Aberrantly low STAT3 and STAT5 responses are associated with poor outcome and an inflammatory gene expression signature in pediatric acute myeloid leukemia

The relapse rate for children with acute myeloid leukemia is nearly 40% despite aggressive chemotherapy and often stem cell transplant. We sought to understand how environment-induced signaling responses are associated with clinical response to treatment. We previously reported that patients whose AML cells showed low G-CSF-induced STAT3 activation had inferior event-free survival compared to patients with stronger STAT3 responses. Here, we expanded the paradigm to evaluate multiple signaling parameters induced by a more physiological stimulus. We measured STAT3, STAT5 and ERK1/2 responses to G-CSF and to stromal cell-conditioned medium for 113 patients enrolled on COG trials AAML03P1 and AAML0531. Low inducible STAT3 activity was independently associated with inferior event-free survival in multivariate analyses. For inducible STAT5 activity, those with the lowest and highest responses had inferior event-free survival, compared to patients with intermediate STAT5 responses. Using existing RNA-sequencing data, we compared gene expression profiles for patients with low inducible STAT3/5 activation with those for patients with higher inducible STAT3/5 signaling. Genes encoding hematopoietic factors and mitochondrial respiratory chain subunits were overexpressed in the low STAT3/5 response groups, implicating inflammatory and metabolic pathways as potential mechanisms of chemotherapy resistance. We validated the prognostic relevance of individual genes from the low STAT3/5 response signature in a large independent cohort of pediatric AML patients. These findings provide novel insights into interactions between AML cells and the microenvironment that are associated with treatment failure and could be targeted for therapeutic interventions (AU)

Aberrantly low STAT3 and STAT5 responses are associated with poor outcome and an inflammatory gene expression signature in pediatric acute myeloid leukemia.

The relapse rate for children with acute myeloid leukemia is nearly 40% despite aggressive chemotherapy and often stem cell transplant. We sought to understand how environment-induced signaling responses are associated with clinical response to treatment. We previously reported that patients whose AML cells showed low G-CSF-induced STAT3 activation had inferior event-free survival compared to patients with stronger STAT3 responses. Here, we expanded the paradigm to evaluate multiple signaling parameters induced by a more physiological stimulus. We measured STAT3, STAT5 and ERK1/2 responses to G-CSF and to stromal cell-conditioned medium for 113 patients enrolled on COG trials AAML03P1 and AAML0531. Low inducible STAT3 activity was independently associated with inferior event-free survival in multivariate analyses. For inducible STAT5 activity, those with the lowest and highest responses had inferior event-free survival, compared to patients with intermediate STAT5 responses. Using existing RNA-sequencing data, we compared gene expression profiles for patients with low inducible STAT3/5 activation with those for patients with higher inducible STAT3/5 signaling. Genes encoding hematopoietic factors and mitochondrial respiratory chain subunits were overexpressed in the low STAT3/5 response groups, implicating inflammatory and metabolic pathways as potential mechanisms of chemotherapy resistance. We validated the prognostic relevance of individual genes from the low STAT3/5 response signature in a large independent cohort of pediatric AML patients. These findings provide novel insights into interactions between AML cells and the microenvironment that are associated with treatment failure and could be targeted for therapeutic interventions.

Copy-neutral loss of heterozygosity is prevalent and a late event in the pathogenesis of FLT3/ITD AML.

Patients with high FLT3 internal tandem duplication allelic ratios (FLT3/ITD-ARs) have a poor prognosis. Single-nucleotide polymorphism/comparative genomic hybridization, single-cell PCR and colony-forming assays were used to evaluate genotypic evolution of high FLT3/ITD-ARs in 85 acute myeloid leukemia (AML) patients. Microarrays were used to examine molecular pathways disrupted in leukemic blasts with high FLT3/ITD-ARs. Copy-neutral loss of heterozygosity (CN-LOH) was identified at the FLT3 locus in diagnostic samples with high FLT3/ITD-ARs (N=11), but not in samples with low FLT3/ITD-ARs (N=24), FLT3-activating loop mutations (N=11) or wild-type FLT3 (N=39). Single-cell assays showed that homozygous FLT3/ITD genotype was present in subsets of leukemic blasts at diagnosis but became the dominant clone at relapse. Less differentiated CD34(+)/CD33(-) progenitor colonies were heterozygous for FLT3/ITD, whereas more differentiated CD34(+)/CD33(+) progenitor colonies were homozygous for FLT3/ITD. Expression profiling revealed that samples harboring high FLT3/ITD-ARs aberrantly expressed genes within the recombination/DNA repair pathway. Thus, the development of CN-LOH at the FLT3 locus, which results in high FLT3/ITD-ARs, likely represents a late genomic event that occurs after the acquisition of the FLT3/ITD. Although the etiology underlying the development of CN-LOH remains to be clarified, the disruption in recombination/DNA repair pathway, which is present before the development of LOH, may have a role.

Heterogeneity of clonal expansion and maturation-linked mutation acquisition in hematopoietic progenitors in human acute myeloid leukemia.

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML), but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34(+)/CD33(-) cells revealed polyclonal growth in highly curable AMLs, suggesting that mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34(+)/CD33(-) cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications.

NUP98/JARID1A is a novel recurrent abnormality in pediatric acute megakaryoblastic leukemia with a distinct HOX gene expression pattern.

Cytogenetic abnormalities and early response to treatment are the main prognostic factors in acute myeloid leukemia (AML). Recently, NUP98/NSD1 (t(5; 11)(q35; p15)), a cytogenetically cryptic fusion, was described as recurrent event in AML, characterized by dismal prognosis and HOXA/B gene overexpression. Using split-signal fluorescence in situ hybridization, other NUP98-rearranged pediatric AML cases were identified, including several acute megakaryoblastic leukemia (AMKL) cases with a cytogenetically cryptic fusion of NUP98 to JARID1A (t(11;15)(p15;q35)). In this study we screened 105 pediatric AMKL cases to analyze the frequency of NUP98/JARID1A and other recurrent genetic abnormalities. NUP98/JARID1A was identified in 11/105 patients (10.5%). Other abnormalities consisted of RBM15/MKL1 (n=16), CBFA2T3/GLIS2 (n=13) and MLL-rearrangements (n=13). Comparing NUP98/JARID1A-positive patients with other pediatric AMKL patients, no significant differences in sex, age and white blood cell count were found. NUP98/JARID1A was not an independent prognostic factor for 5-year overall (probability of overall survival (pOS)) or event-free survival (probability of event-free survival (pEFS)), although the 5-year pOS for the entire AMKL cohort was poor (42 ± 6%). Cases with RBM15/MLK1 fared significantly better in terms of pOS and pEFS, although this was not independent from other risk factors in multivariate analysis. NUP98/JARID1A cases were characterized by HOXA/B gene overexpression, which is a potential druggable pathway. In conclusion, NUP98/JARID1A is a novel recurrent genetic abnormality in pediatric AMKL.

Molecular alterations of the IDH1 gene in AML: a Children's Oncology Group and Southwest Oncology Group study.

Recent whole-genome sequencing efforts led to the identification of IDH1(R132) mutations in acute myeloid leukemia (AML) patients. We studied the prevalence and clinical implications of IDH1 genomic alterations in pediatric and adult AML. Diagnostic DNA from 531 AML patients treated on Children's Oncology Group trial COG-AAML03P1 (N=257), and Southwest Oncology Group trials SWOG-9031, SWOG-9333 and SWOG-9500 (N=274), were tested for IDH1 mutations. Codon R132 mutations were absent in the pediatric cohort, but were found in 12 of 274 adult patients (4.4%, 95% CI 2.3-7.5). IDH1(R132) mutations occurred most commonly in patients with normal karyotype, and those with FLT3/ITD and NPMc mutations. Patients with IDH1(R132) mutations trended toward higher median diagnostic white blood cell counts (59.2 x 10(9) vs 29.1 x 10(9) per liter, P=0.19) than those without mutations, but the two groups did not differ significantly in age, bone marrow blast percentage, overall survival or relapse-free survival. Eleven patients (2.1%) harbored a novel V71I sequence alteration, which was found to be a germ-line polymorphism. IDH1 mutations were not detected in pediatric AML, and are uncommon in adult AML.

DNA repair polymorphisms and outcome of chemotherapy for acute myelogenous leukemia: a report from the Children's Oncology Group.

Polymorphisms of DNA repair genes RAD51 and XRCC3 increase susceptibility to acute myeloid leukemia (AML) in adults, an effect enhanced by deletion of the glutathione-S-transferase M1 (GSTM1) gene. In this study, we genotyped 452 children with de novo AML treated on CCG protocols 2941 and 2961 and compared genotype frequencies with those of normal blood donors, and analyzed the impact of genotype on outcome of therapy. XRCC3 Thr241Met, RAD51 G135C and GSTM1 genotypes did not increase susceptibility to AML when assessed singly. In contrast, when XRCC3 and RAD51 genotypes were examined together a significant increase in susceptibility to AML was seen in children with variant alleles. Analysis of outcome of therapy showed that patients heterozygous for the XRCC3 Thr241Met allele had improved post-induction disease-free survival compared to children homozygous for the major or minor allele, each of whom had similar outcomes. Improved survival was due to reduced relapse in the heterozygous children, and this effect was most marked in children randomized to therapy likely to generate DNA double-strand breaks (etoposide, daunomycin), compared with anti-metabolite (fludarabine, cytarabine) based therapy. In contrast, RAD51 G135C and the GSTM1 deletion polymorphism did not influence outcome of AML therapy in our study population.

Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia.

Activating mutations in RAS and receptor tyrosine kinases such as KIT and FLT3 are hypothesized to cooperate with chimeric transcription factors in the pathogenesis of acute myeloid leukemia (AML). To test this hypothesis, we genotyped 150 pediatric AML samples for mutations in KIT (exons 8, 17), NRAS and KRAS (exons 1, 2) and FLT3/ITD. This is the largest cohort of pediatric AML patients reported thus far screened for all four mutations. Of the children with AML, 40% had a mutation in KIT (11.3%), RAS (18%) or FLT3/ITD (11.1%), and 70% of cases of core-binding factor (CBF) leukemia were associated with a mutation of KIT or RAS. Mutations in RAS or FLT3/ITD were frequently found in association with a normal karyotype. Patients with a FLT3/ITD mutation had a significantly worse clinical outcome. However, the presence of a KIT or RAS mutation did not significantly influence clinical outcome. We demonstrate that KIT exon 8 mutations result in constitutive ligand-independent kinase activation that can be inhibited by clinically relevant concentrations of imatinib. Our results demonstrate that abnormalities of signal transduction pathways are frequent in pediatric AML. Future clinical studies are needed to determine whether selective targeting of these abnormalities will improve treatment results.

PTPN11 mutations in pediatric patients with acute myeloid leukemia: results from the Children's Cancer Group.

The PTPN11 gene encodes SHP-2, a nonreceptor protein tyrosine phosphatase that relays signals from activated growth factor receptors to p21(ras) (Ras) and other signaling molecules. Somatic PTPN11 mutations are common in patients with juvenile myelomonocytic leukemia (JMML) and have been reported in some other hematologic malignancies. We analyzed specimens from 278 pediatric patients with acute myelogenous leukemia (AML) who were enrolled on Children's Cancer Group trials 2941 and 2961 for PTPN11 mutations. Missense mutations of PTPN11 were detected in 11 (4%) of these samples. None of these patients had mutations in NRAS; however, one patient had evidence of a FLT3 alteration. Four of the patients with PTPN11 mutations (36%) were boys with French-American-British (FAB) morphology M5 AML (P=0.012). Patients with mutations also presented with elevated white blood cell counts. There was no difference in clinical outcome for patients with and without PTPN11 mutations. These characteristics identify a subset of pediatric AML with PTPN11 mutations that share clinical and biologic features with JMML.

FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia.

The prevalence and significance of genetic abnormalities in older patients with acute myeloid leukemia (AML) are unknown. Polymerase chain reactions and single-stranded conformational polymorphism analyses were used to examine 140 elderly AML patients enrolled in the Southwest Oncology Group study 9031 for FLT3, RAS, and TP53 mutations, which were found in 34%, 19%, and 9% of patients, respectively. All but one of the FLT3 (46 of 47) mutations were internal tandem duplications (ITDs) within exons 11 and 12. In the remaining case, a novel internal tandem triplication was found in exon 11. FLT3 ITDs were associated with higher white blood cell counts, higher peripheral blast percentages, normal cytogenetics, and less disease resistance. All RAS mutations (28 of 28) were missense point mutations in codons 12, 13, or 61. RAS mutations were associated with lower peripheral blast and bone marrow blast percentages. Only 2 of 47 patients with FLT3 ITDs also had a RAS mutation, indicating a significant negative association between FLT3 and RAS mutations (P =.0013). Most TP53 mutations (11 of 12) were missense point mutations in exons 5 to 8 and were associated with abnormal cytogenetics, especially abnormalities in both chromosomes 5 and 7. FLT3 and RAS mutations were not associated with inferior clinical outcomes, but TP53 mutations were associated with a worse overall survival (median 1 versus 8 months, P =.0007). These results indicate that mutations in FLT3, RAS, or TP53 are common in older patients with AML and are associated with specific AML phenotypes as defined by laboratory values, cytogenetics, and clinical outcomes. (Blood. 2001;97:3589-3595)

Prevalence and prognostic significance of Flt3 internal tandem duplication in pediatric acute myeloid leukemia.

The Flt3 gene encodes a tyrosine kinase receptor that regulates proliferation and differentiation of hematopoietic stem cells. An internal tandem duplication of the Flt3 gene (Flt3/ITD) has been reported in acute myelogenous leukemia (AML) and may be associated with poor prognosis. We analyzed diagnostic bone marrow specimens from 91 pediatric patients with AML treated on Children's Cancer Group (CCG)-2891 for the presence of the Flt3/ITD and correlated its presence with clinical outcome. Fifteen of 91 samples (16.5%) were positive for the Flt3/ITD. Flt3/ITD-positive patients had a median diagnostic white count of 73 800 compared with 28 400 for the Flt3/ITD-negative patients (P =.05). The size of the duplication ranged from 21 to 174 base pairs (bp). Nucleotide sequencing of the abnormal polymerase chain reaction products demonstrated that all duplications involved exon 11 of the Flt3 gene and also preserved the reading frame. Lineage restriction analysis revealed that Flt3/ITD was not present in the lymphocytes, suggesting a lack of stem cell involvement for this mutation. None of the Flt3/ITD-positive patients had unfavorable cytogenetic markers, and there was no predominance of a particular FAB class. The remission induction rate was 40% in Flt3/ITD-positive patients compared with 74% in Flt3/ITD-negative ones (P =.005). The Kaplan-Meier estimates of event-free survival at 8 years for patients with and without Flt3/ITD were 7% and 44%, respectively (P =.002). Multivariate analysis demonstrated that presence of the Flt3/ITD was the single most significant, independent prognostic factor for poor outcome (P =.009) in pediatric AML.

Comparison of multidimensional flow cytometry with standard morphology for evaluation of early marrow response in pediatric acute lymphoblastic leukemia.

PURPOSE: We compared multidimensional flow cytometry (MDF) with morphology in evaluating early marrow response to induction chemotherapy in pediatric ALL. METHODS: Chemotherapy response was determined by standard morphology or by MDF assessed by residual leukemic cell percentage remaining in the marrow on days 7, 14, and 28 of induction. Bone marrow response was classified as M3 (>25% leukemic blasts) or M1/M2 (< or = 25% leukemic blasts). Multidimensional flow cytometry evaluation was compared with that of standard morphology. Available day-7 and day-14 marrow slides were also reevaluated by a single pathologist without patients' clinical information. RESULTS: Of 46 day-7 specimens, eight (17%) had discordant MDF and morphologic results (P < 0.001), including six classified as M3 by morphology but were M1/M2 by MDF, and two were classified as M3 by MDF but were M1/M2 by morphology. Of 24 day-14 bone marrow specimens, five (20.5%) were discordant (P < 0.001), including two classified as M3 by morphology but were M1/M2 by MDF, and three were classified as M3 by MDF but were M1/M2 by morphology. Reevaluation of the blinded day-7 and day-14 marrow slides yielded discordance between repeated pathology readings of 11% (P < 0.001) and 6% (P = 0.04), respectively. CONCLUSION: Our data show significant discordance between the morphologic and MDF evaluation of early marrow response. Early response to therapy is a significant prognostic indicator in pediatric acute lymphoblastic leukemia and is used to alter subsequent treatment; thus, precise assessment of response is important. A larger comparison of MDF with morphology for the evaluation of early response, including correlation with clinical outcome, is warranted.

Purification of the endogenous glucocorticoid receptor stabilizing factor.

A ubiquitous, low molecular weight, heat-stable component of cytosol stabilizes the glucocorticoid receptor in its untransformed state in association with hsp90. This heat-stable factor mimics molybdate in its effects on receptor function, and it has the heat stability, charge, and chelation properties of a metal oxyanion [Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., & Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817]. In this paper, we describe the further purification of the endogenous factor from rat liver cytosol by anion-exchange HPLC (Ion-110) after prepurification by molecular sieving, cation absorption, and charcoal absorption. Elution of the factor with an isocratic gradient of ammonium bicarbonate results in recovery of all of the bioactivity in a single peak which coelutes with inorganic phosphate and contains all of the endogenous molybdenum. The bioactivity can be separated from inorganic phosphate by chromatography of the partially purified endogenous factor on a metal-chelating column of Chelex-100. The chelating procedure results in complete loss of bioactivity with recovery of 98% of the inorganic phosphate in both the column drop-through and a subsequent 1 M NaCl wash. The factor preparation purified through the Ion-110 HPLC step inhibits temperature-mediated dissociation of the immunopurified glucocorticoid receptor-hsp90 complex, but it is considerably more effective at stabilizing the unpurified receptor-hsp90 complex in a Chelex-treated cytosol system that has been depleted of metal components. These observations support the proposal that an endogenous metal can stabilize the binding of hsp90 to the receptor but it is likely that other cytosolic components that are not present in the immunopurified complex must contribute to the stability of the soluble protein-protein complex in cytosol.

Redox manipulation of DNA binding activity and BuGR epitope reactivity of the glucocorticoid receptor.

Treatment of the transformed glucocorticoid receptor with hydrogen peroxide promotes the formation of disulfide bonds and inhibits the ability of the receptor to bind to DNA (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). It has not been determined whether the inhibition of DNA binding activity is due to disulfide bonds formed within the DNA binding domain or between the DNA binding domain and another region of the receptor. In this paper, we examined the ability of hydrogen peroxide to inactivate the DNA binding activity of the mouse glucocorticoid receptor. We show that inhibition of DNA binding activity caused by hydrogen peroxide can be accounted for entirely by the formation of disulfide bonds between cysteine residues lying within the 15-kDa tryptic fragment containing the DNA binding domain of the receptor. Reversal of the peroxide-induced inactivation of DNA binding activity requires both zinc and a thiol-disulfide exchange reagent, such as dithiothreitol. Peroxide also eliminates recognition of the intact receptor and the 15-kDa tryptic fragment by the BuGR monoclonal antibody, and the reactivity of the BuGR epitope is restored by reduction without a requirement for zinc. Pretreatment of the receptor with methyl methanethiosulfonate inhibits much of the peroxide-mediated inactivation of the BuGR epitope but pretreatment with N-ethylmaleimide does not. Similarly, DNA binding activity of the receptor is inhibited by methyl methanethiosulfonate but not by N-ethylmaleimide. These results are consistent with the proposal that peroxide promotes the formation of disulfide bonds between thiols that lie spatially close to one another in the 15-kDa tryptic fragment, resulting in rapid elimination of zinc. Restoration of the zinc finger structure restores DNA-binding activity but restoration of the BuGR epitope requires only reduction without restoration of the zinc fingers.

The relationship between glucocorticoid receptor binding to Hsp90 and receptor function.

In this minireview we summarize evidence that the association of the glucocorticoid receptor (GR) with hsp90 may determine three functional states of the receptor. First, there is a direct correlation between hsp90 binding to the receptor and repression of DNA binding activity. Temperature-dependent dissociation of hsp90 from the cytosolic GR-hsp90 complex is promoted by hormone with simultaneous conversion of the receptor to the DNA binding state. GR that is translated in rabbit reticulocyte lysate binds to hsp90 at or near the termination of receptor translation and is in the non-DNA-binding form. Second, there is a direct correlation between binding of the immunopurified GR to hsp90 and the presence of a high affinity steroid binding conformation of the receptor. GR translated in reticulocyte lysate binds steroid with high affinity, but GR translated in wheat germ extract is not bound to hsp90, does not bind steroid with high affinity, and is in the DNA-binding form. When immunopurified, hsp90-free GR is incubated with rabbit reticulocyte lysate, hsp90 associates with the receptor and high affinity steroid binding capacity is completely reactivated. Third, there is a correlation between binding of hsp90 to steroid receptors and their retention in an inactive "docking" state until the binding of hormone in the intact cell triggers a progression to high affinity nuclear binding sites where the primary events involved in transcriptional activation occur. In contrast to the receptors that are retained in the docking state, the unliganded thyroid hormone receptor proceeds directly to high affinity nuclear binding sites. Consistent with this difference in behavior, the thyroid hormone receptor is translated in reticulocyte lysate in its DNA binding form and is not associated with hsp90.