the promyelocyte-retinoic acid receptor PML-RARalpha fusion gene transforms haematopoietic progenitors in vitro and induces acute leukaemias.

The promyelocyte (PML)-retinoic acid receptor alpha (RARalpha) fusion gene results from a t(15;17) chromosome translocation in acute promyelocytic leukaemia. We have analysed the oncogenic potential of the human fusion PML-RARalpha product in chicken using retrovirus vectors. We show that PML-RARalpha transforms very early haematopoietic progenitor cells in vitro and induces acute leukaemias. Neither PML nor RARalpha domains alone achieve such a transformation. The PML-RARalpha viruses recovered from the transformed cells carry two point mutations in the PML domain, one of which alters both the pattern of intracellular localization of the fusion protein and its functional interference with AP-1, thus defining an essential domain in PML for oncogenic transformation.

Retroviral vectors for the transduction of the PML-RARalpha fusion product of acute promyelocytic leukemia.

We have designed several retroviral constructs to transduce the PML-RA Ralpha fusion product of human acute promyelocytic leukemia. Our aim to generate high-titer stable vector-producing cell lines was hindered by a toxic effect of PML-RARalpha expression on packaging cells. To circumvent this, we tested retroviral vectors expressing the transgene from several internal promoters including inducible and myelospecific promoters. To compare efficiency of these constructs in their ability to generate protein expression in the appropriate target cells and optimal viral titers, we used the BOSC23 transient packaging cell line. We found that the direct-oriented vector did not ensure tissue-specificity of PML-RARalpha expression, while the reverse-oriented retroviral vector did. The latter construct, however, failed to generate high-titer recombinant virus. This study exemplifies the unpredictable behavior of retroviral constructs and the superiority of transient systems for transduction of a toxic product.

The acute promyelocytic leukaemia-associated PML gene is induced by interferon.

The PML protein concentrates within discrete nuclear structures known as nuclear bodies, also called NDs or PODs, which contain several proteins including the interferon (IFN)-inducible SP100 product. The function of these structures remains elusive. We and others have shown recently that they represent specific targets for adenovirus and herpes simplex virus. This prompted us to investigate whether PML, like SP100, might be induced by IFN and to explore the role of PML in viral infection. Here we report that PML mRNA levels increase rapidly in response to interferon treatment. This accumulation of PML transcripts is a primary IFN response since it does not require de novo protein synthesis. The IFN-induced activation of the PML gene is accompanied by enhanced protein expression as revealed by immunolabelling. Both the intensity of the staining and the number of labelled structures increased upon interferon exposure. To probe the role of PML in IFN action, we compared the antiviral state established by alpha-interferon in embryonic fibroblasts (EFs) derived from null mutant mice for PML and from wild-type control mice. The resistance to viral infection conferred by IFN-alpha was identical in both PML+/+ and PMLm/m fibroblasts indicating that PML is not an essential mediator of the antiviral effect of interferon. We also noted that DNA-binding factors are normally activated by IFN in PMLm/m cells.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

The t(15;17) translocation in acute promyelocytic leukemia.

Retinoic acid (RA) is a vitamin A derivative with striking effects on development and cell differentiation. The identification of three RA receptors (RAR alpha, beta and gamma) as members of the nuclear receptor superfamily led to important insights into the molecular mechanism of action of retinoids. The nuclear receptors, that also include receptors for steroid hormone, vitamin D3 and thyroid hormone act as ligand-inducible transcription factors and are characterized by the presence of two well conserved DNA- and hormone-binding domains. One of the most intriguing properties of RA is its ability to induce in vivo differentiation of acute promyelocytic leukaemia (APL) cells into mature granulocytes, leading to morphological complete remissions. We and others have shown that the t(15;17) translocation specifically associated with APL fuses an as yet unidentified gene, named PML, to the retinoic acid receptor alpha locus. The resulting PML-RAR alpha hybrid protein that retains most of the functional domains of parental proteins exhibits altered transactivating functions when compared to the wild-type receptor; however the biological significance of this property in the transforming phenotype is still obscure. PML, whose function is unknown, belongs to a novel family of nuclear proteins characterized by the presence of a Cys/His-rich motif, named a RING finger, that include RNA-binding proteins, transcription factors and oncoproteins. A dimerization domain within PML is able to mediate the formation of PML-RAR alpha homodimers that can bind to target sequences with distinct DNA binding properties if compared with RAR alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

The t(15;17) translocation in acute promyelocytic leukemia.

Acute promyelocytic leukemia is characterized by a specific t(15;17) chromosomal translocation and a particular sensitivity to retinoic acid. The translocation fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene resulting in the production of a PML-RAR alpha fusion protein. The hybrid molecule retains most of the functional domains of both native products, PML and RAR alpha, but it has novel features. Its cellular distribution is completely reorganized when compared to that of PML: the hybrid is found in multiple small nuclear substructures and upon retinoic acid treatment, it goes back to the normal PML localization, that is in typical well organized nuclear bodies. PML-RAR alpha also acquires novel transcriptional properties if compared to RAR alpha, it does so either by direct binding to target gene regulatory sequences or by protein interaction with cofactors. Expression of PML-RAR alpha in HL60 or U937 cell has been shown to block their maturation while it can force their differentiation at high doses of retinoic acid. Different mechanisms are proposed to explain how PML-RAR alpha blocks differentiation and how this may be reversed by retinoic acid. A likely hypothesis might be the delocalization of critical cofactors into the aberrant PML-RAR alpha substructures while the therapeutic effect of retinoic acid would be correlated to its capacity to restore a normal nuclear organization.

Retinoic acid regulates aberrant nuclear localization of PML-RAR alpha in acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation that fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene product, PML. The involvement of RAR alpha is particularly intriguing in view of the efficient therapeutic effect of retinoic acid (RA) in this disease. In this report, we show that PML is specifically localized within a discrete subnuclear compartment corresponding to nuclear bodies recognized by patient autoimmune sera. In APL cells, the PML-RAR alpha hybrid displays an abnormal localization and directs RXR and other nuclear antigens into aberrant structures that are tightly bound to chromatin. This suggests that the hybrid could exert a dominant negative effect by diverting a subset of proteins from their natural sites of action. Interestingly, treatment of APL cells with RA induces a complete relocalization of each of these proteins. We propose that the beneficial role of RA in promoting myeloid differentiation in APL might be related to its ability to restore a normal subnuclear organization.

The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR.

We have previously shown that the t(15;17) translocation specifically associated with acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR alpha) locus to an as yet unknown gene, initially called myl and now renamed PML. We report here that this gene product contains a novel zinc finger motif common to several DNA-binding proteins. The PML-RAR alpha mRNA encodes a predicted 106 kd chimeric protein containing most of the PML sequences fused to a large part of RAR alpha, including its DNA- and hormone-binding domains. In transient expression assays, the hybrid protein exhibits altered transactivating properties if compared with the wild-type RAR alpha progenitor. Identical PML-RAR alpha fusion points are found in several patients. These observations suggest that in APL, the t(15;17) translocation generates an RAR mutant that could contribute to leukemogenesis through interference with promyelocytic differentiation.