Inactivation of KLF4 promotes T-cell acute lymphoblastic leukemia and activates the MAP2K7 pathway.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a high incidence of relapse in pediatric ALL. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate the onset of leukemia and worsen disease progression are largely unknown. Here, we show that the gene encoding the transcription factor KLF4 is inactivated by DNA methylation in children with T-ALL. In mice, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL by enhancing the G1-to-S transition in leukemic cells and promoting the expansion of leukemia-initiating cells. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7. Our results showed that in murine and pediatric T-ALL, loss of KLF4 leads to aberrant activation of MAP2K7 and of the downstream effectors JNK and ATF2. As a proof-of-concept for the development of a targeted therapy, administration of JNK inhibitors reduced the expansion of leukemia cells in cell-based and patient-derived xenograft models. Collectively, these data uncover a novel function for KLF4 in regulating the MAP2K7 pathway in T-ALL cells, which can be targeted to eradicate leukemia-initiating cells in T-ALL patients.

Tumor promoting properties of the ETS protein MEF in ovarian cancer.

We have previously shown that MEF (myeloid ELF1-like factor, also known as ELF4) functions as a transcriptional activator of the interleukin (IL)-8, perforin, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-3 genes in hematopoietic cells. MEF is also expressed in non-hematopoietic tissues including certain ovarian cancer cells. To define the function of MEF in these cells, we examined primary human ovarian epithelial tumors and found that MEF is expressed in a significant proportion of ovarian carcinomas, and in the CAOV3 and SKOV3 ovarian cancer cell lines, but not in normal ovarian surface epithelium. Manipulating MEF levels in these cell lines altered their behavior; reducing MEF levels, using short hairpin RNA expressing vectors, significantly inhibited the proliferation of SKOV3 and CAOV3 cells in culture, and impaired the anchorage-independent growth of CAOV3 cells. Overexpression of MEF in SKOV3 cells (via retroviral transduction) significantly increased their growth rate, enhanced colony formation in soft agar and promoted tumor formation in nude mice. The oncogenic activity of MEF was further shown by the ability of MEF to transform NIH3T3 cells, and induce their tumor formation in nude mice. MEF is an important regulator of the tumorigenic properties of ovarian cancer cells and could be used a therapeutic target in ovarian cancer.

Dysregulated expression of pre-Talpha reveals the opposite effects of pre-TCR at successive stages of T cell development.

The pre-TCR complex (TCRbeta-pre-TCRalpha chain (pTalpha)), first expressed in a fraction of CD8(-)4(-)CD44(-)25(+) (DN3) cells, is believed to facilitate or enable an efficient transition from the CD8(-)4(-) double-negative (DN) to the CD8(+)4(+) double-positive (DP) developmental stage. Subsequent to pre-TCR expression, DN3 thymocytes receive survival, proliferation, and differentiation signals, although it is still unclear which of these outcomes are directly induced by the pre-TCR. To address this issue, we generated mice bearing a range of pTalpha transgene copy number under the transcriptional control of the p56(lck) proximal promoter. All lines exhibited increased DN3 cycling, accelerated DN3/4 transition, and improved DN4 survival. However, the high copy number lines also showed a selective reduction in thymic cellularity due to increased apoptosis of DP thymocytes, which could be reversed by the ectopic expression of Bcl-2. Our results suggest that transgenic pTalpha likely caused apoptosis of DP thymocytes due to competitive decrease in surface TCRalphabeta formation. These results highlight the critical importance of precise temporal and stoichiometric regulation of pre-TCR and TCR component expression.

Premature TCR alpha beta expression and signaling in early thymocytes impair thymocyte expansion and partially block their development.

In thymocyte ontogeny, Tcr-a genes rearrange after Tcr-b genes. TCR alpha beta transgenic (Tg) mice have no such delay, consequently expressing rearranged TCR alpha beta proteins early in the ontogeny. Such mice exhibit reduced thymic cellularity and accumulate mature, nonprecursor TCR(+)CD8(-)4(-) thymocytes, believed to be caused by premature Tg TCR alpha beta expression via unknown mechanism(s). Here, we show that premature expression of TCR alpha beta on early thymocytes curtails thymocyte expansion and impairs the CD8(-)4(-) --> CD8(+)4(+) transition. This effect is accomplished by two distinct mechanisms. First, the early formation of TCR alpha beta appears to impair the formation and function of pre-TCR, consistent with recently published results. Second, the premature TCR alpha beta contact with intrathymic MHC molecules further pronounces the block in proliferation and differentiation. These results suggest that the benefit of asynchronous Tcr-a and Tcr-b rearrangement is not only to minimize waste during thymopoiesis, but also to simultaneously allow proper expression/function of the pre-TCR and to shield CD8(-)4(-) thymocytes from TCR alpha beta signals that impair thymocyte proliferation and CD8(-)4(-) --> CD8(+)4(+) transition.

Failure of rearranged TCR transgenes to prevent age-associated thymic involution.

After puberty, the thymus undergoes a dramatic loss in volume, in weight and in the number of thymocytes, a phenomenon termed age-associated thymic involution. Recently, it was reported that age-associated thymic involution did not occur in mice expressing a rearranged transgenic (Tg) TCRalphabeta receptor. This finding implied that an age-associated defect in TCR rearrangement was the major, if not the only, cause for thymic involution. Here, we examined thymic involution in three other widely used MHC class I-restricted TCRalphabeta Tg mouse strains and compared it with that in non-Tg mice. In all three TCRalphabeta Tg strains, as in control mice, thymocyte numbers were reduced by approximately 90% between 2 and 24 mo of age. The presence or absence of the selecting MHC molecules did not alter this age-associated cell loss. Our results indicate that the expression of a rearranged TCR alone cannot, by itself, prevent thymic involution. Consequently, other presently unknown factors must also contribute to this phenomenon.

Involvement of histidine 134 in the binding of alpha-bungarotoxin to the nicotinic acetylcholine receptor.

Peptides corresponding to the sequence alpha 124-147 of the Torpedo californica and Homo sapiens nicotinic cholinergic receptors were synthesized. The His residue at position 134 was ethoxyformylated or substituted by Ala. Effects of such modifications were studied by: (a) a toxin blot assay and (b) a competition assay between each peptide and the Discopyge Ischudii receptor for 125I alpha-bungarotoxin, in solution. Apparent Kd values were 0.1 and 0.8 microM for Torpedo californica and Homo sapiens native peptides, respectively, and no binding was observed when the His residue was modified or substituted by Ala. ic50 values for the Torpedo californica and Homo sapiens fragments were 1.0 and 0.8 microM, respectively, and no significant displacement occurred when His 134 was ethoxyformylated or substituted by Ala. Hydroxylamine treatment restored 80-100% of their binding ability. Results strongly support the involvement of His 134 in the binding of alpha-bungarotoxin either to the Torpedo californica or the Homo sapiens receptor.

Localization of histidine residues relevant for the binding of alpha-bungarotoxin to the acetylcholine receptor alpha-subunit in V8-proteolytic fragments.

Histidine residues have been shown to be critical for alpha-BgTx binding to the acetylcholine receptor (Lacorazza et al., 1992; Bouzat et al., 1993; Lacorazza et al., 1995). Receptor subunits from Discopyge tschudii were modified with diethylpyrocarbonate (DEP). DEP treatment produces a concentration-dependent decrease of [125I] alpha-BgTx binding to the alpha-subunit. The neurotoxin binding capacity was fully restored by adding the nucleophile hydroxylamine. By proteolytic mapping of the alpha-subunit with V8-protease, we determined that the binding capacity to the fragment alpha V8-19 decreased 80% by DEP treatment. In addition, the [125I] alpha-BgTx binding to the same fragment decreased by 70% when the subunits were reduced and affinity-alkylated. We report the N-terminal sequence of both subunits and V8-fragments (alpha V8-10, alpha V8-13, and alpha V8-18), which constitute a first contribution to the knowledge of the primary structure of the Discopyge tschudii receptor. We propose that the fragment alpha V8-19 contains one or more of the histidine residues involved in the alpha-BgTx binding and probably includes the Cys alpha 192-193 disulfide bond. Only two histidine residues are present in the extracellular sequence of Torpedo californica for such fragments: His alpha 186 and alpha 204.

Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells.

In humans, beta-hexosaminidase alpha-subunit deficiency prevents the formation of a functional beta-hexosaminidase A heterodimer resulting in the severe neurodegenerative disorder, Tay-Sachs disease. To explore the feasibility of using ex vivo gene transfer in this lysosomal storage disease, we produced ecotropic retroviruses encoding the human beta-hexosaminidase alpha-subunit cDNA and transduced multipotent neural cell lines. Transduced progenitors stably expressed and secreted high levels of biologically active beta-hexosaminidase A in vitro and cross-corrected the metabolic defect in a human Tay-Sachs fibroblasts cell line in vitro. These genetically engineered CNS progenitors were transplanted into the brains of both normal fetal and newborn mice. Engrafted brains, analyzed at various ages after transplant, produced substantial amounts of human beta-hexosaminidase alpha-subunit transcript and protein, which was enzymatically active throughout the brain at a level reported to be therapeutic in Tay-Sachs disease. These results have implications for treating neurologic diseases characterized by inherited single gene mutations.

In situ assessment of beta-hexosaminidase activity.

We have adapted two methods to evaluate the beta-hexosaminidase (HEX) enzymatic activity in cultured cells, based on the use of (i) the fluorogenic substrate 4-methylumbelliferyl-6-sulfo-2-acetamido-2- deoxy-beta-D-glucopyranoside (MU-GlcNAc-6-SO4) and (ii) the naphthol AS-BI-N-acetyl-beta-D-glucosaminide and hexazotized pararosaniline. We demonstrate that both methods could be used for the HEX isoenzymes by comparing wild-type and mutant human fibroblast cell lines, deficient for either an alpha or beta subunit from Tay-Sachs and Sandhoff patients. This in situ cytochemical assessment of HEX activity offers a rapid evaluation to study the expression of this enzyme in a heterogeneous cell population such as in gene transfer experiments.

Correction of ornithine-delta-aminotransferase deficiency in a Chinese hamster ovary cell line mediated by retrovirus gene transfer.

Gyrate atrophy (GA) of the choroid and retina is an autosomal recessive chorioretinal degeneration, caused by deficiency of the mitochondrial matrix enzyme ornithine-delta-aminotransferase (OAT). This deficiency results in the accumulation of ornithine in the body fluids and leads to hyperornithinemia. Although the clinical phenotype is largely confined to the eye, OAT deficiency is a systemic disorder. With the final goal of applying gene therapy to this human genetic disease, we have established an in vitro model to test the correction of OAT enzymatic deficiency in mammalian cells, using OAT recombinant retroviruses. We report the construction of several Moloney murine leukemia virus (MoMLV)-based recombinant retrovirus vectors, in which the human OAT cDNA was placed under the transcriptional control of the mouse phosphoglycerate kinase (PGK) promoter or under the enhancer-promoter regulatory element derived from the MoMLV long terminal repeat (LTR). The retrovirus constructs were packaged in the PG13-GALV cell line and used to transduce C9, an OAT deficient cell line derived from Chinese hamster ovary cells (CHO-K1). We show that the recombinant retrovirus transfers the human OAT (hOAT) gene into C9. Expression of the hOAT gene in the transduced C9 deficient cell line exceeded the OAT mRNA level and enzymatic activity of endogenous human fibroblasts.

Retrovirus-mediated gene transfer and expression of human ornithine delta-aminotransferase into embryonic fibroblasts.

Ornithine delta aminotransferase (OAT) is a nuclear-encoded mitochondrial matrix enzyme that catalyzes the reversible transamination of ornithine to glutamate semialdehyde. In humans, genetic deficiency of OAT results in gyrate atrophy of the choroid and retina, a blinding chorioretinal degeneration usually beginning in late childhood. This disorder has been shown to be autosomal recessive, and is often caused by missense, nonsense, and/or frameshift mutations in the OAT gene. With the view of applying gene therapy, a Moloney murine leukemia virus (MoMLV)-based recombinant retrovirus vector has been constructed. The human OAT cDNA was placed under the control of the enhancer-promoter regulatory elements derived from the MoMLV long terminal repeat (LTR). The construct was transfected into the retroviral packaging cell lines GP + E - 86 and psi CRIP to produce virus particles. Supernatant from these OAT retrovirus producer cell lines were used to transduce mouse C57B1/6 embryonal fibroblasts. We showed that the recombinant retrovirus transfers the OAT gene to the recipient cells, which produce an OAT RNA transcript when analyzed by Northern blot. Western blot analysis and enzymatic assays confirmed the presence of an OAT polypeptide that has a high enzymatic activity in the transduced cell lines, even after a long period of time in vitro.

Effect of chemical modification of extracellular histidyl residues on the channel properties of the nicotinic acetylcholine receptor.

We have examined the effect of chemical modification with diethyl pyrocarbonate (DEP) on the properties of acetylcholine (ACh)-activated channels in the cloned muscle-cell line BC3H-1. After protein modification, patch-clamp recordings showed alterations in the kinetics of the nicotinic acetylcholine receptor (AChR) channel. The major effect was observed in the channel mean open time, which was reduced up to about 12-fold at 466 microM DEP. The specificity of the effect was first established through comparison with both untreated cells and cells treated with inactivated DEP. Consistent with an increase in the number of unprotonated histidine residues (pKa = 6.0), this effect increased concomitantly with the pH of the reaction medium, being faster at pH 8 than at pH 6. The changes were dependent on time and DEP concentration, with an apparent EC50 = 114 microM. Modified channels also showed an increase in the number of events per burst of openings together with a decrease in burst durations. The amplitude of the channel-closed time component of about 1 ms increased with respect to the longest-duration-closed component. The number of alpha-bungarotoxin sites was slightly reduced after the modification, without affecting ligand binding affinity. The results suggest that DEP affects extracellular histidine residues involved in the ion translocation function of the AChR, but not its toxin-recognition ability. DEP could, therefore, induce a dissociation between toxin and agonist binding, as is often observed in neuronal AChR.

Involvement of a disulfide bond in the binding of flunitrazepam to the benzodiazepine receptor from bovine cerebral cortex.

The effects of chemical modification of a disulfide bond(s) (-SS-) or sulfhydryl group(s) (-SH) on the [3H]-flunitrazepam ([3H]FNZ) binding to membrane-bound or immunoprecipitated benzodiazepine (BZD) receptors (BZD-R) from bovine cerebral cortex were examined. Reduction of -SS- with dithiothreitol (DTT) brought about a reversible, time- and dose-dependent inhibition of [3H]FNZ binding to the membrane-bound BZD-R. Alkylation of the membranes with the -SH-modifying reagent iodoacetamide (IAA) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) produced a slight inhibition of [3H]FNZ binding in a dose-dependent manner. Scatchard analysis of saturation curves of [3H]FNZ binding in the presence and absence of 5 mM DTT revealed changes in affinity without modification in the maximal binding capacity, thus indicating a competitive mode of interaction. DTT pretreatment of both the membrane-bound and the immunoprecipitated BZD-R led to [3H]FNZ binding inhibition. Consistent with the modification of a binding site is the observation that reduction of -SS- does not bear on the binding affinity, but rather reduces the number of sites. Complete protection from DTT inhibition of [3H]FNZ binding by FNZ (an agonist) or by Ro 15-1788 (an antagonist) suggests the presence of -SS- at, or very close to, the BZD recognition binding site. No protection against IAA or DTNB inhibition was provided by FNZ. Photoaffinity labeling experiments with [3H]FNZ revealed a clear-cut band of 50 kDa in native and alkylated membranes but an extremely weak label in 5 mM DTT/IAA-treated membranes. The present results provide evidence for the participation of a disulfide bond in the recognition binding site of the bovine cerebral cortex BZD-R.

Role of histidine residues in the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.

This paper studies the effect of histidine chemical modification of the membrane-bound acetylcholine receptor from Discopyge tschudii on its specific alpha-bungarotoxin binding. The acylating reagent ethoxyformic anhydride (diethyl pyrocarbonate, DEP), was used. DEP-treatment induces a loss of binding capacity, time and DEP-concentration dependent. After a 30 min period of derivatization with 2 mM final DEP-concentration, at pH 7.4, the decrease reaches 70%; the loss of binding capacity is faster at pH 7.4 than at pH 6.0, as expected, since the amount of unprotonated species is higher under the first condition. Moreover, when ethoxyformylation is carried out at different pH values, the most important neurotoxin binding decrease occurs between pH 6.0 and 8.0. Furthermore, ethoxyformylation reversion restores such capacity. Consistent with the modification of a binding site, the ethoxyformylation does not bear on the affinity but reduces the number of receptors. Ethoxyformylation in the presence of carbamylcholine shows some ligand protective effect. These results, as a whole, strongly indicate a relevant role for histidine residues at the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.