Pediatric Oncology Group Study of in vitro clonal growth patterns of leukemic cells in childhood acute nonlymphocytic leukemia as a predictor of induction response.

Previous studies have shown that clonal growth patterns of leukemic cells from adult patients with acute nonlymphocytic leukemia (ANLL) have prognostic significance for achieving complete remission (CR). In order to determine if a similar correlation between clonal growth patterns and response to chemotherapy exists in childhood ANLL, bone marrow cells from 189 children with newly diagnosed ANLL were cultured in agar. After 7 days of incubation, colonies (greater than 50 cells), large clusters (20 to 50 cells), and small clusters (4 to 20 cells) were counted. Cultures were analyzed for frequency of clusters and colonies as well as for size of clusters. Two growth patterns significantly associated with poor prognosis for achieving CR were large-cluster growth and high cluster incidence (defined as greater than 400 clusters/10(5) bone marrow cells). The CR rate for the former was 53% (versus 79% for non-Group 1 patients; P = 0.03); the CR rate for the latter was 46% (versus 81% for non-Group 2 patients; P = 0.004). These findings indicate that clonal growth characteristics of leukemic cells from childhood ANLL patients are significantly correlated with response to induction chemotherapy and are useful in identifying a subset of patients with poor prognosis.

The clinical significance of Fas expression in leukemia: questions and controversies.

Binding of agonistic anti-Fas (APO-1/CD95) antibodies or Fas ligand (Fas-L) induces apoptosis in some Fas+ leukemias, and anti-Fas antibody was originally investigated as a possible therapeutic reagent. More recently, a number of studies have examined a potential role for the Fas/Fas-L pathway in chemotherapy-induced apoptosis as well as the effect of Bcl-2 on this pathway. These studies are briefly reviewed here.

Characterization of a myeloperoxidase mRNA(+) acute lymphoblastic leukemia cell line (EU-1/ALL) established from a child with an apparent case of ALL.

A myeloid-antigen-positive acute lymphoblastic leukemia (My+ALL) cell line (EU-1) was established from the bone marrow cells of a child with apparent ALL. By morphology, cytochemistry and fluorescent-antibody phenotyping, EU-1 cells appeared to be lymphoblastic (L1 morphology, TdT+, CD10+, CD19+). However, by a sensitive immunocytochemical assay, EU-1 cells additionally displayed several myeloid antigens (CD13, CD14, CD33) not detected by flow-cytometry. Furthermore, EU-1 cells were cytochemically and immunocytochemically negative for myeloperoxidase (MPO) but positive for MPO mRNA by Northern blot analysis. After incubation with dimethylsulfoxide (DMSO), myeloid cell surface antigens were detected on EU-1 cells by flow cytometry, and a marked decrease in MPO mRNA expression was observed. These results demonstrate that EU-1 is a unique ALL cell line representing a significant subset of pediatric ALL patients who also express myeloid antigens and have poor prognosis.

Sensitivity to Fas-mediated apoptosis in pediatric acute lymphoblastic leukemia is associated with a mutant p53 phenotype and absence of Bcl-2 expression.

Fas (APO-1/CD95) is a cell-surface protein that can mediate apoptosis upon specific ligand or antibody binding. The Bcl-2 protein may function as a modulator of Fas-induced apoptosis by blocking a downstream activation step, and Bcl-2 expression in acute lymphoblastic leukemia (ALL) cells appears to depend partly on expression of a wild-type (wt) p53 tumor suppressor gene (Findley et al, Blood 1997; 89: 2986). We therefore investigated the relationship between sensitivity to Fas-mediated apoptosis and (1) Fas expression, (2) p53 status, and (3) Bcl-2 protein levels in pediatric ALL cell lines and primary leukemic cells. Cell lines included 21 B cell precursor (BCP)-ALL and four T-ALL lines; in five cases, cryopreserved primary leukemic cells from which these lines were established were also examined. Additionally, we evaluated the effect of anti-Fas monoclonal antibody on the activation of protease CPP32 and induction of apoptosis in these lines. By SSCP analysis and DNA sequencing, we detected p53 mutations (mt) in eight out of 25 ALL cell lines (exon-7, codon 248 n=6; exon-8, codon 273, n=2). The expression of Fas and Bcl-2 was examined by immunofluorescence staining and quantified as the number of molecules of equivalent soluble fluorochrome (MESF). Elevated levels of Fas were expressed in all six lines with a mutation of p53 in codon 248 (1500 to 10800 MESF). Although Fas was detectable in seven of the 17 lines with wt-p53, expression was lower (150-900 MESF) compared with mt-p53+ lines. Bcl-2 was expressed in 10 of the 25 lines. Most (9/10) wt-p53+ lines expressed Bcl-2, whereas only one of eight mt-p53+ lines and no p53-null lines expressed this protein. Treatment of Fas-positive lines with anti-Fas monoclonal antibody (200 ng/ml) for 6 h induced activation of CPP32 and apoptosis in eight of 13 Fas+ lines. Sensitivity to Fas-mediated apoptosis was associated with a mt-p53 phenotype and absence of Bcl-2 expression. Six of eight Fas+/Fas-sensitive (S) lines were mt-53+/Bcl-2-, whereas only two Fas+/Fas-S lines were wt-p53+/Bcl-2+; both of these latter lines expressed low levels of Bcl-2 compared to Fas-resistant lines. In contrast, four of five Fas+/Fas-resistant (R) lines were wt-p53+/Bcl-2+; the exception was p53-null/Bcl-2- but expressed a low level of Fas (150 MESF). Activation of the cysteine protease CPP32 and cleavage of its substrate poly(ADP-ribose)polymerase (PARP) was also detected in Fas-S but not Fas-R lines. We obtained similar results from both the primary leukemic cells and the corresponding cell lines in five cases: overexpression of Fas and Fas-sensitivity were present in mt-p53+/Bcl-2- but not wt-p53+/Bcl-2+ cells. These results suggest that some pediatric ALL cells expressing mt-p53+ may be sensitive to Fas-mediated apoptosis due to high levels of Fas expression and lack of Bcl-2, and further suggest that molecular methods of activating Fas may be useful for therapy of refractory ALL with the Fas+/mt-p53+ phenotype.

Expression of myeloperoxidase mRNA by leukemic cells from childhood acute lymphoblastic leukemia.

The frequency of acute lymphoblastic leukemia (ALL) expressing the myeloperoxidase (MPO) gene and other myeloid-associated characteristics was determined in 26 (20 B-lineage, six T-lineage) children at diagnosis. All cases were diagnosed as ALL by standard morphological and cytochemical criteria. In the 26 cases, leukemic blast cells from four B-lineage and two T-lineage ALL patients were simultaneously expressing myeloid-associated antigens. By Northern blot analysis, MPO mRNA was detected in leukemic cells from five out of six cases expressing both lymphoid and myeloid antigens, and from four out of 16 B-lineage and one out of four T-lineage ALL without myeloid antigens. There was no detectable MPO protein or enzymatic activity in the leukemic cells of these cases as examined by immunocytochemistry and cytochemistry. MPO mRNA expression in ALL cells was significantly associated with age < 1 year: leukemic blast cells from all five infant ALL patients expressed MPO mRNA, compared to five out of 21 ALL patients > 1 year of age whose leukemic cells were MPO mRNA(+) (p < 0.01). These results suggest that MPO gene transcription in the absence of translation may characterize a recently described subset of pediatric ALL patients who also express myeloid markers, and may serve as a useful marker for this entity.

The effect of recombinant GM-CSF and G-CSF on the bone marrow cells of children with acute lymphoblastic leukemia.

Colony stimulating factors (CSFs) are glycoprotein hormones that regulate growth and differentiation of hematopoietic progenitor cells. Their use to stimulate granulocyte precursors during periods of neutropenia in patients with acute myeloid leukemia (AML) is limited by their concomitant stimulation of the proliferation of myeloblasts. The effects of these agents on leukemic lymphoblasts is not entirely known. We have investigated the in vitro effects of granulocyte-CSF (G-CSF) and granulocyte/macrophage-CSF (GM-CSF) on leukemic cells from children with acute lymphoblastic leukemia (ALL). DNA synthesis of bone marrow cells from 22 children with ALL, either at diagnosis or in relapse, was examined with and without CSFs. Proliferative potential was also tested in a clonogenic assay with 13 bone marrow specimens. These factors did not stimulate the growth of ALL cells in either assay. Our results indicate that G-CSF and GM-CSF should be able to stimulate granulocyte proliferation without enhancing leukemic proliferation during periods of neutropenia in children with ALL.

CD40 ligand upregulates expression of the IL-3 receptor and stimulates proliferation of B-lineage acute lymphoblastic leukemia cells in the presence of IL-3.

The proliferative response of B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells to IL-3 is dependent on the expression of functional IL-3 receptors (IL-3R). Here we report that CD40 ligand (CD40L) in the presence of recombinant IL-3 increased proliferation of BCP-ALL cells by upregulating expression of IL-3R. Upregulation of IL-3R in BCP-ALL cells was observed as early as 1 h after treatment with CD40L, and a 50- to 500-fold increase of IL-3R expression after 24 h was detected in all 12 cases studied. Moreover, expression of receptors for IL-7 (IL-7R) and stem cell factor (SCF-R, c-Kit) was also induced by CD40L in the majority of BCP-ALL cases examined; however, levels of induction were low compared to those for IL-3R. To test the functional activity of upregulated receptors for IL-3, SCF and IL-7, we evaluated the proliferation and growth of BCP-ALL cells cultured in serum-free media with CD40L plus these factors. When CD40L was added with either a single cytokine (IL-3, SCF and IL-7) or their combinations, cell proliferation was significantly increased as detected by DNA synthesis assay. Combinations of CD40L plus IL-3 and either SCF or IL-7 were able to support long-term growth of BCP-ALL cells for at least 8 weeks in three of the seven cases studied. Immunophenotyping and gene rearrangement studies indicated that cells in long-term cultures were monoclonal and retained their original phenotypes. The leukemic cells remained primarily dependent on the presence of IL-3 and its receptor for long-term growth, as shown by selective withdrawal of growth factors or antibody blockade of receptors. These results suggest an important role for CD40L in upregulating expression of IL-3R on BCP-ALL cells and enabling these cells to proliferate in long-term cultures in the presence of IL-3 and either SCF or IL-7.

Pediatric acute myelogenous leukemia cells express IL-6 receptors and are sensitive to a recombinant IL6-Pseudomonas exotoxin.

We have studied IL-6 receptor (IL-6R) expression on AML cells from 15 pediatric patients by immunocytochemistry/flow cytometry, reverse-transcription polymerase chain reaction, and Scatchard analysis. High-affinity IL-6R were detected on leukemic cells from 12 (80%) patients. Binding sites per cell ranged from 140 to 3580 (median 920; mean 1240), with dissociation constants of 0.26 to 0.71 nM. We therefore assessed the in vitro sensitivity of IL-6R+ AML cells to treatment with a recombinant IL6-Pseudomonas exotoxin fusion protein (IL6-PE4E), using the XTT cytotoxicity assay. Leukemic cells from eight patients had ID50 values (concentration of IL6-PE4E producing a 50% decrease in cell viability) of <1000 ng/ml (median, 87 ng/ml; mean, 262 ng/ml). Sensitivity to IL6-PE4E correlated significantly with receptor number. Normal bone marrow mononuclear cells had undetectable IL6-R expression (<20 receptors/cell) and were relatively resistant to IL6-PE4E. To test the efficacy of IL6-PE4E for ex vivo purging in an autologous stem cell transplantation setting, we incubated primary IL-6R+ AML cells with 10(3) ng/ml IL6-PE4E for 24 h, followed by inoculation into SCID mice. Mice receiving treated cells showed no leukemic engraftment, while all mice receiving untreated or control-treated cells developed leukemia with a median presymptomatic interval of 55 days. In recipients of IL6-PE4E treated cells, no evidence of occult leukemia was detected by PCR analysis of blood and bone marrow cells at 185 days postinoculation. These data suggest that IL-6R are expressed on leukemic cells from a substantial percentage of pediatric AML patients. Furthermore, leukemic cells expressing high numbers of IL6-R may be sensitive to IL6-PE4E in an ex vivo purging protocol.

Identification and characterization of the human myeloperoxidase promoter.

Myeloperoxidase (MPO) is a microbicidal protein present in the primary granules of myeloid cells. Transcription of the MPO gene is turned on only during the late myeloblast and promyelocyte stages of myeloid maturation. Identification of cis-regulatory elements and transcription factors which regulate the MPO gene should, therefore, shed light on myeloid maturation. We report transfection and in vitro transcription experiments which demonstrate promoter activity in the proximal 5'-flanking region of the human MPO gene. Using a chloramphenicol acetyl transferase (CAT) reporter vector system, and segments of the 5'-flanking MPO DNA, we constructed a series of MPO promoter-CAT expression vectors. By electroporation and lipofectin-mediated transient transfection assays, as well as by in vitro transcription studies, a 594-bp MPO DNA sequence (bp -583 to +11) showed promoter activity in a variety of MPO-expressing and non-MPO-expressing cell lines. Compared with the SV40 early promoter, the MPO promoter had greater relative activity in MPO-expressing than in non-MPO-expressing cell lines, suggesting slight tissue specificity. However, a CAT reporter plasmid containing 1099-bp of 5'-flanking MPO DNA showed greater specificity for MPO expressing cell lines. Analysis of a group of promoter deletion mutants showed that the minimal promoter was contained in a DNA fragment extending from bp-128 to +11. The remainder of the promoter region contained several segments which appeared to enhance the activity of the minimal promoter. One such enhancer sequence was homologous to an enhancer previously described in the human elastase promoter. Activity of the 594-bp MPO promoter in HL-60 was reduced by only approximately 30% following treatment of the cells with chemical inducers of maturation, but the 1099-bp MPO promoter showed 60% reduction in activity after DMSO treatment. A previously described enhancer region in intron 9 of the MPO gene had little or no effect on activity of the 594-bp MPO promoter. The availability of the MPO promoter will facilitate determination of other factors involved in the regulation of this myeloid-specific gene.

Homozygous deletions of the CDKN2 (MTS1/p16ink4) gene in cell lines established from children with acute lymphoblastic leukemia.

Homozygous deletions of the CDKN2 (MTS1/p16ink4) gene have been found at high frequency in cell lines derived from a variety of adult solid tumors. In order to investigate the status of the CDKN2 gene in cell lines established from childhood acute lymphoblastic leukemia (ALL), we surveyed 25 lines representing the major pediatric ALL phenotypes for the presence of this gene by Southern blot analysis. Homozygous deletions of all or part of the CDKN2 gene were detected in 21 (84%) cell lines, including 11 of 14 (79%) early-pre-B-ALL, four of five (80%) pre-B-ALL, and six of six T-ALL lines. CDN2 mRNA was detected by Northern blotting in each of the four lines containing an intact CDKN2 gene. These data suggest an important role for CDKN2 deletion in the cause and/or progression of pediatric ALL.

Incidence and prognostic significance of MDM2 oncoprotein overexpression in relapsed childhood acute lymphoblastic leukemia.

MDM2 overexpression by pediatric ALL cells at initial diagnosis has been linked to poor response to therapy. In the present study, we evaluated the incidence of MDM2 overexpression by ALL cells from pediatric patients at first relapse and compared MDM2 protein levels with in vitro response to adriamycin and with duration of initial complete remission (CR1). Since an important role of MDM2 in enhancing cell proliferation and survival appears to be inhibition of p53 activity, we also evaluated the status of p53 in these patients' leukemic cells. MDM2 protein levels were determined by Western blot analysis of leukemic bone marrow cells obtained from 42 patients with B cell precursor (BCP) ALL who relapsed during or following therapy on standard POG ALL protocols. Twelve of 42 (29%) cases have MDM2 levels >/=10-fold higher than those detected in normal bone marrow mononuclear (NMMC) cells, which express relatively low levels of protein. Thirty cases (71%) expressed MDM2 at levels <10-fold those in NMMC, including 24 MDM2-negative cases (57%). P53 mutations were detected by single-strand conformation polymorphism analysis in two cases. Overexpression of MDM2 (>/=10-fold) was significantly correlated with adriamycin resistance and decreased duration of CR1. Eight of 12 (75%) overexpressers showed high levels of in vitro resistance to adriamycin, compared to four of 30 (13%) non-overexpressers (P < 0.005). The median CR1 for MDM2 overexpressers was 20.5 months (range: 3-75 months) compared to 41 months (range: 8-98 months) for non-overexpressers (P < 0.01). Four of 42 patients failed to achieve CR following re-induction: leukemic cells from three of these patients either overexpressed MDM2 or contained a mutant p53. These results indicate that overexpression of MDM2 plays a significant role in refractory pediatric ALL and is associated with early relapse, adriamycin resistance, and failure to respond to re-induction therapy. Leukemia (2000) 14, 61-67.

Expression of interleukin-6 receptors by pediatric acute lymphoblastic leukemia cells with the t(4;11) translocation: a possible target for therapy with recombinant IL6-Pseudomonas exotoxin.

We have detected expression of interleukin-6 receptors (IL-6R) by primary leukemic cells from three of six patients with t(4;11)+ ALL. Scatchard analysis revealed from 960 to 2100 high-affinity IL-6R/cell on these cells (median, 1560; mean, 1540). All three IL-6R+ cases also expressed CD33, which was not expressed on IL-6R-negative cases. To determine if these receptors could serve as a target for a recombinant ligand-toxin, we examined the sensitivity of primary IL-6R+ ALL cells to a recombinant IL6-Pseudomonas exotoxin (IL6-PE4E) fusion protein, in which the toxicity and specificity of the chimeric toxin was enhanced by substitution of four glutamine residues for naturally occurring amino acids in PE domain I. Primary cells from IL-6R+ cases were sensitive to IL6-PE4E in a 48-h cytotoxicity assay, with ID50 values (concentrations causing 50% decrease in viability) ranging from 23 ng/ml to 92 ng/ml (median, 61; mean, 58). Furthermore, incubation of these cells with 10(3) ng/ml IL6-toxin for 24 h prevented their subsequent engraftment in SCID mice. Thus, IL6-PE4E may be useful for ex vivo purging of IL-6R+ leukemic cells in an autologous bone marrow transplantation setting and possibly for therapy of residual, chemotherapy-resistant disease.

The effect of leukemic human serum on normal granulopoiesis.

Acute lymphoblastic leukemia (ALL) in children is often characterized by defective granulopoiesis during initial and relapse stages of the disease, resulting in absolute neutropenia in vivo and in low or absent production of granulocyte-macrophage colonies in vitro. The purpose of this study was to determine if serum from leukemic children with ALL could inhibit normal granulopoiesis. Several concentrations of serum from 12 leukemic patients were mixed with normal bone marrow cells and co-cultured by the double layer agar technique. Also, serum from normal individuals and from ALL patients in remission and relapse was examined for inhibitors. Cultures without serum served as controls. The majority of ALL (initial, relapse and remission) and normal sera either stimulated or had no effect on colony formation. These groups also had similar percentages of inhibitory sera, with the exception of the somewhat higher levels of inhibition found in the remission group. Thus, ALL serum resembled normal serum in its effect on in vitro granulopoiesis at the committed granulocyte-macrophage stem cell level. It is therefore unlikely that inhibition of in vivo granulopoiesis at the committed level by serum inhibitors is responsible for ALL-associated neutropenia.

The effect of leukemic lymphoblasts on normal granulopoiesis.

Acute lymphoblastic leukemia (ALL) in children is often characterized by defective granulopoiesis during initial and relapse stages of the disease, resulting in absolute neutropenia in vivo and in low or absent production of granulocyte-macrophage colonies in vitro. The purpose of this study was to determine if leukemic lymphoblasts from untreated ALL patients could inhibit normal granulopoiesis. Several concentrations of leukemic bone marrow cells from nine patients were mixed with either normal bone marrow cells or with autologous (HLA-identical) remission bone marrow cells, incubated for 1 hour, and co-cultured by the double layer agar technique. The cells were also cultured separately as controls. No statistically significant differences occurred between observed and expected colony counts in the majority of experiments. With three patients, slight inhibition occurred at some but not all leukemic cell concentrations tested; this inhibition was not correlated with the leukemic cell concentration. These results indicate that leukemic cells from untreated ALL patients do not significantly inhibit normal in vitro granulopoiesis at the committed stem cell level or at later levels of differentiation; therefore, such inhibition does not appear to be responsible for ALL-associated neutropenia.

Effect of retinoic acid on the clonal growth of childhood myeloid and lymphoid leukemias: a pediatric oncology group study.

We have studied the effects of retinoic acid (RA) on bone marrow leukemic cells from children with acute nonlymphocytic leukemia (ANLL) at the time of diagnosis, and on cells from four ALL/lymphoma cell lines (common-ALL, pre-B-ALL, T-ALL, and Burkitt's lymphoma) derived from children with these diseases. Cells were cultured in methylcellulose medium with clinically attainable concentrations (0.25-2.0 microM) of RA for two weeks prior to colony and cluster quantitation. Myeloid progenitor cells (CFU-GM) obtained from children with hematologically normal bone marrows were also cultured with RA. Of 19 patients with ANLL whose cells formed colonies, 16 (84%) were inhibited by RA; three patients showed either increased or unchanged colony numbers with RA. RA had a similar effect on both ANLL cluster and colony growth. RA (1-2 microM) also inhibited colony growth of the pre-B-ALL, common-ALL, and Burkitt's lymphoma lines; the T-ALL line and normal bone marrow CFU-GM were not inhibited. The inhibitory effects of RA on pediatric ANLL bone marrow cells and on some ALL/lymphoma cell lines compared with CFU-GM indicate that RA may be of value in the treatment of these malignancies in children.

Phase II study of 13-cis-retinoic acid in pediatric patients with acute nonlymphocytic leukemia--a Pediatric Oncology Group study.

Forty-one patients with refractory acute non-lymphocytic leukemia (ANLL) in relapse were treated with 13-cis-retinoic acid (cRA) as salvage therapy. The cRA was given as a single oral dose of 100 mg/m2/day for 4 weeks. One patient achieved a complete remission and two patients achieved a partial remission with reduction of the bone marrow blast count from 40 to 20% after the first course. We recommend further study of cRA in combination with other agents in the treatment of ANLL in children.

Effect of retinoic acid on myeloid antigen expression and clonal growth of leukemic cells from children with acute non lymphocytic leukemia--a Pediatric Oncology Group Study.

We have used a panel of 5 monoclonal antibodies against normal myeloid-differentiation antigens to determine retinoic acid-induced changes in cell surface antigens on ANLL bone marrow cells from 24 children at the time of diagnosis. Two of these antibodies (T5A7 and 5F1) detect antigens expressed on normal mature granulocytes and on all monocytes, respectively. The percentage of positive cells for each monoclonal antibody was determined by indirect immunofluorescence. After 5 days incubation with 1 microM RA in liquid culture, cells from 11 of 24 patients showed substantially increased expression of one or both antigens detected by T5A7 and 5F1. Leukemic bone marrow cells from these patients were also cultured in methylcellulose medium with and without 1 microM RA for one week, and cells from 16 of 24 patients showed clonal growth. Cultures from 10 of these 16 patients showed RA-induced inhibition of colony growth; of these 10 patients, cultures from six patients showed RA-induced increases in antigens associated with maturing myeloid cells. This suggests that the RA-induced inhibition of clonal growth observed with leukemic cells from these patients may be accompanied by the increased expression of maturation-associated myeloid antigens by these cells in the presence of RA.

Characterization of a new chromosomal marker for acute lymphoblastic leukemia from a long-term cell line.

A bone marrow aspirate from a child with acute lymphoblastic leukemia (ALL) at first relapse was used to establish cell line # 697. The cultured line and marrow aspirates taken at initial diagnosis and first relapse were examined and compared. Similarities in all patterns evaluated confirmed the leukemic origin of the line. Morphologically, the cells were typically lymphoblastic. Cytochemically, they were slightly acid phosphatase-positive and negative for peroxidase, ASD chloroacetate esterase, and nonspecific esterase. Immunologically, they were found positive for common-ALL antigen (CALLA), Ia-associated antigen, terminal deoxynucleotidyl transferase (TdT), and cytoplasmic IgM (cIgM) and slightly positive for surface IgM (sIgM). Testing for Epstein-Barr virus (EBV) capsid antigen was also positive. Cytogenetic evaluations performed on initial, relapse, and cell line specimens each revealed the presence of a pseudodiploid cell line characterized by a consistent marker chromosome. GTG-, QFQ-, and RF-banding identified the marker as being derived from a translocation involving chromosomes #7 and #19: t(7;19) (q11;q13). Iso 7q, -5, -9, and +2 were also found in significant association with the marker and were viewed as demonstrating continued karyotypic evolution within the cell line. From these data, cell line #697 has been classified as a leukemic line of B-cell lineage in a transitional stage between pre-B and mature B cells.

MDM2 antagonist nutlin-3 is a potent inducer of apoptosis in pediatric acute lymphoblastic leukemia cells with wild-type p53 and overexpression of MDM2.

In pediatric acute lymphoblastic leukemia (ALL), overexpression of murine double minute 2 (MDM2) protein by leukemic cells is typically associated with a wild-type (wt)-p53 phenotype and chemoresistance. A recently developed small-molecule antagonist of MDM2, nutlin-3, inhibits the MDM2-p53 interaction, resulting in induction of p53 activity and apoptosis. In this study, we evaluated the cytotoxic effect of nutlin-3 on ALL cells with different p53 status and MDM2 expression, using 18 cell lines and 30 primary leukemia samples. We found that both ALL cell lines and primary ALL samples with wt-p53 are sensitive to nutlin-3. No cytotoxic effect of nutlin-3 was detected in ALL cells with either p53-mutant or -null phenotype. In wt-p53 ALL cells, there was a significant positive correlation between MDM2 expression levels and sensitivity to nutlin-3. Nutlin-3-induced cell death was mediated by p53-induced activation of proapoptotic proteins and by p53-induced repression of the anti-apoptotic protein survivin. As p53 function is inhibited by MDM2 in chemoresistant, MDM2-overexpressing ALL cells, potent killing of these cells by nutlin-3 suggests that this agent may be a novel therapeutic for refractory ALL.