The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells.

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/- stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Design and synthesis of potent and selective inhibitors of integrin VLA-4.

The synthesis and identification of a novel series of inhibitors of integrin VLA-4 are described. Their in vitro activity and selectivity against closely related integrins are also presented.

A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates.

We have developed a homogeneous high-capacity assay format for measuring integrin- and selectin-dependent cell binding to immobilized ligand using V-well microtiter plates. 2',7'-Bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescence, acetoxymethylester-labeled cells are added to ligand-coated V-shaped microtiter wells. Bound cells are separated from free cells using centrifugal force to produce shear stress. Nonadherent cells accumulate in the nadir of the well and are measured using a fluorescence plate reader. Antibody or low-molecular-weight inhibitors of either the ligand or the cell surface receptor result in less cell binding, more cells in the pellet, and increased signal. The optimization and validation of the very late antigen-4/vascular cell adhesion molecule-1 assay is described in detail. We demonstrate that this assay can be rapidly adapted to measure other integrin- and selectin-mediated interactions. This assay format has several advantages over conventional assays. The centrifugal process is biologically relevant and eliminates the washing steps to remove nonadherent cells that can cause well-to-well and plate-to-plate variation. Because the assay is robust with a high signal-to-noise ratio and low variability, it is ideally suited for studying multiple parameters of cell adhesion and for high capacity screening.

A high-capacity quantitative mouse model of drug-mediated immunosuppression based on rejection of an allogeneic subcutaneous tumor.

We describe a high-capacity in vivo assay to measure drug-mediated transplant immunosuppression using a mouse model of Sa1 tumor rejection. Sa1 grew poorly and was rejected by 14 days in immunocompetent allogeneic recipient mice. In nude (nu/nu) mice, Sa1 grew more rapidly and was not rejected, confirming the T cell dependence of this response. In immunocompetent animals, administration of immunosuppressive agents resulted in increased tumor growth relative to vehicle-treated animals. Treatment with immunosuppressive drugs such as cyclosporin A (CsA), 40-O-(2-hydroxyethyl)-rapamycin (SDZ RAD), or 2-amino-2-[2-(4-octylphenyl)ethyl]-1,2-propanediol hydrochloride (FTY720) produced dose-dependent inhibition of tumor rejection. By contrast, the drugs did not affect Sa1 tumor growth in nu/nu mice, which is consistent with their predicted indirect effect on tumor size by suppressing immunity, rather than by directly stimulating Sa1 growth. Drug potency, which is usually not described for immunosuppressive agents, was calculated from the linear relationship between drug dose and tumor volume. The potency of CsA was inversely related to the stringency of the histocompatibility barrier. Another advantage of this assay is that the endpoint is an objective size measurement over a short time period, compared with transplant models where the endpoint may not be reached for many weeks and may be more subjective. In addition, this model can measure the potency of combination drug treatments and compare new immunosuppressive drug regimens. For example, the administration of SDZ RAD or FTY720 with CsA resulted in a more than additive increase in potency, compared with the sum of the drugs as single agents.

Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion.

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.

Combinatorial approaches to inhibitors of VLA-4: piperazine-peptoid-bisarylureas.

A combinatorial approach towards identifying inhibitors of VALA-4 was investigated. A library of piperazine-peptoid-bisarylureas was assembled in solid phase and screening in the novel v-well assay enabled the identification of active compounds.

Absorption and disposition of a tripeptoid and a tetrapeptide in the rat.

The present study is concerned with the absorption and disposition of a tripeptoid (N-substituted glycine derivative) and a tetrapeptide in the rat. The two compounds have similar backbone structures but differ with respect to the presence or absence of peptide bond. [3H]tripeptoid and [3H]tetrapeptide were administered orally (30 mg kg(-1)) and intravenously (i.v.) (30 or 3 mg kg(-1)) to Sprague Dawley rats. Blood, urine and feces were collected at designated times for radioactivity and parent drug analysis. The intestinal absorptive clearances of the tripeptoid and tetrapeptide were studied using an in situ rat intestinal perfusion model. The octanol/water partition coefficient of these two compounds was also determined. The results showed that the peptoid and peptide have similar absorptive clearance and octanol/water partitioning, but different in vivo absorption and disposition characteristics. The absorptive clearances of the tripeptoid and tetrapeptide were 6.7 and 4.8 x 10(-4) mL min(-1) cm(-1), respectively, and the corresponding octanol/water partition coefficients were 0.39 and 0.30. The extent of oral absorption of the tripeptoid was only 3-8%, consistent with its low absorptive clearance. In contrast, the apparent absorption of the tetrapeptide was > 75% of the radioactive dose. The peptide was completely metabolized within 2 h after an i.v. dose, whereas the peptoid was stable in blood and was primarily eliminated in feces as intact drug. In conclusion, the difference in in vivo absorption and disposition between the peptoid and peptide was apparently due to the presence or absence of a peptide bond. The tetrapeptide was subject to rapid metabolism in the body. Its relatively high absorption appeared to represent the absorption of metabolized radioactive fragments. The peptoid appears to have advantages over the peptide in term of metabolic stability, but its low oral absorption and rapid biliary excretion present additional challenges in the selection of an optimal drug candidate.

Heterologous desensitization of the high affinity receptor for IgE (Fc epsilon R1) on RBL cells.

To study the properties of Fc epsilon RI desensitization induced by aggregation of that receptor, RBL cells were sensitized with a mixture of two different IgE mAb to create two different populations of IgE-receptor complexes. Cross-linking of one receptor population containing anti-dinitrophenyl (DNP) IgE with a bivalent Ag, 1-DNP-amino-12-biotinamidododecane)2-avidin ((DNP)2-avidin), results in desensitization of a subsequent response, both of the same receptor population (homologous desensitization) and of the second receptor population containing anti-5-dimethylaminonaphthalene-1-sulfonyl (dansyl) IgE (heterologous desensitization). The extent of heterologous desensitization is dependent on several parameters, including the concentration of both the first and the second Ag and the densities of the respective IgE populations. Heterologous desensitization of the Ca2+ response is more sensitive to the concentration of the second stimulus (dansyl-BSA) than heterologous desensitization of the degranulation response. AlF4-, which activates GTP-binding proteins, can effectively replace (DNP)2-avidin as the initial stimulant and desensitizing agent. Other agents that mobilize intracellular Ca2+ including thrombin and a Ca2+ ionophore are less effective at replacing (DNP)2-avidin. Because prestimulation with Ag does not desensitize subsequent responses to AlF4- or Ca2+ ionophore, it appears that signal transduction via Fc epsilon RI is impaired at an early step. Addition of monovalent DNP hapten within approximately 10 min after cross-linking by (DNP)2-avidin completely prevents the desensitization of the subsequent Ca2+ or degranulation response to dansyl-BSA. After longer times of incubation with DNP Ag, the DNP hapten becomes increasingly less effective at preventing the desensitization of the dansyl-BSA response, even though ongoing signal transduction by the DNP Ag is halted. These results suggest a form of cellular memory for the desensitized state.

Recombinant human IgG1-murine IgE chimeric Ig. Construction, expression, and binding to human Fc gamma receptors.

We have constructed a set of chimeric Ig by exchanging corresponding H chain C domains between human (hu) IgG1 and murine (m) IgE. We used this set of Ig to dissect the interaction of individual Ig domains with human Fc gamma receptors. Only one of the chimeras, epsilon/C gamma 2,3 (an mIgE with C epsilon 3 and C epsilon 4 replaced by C gamma 2 and C gamma 3 from huIgG1), binds tightly to the human Fc gamma RI on U937 cells. We found that epsilon/C gamma 2,3 has only twofold lower affinity for Fc gamma RI as compared to huIgG1. The gamma/C epsilon 4 (huIgG1 with C epsilon 4 replacing C gamma 3) binds weakly to Fc gamma RI. The other chimeric Ig, epsilon/C gamma 3, epsilon/C gamma 2, and gamma/C epsilon 3, as well as mIgE do not bind detectably to Fc gamma RI. From these data we conclude that the C gamma 2 domain is crucial for binding and contains the majority of the binding site for Fc gamma RI on IgG1. The C gamma 3 domain makes a smaller contribution to the binding, and the C gamma 1 domain and the hinge region have very little effect on the Fc gamma RI-IgG1 interaction. The chimeric epsilon/C gamma 2,3 and huIgG1 both mediate the formation of rosettes between K562 cells and antigen-sensitized E with similar concentration dependences. These results suggest similar ability to bind to Fc gamma RII. The other chimeric Ig do not cause rosettes in this assay system. Hence, both C gamma 2 and C gamma 3 seem to be required for binding to Fc gamma RII, but the C gamma 1-hinge region has no detectable effect.

Mapping the site of interaction between murine IgE and its high affinity receptor with chimeric Ig.

We have investigated the interaction of mouse (m) IgE with its Fc epsilon RI on rat basophilic leukemia cells using a set of chimeric Ig that were constructed by exchanging homologous H chain C domains between human (hu) IgG1 and mIgE. Binding affinities were examined with equilibrium and kinetic measurements, and we found that epsilon/C gamma 3 (mIgE with C epsilon 4 replaced by C gamma 3) was indistinguishable from mIgE. The huIgG1 and the other chimeric Ig, which did not contain both C epsilon 2 and C epsilon 3, did not bind detectably to rat basophilic leukemia cells (Ka less than 10(6) M-1). The ability of these chimeric Ig to stimulate a cellular response (degranulation) in the presence of multivalent Ag was also tested. The epsilon/C gamma 3 was indistinguishable from mIgE in eliciting a high level of degranulation, whereas the other chimeric Ig stimulated no response even when they were preaggregated to enhance their binding avidity. These results demonstrate that C epsilon 4 may be replaced by C gamma 3 without affecting the binding and cell activating properties of mIgE. The lack of binding by the other chimeric Ig indicates that both C epsilon 2 and C epsilon 3 are necessary for the binding interaction.