RAS pathway mutations as a predictive biomarker for treatment adaptation in pediatric B-cell precursor acute lymphoblastic leukemia.

RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50-70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27-4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: an international collaborative study.

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4-7 and exons 1-8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case-control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2-7 (P=0.03), DEL 2-8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4-7 and DEL 1-8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

PKCζ and PKMζ are overexpressed in TCF3-rearranged paediatric acute lymphoblastic leukaemia and are associated with increased thiopurine sensitivity.

Both tumour suppressor and oncogenic functions have been ascribed to the atypical zeta isoform of protein kinase C (PKCζ), whereas its constitutively active form PKMζ is almost exclusively expressed in the brain where it has a role in long-term memory. Using primers unique for either isoform, we found that both PKCζ and PKMζ were expressed in a subset of paediatric acute lymphoblastic leukaemia (ALL) cases carrying a TCF3 (E2A) chromosomal rearrangement. Combined PKCζ and PKMζ (PKC/Mζ) protein as well as phosphorylation levels were elevated in ALL cases, especially TCF3-rearranged precursor B-ALL cases, compared with normal bone marrow (P<0.01). Furthermore, high PKC/Mζ expression in primary ALL cells was associated with increased sensitivity to 6-thioguanine and 6-mercaptopurine (P<0.01), thiopurines used in ALL treatment. PKCζ is believed to stabilize mismatch-repair protein MSH2, facilitating thiopurine responsiveness in T-ALL. However, PKC/Mζ knockdown in a TCF3-rearranged cell line model decreased MSH2 expression but did not induce thiopurine resistance, indicative that the link between high PKC/Mζ levels and thiopurine sensitivity in paediatric precursor B-ALL is not directly causal. Collectively, our data indicate that thiopurine treatment may be effective, especially in paediatric TCF3-rearranged ALL and other patients with a high expression of PKC/Mζ.

EMP1, a novel poor prognostic factor in pediatric leukemia regulates prednisolone resistance, cell proliferation, migration and adhesion.

Still 20% of pediatric acute lymphoblastic leukemia (ALL) patients relapse on or after current treatment strategies. Treatment failure is associated with resistance to prednisolone. We aimed to find new druggable targets that modulate prednisolone resistance. We generated microarray gene expression profiles of 256 pediatric ALL patient samples and identified a 3.4-fold increase in epithelial membrane protein 1 (EMP1) expression in in vitro prednisolone-resistant compared with -sensitive patients (P=0.003). EMP1 silencing in six precursor-B ALL (BCP-ALL) and T-ALL cell lines induced apoptosis and cell-cycle arrest leading to 84.1±4.5% reduction in survival compared with non-silencing control transduced cells (non-silencing control short hairpin, shNSC) (P=0.014). Moreover, EMP1 silencing sensitized to prednisolone up to 18.8-fold (P<0.001). EMP1 silencing also abrogated migration and adhesion to mesenchymal stromal cells (MSCs) by 78.3±9.0 and 29.3±4.1% compared with shNSC (P<0.05). We discovered that EMP1 contributes to MSC-mediated prednisolone resistance. Pathway analysis indicated that EMP1 signals through the Src kinase family. EMP1-high BCP-ALL patients showed a poorer 5-year event-free survival compared with EMP1-low patients (77±2 vs. 89±2%, P=0.003). Multivariate analysis taking along white blood cell count, age, prednisolone resistance and subtype identified EMP1 as an independent predictor for poor outcome in BCP-ALL (P=0.004, hazard ratio: 2.36 (1.31-4.25). This study provides preclinical evidence that EMP1 is an interesting candidate for drug development to optimize treatment of BCP-ALL.

miR-9 is a tumor suppressor in pediatric AML with t(8;21).

MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and development of leukemia. Great interest emerged in modulating miRNA expression for therapeutic purposes. In order to identify miRNAs, which specifically suppress leukemic growth of acute myeloid leukemia (AML) with t(8;21), inv(16) or mixed lineage leukemia (MLL) rearrangement by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases. Four miRNAs, specifically downregulated in MLL-rearranged, t(8;21) or inv(16) AMLs, were characterized by their tumor-suppressive properties in cell lines representing those respective cytogenetic groups. Among those, forced expression of miR-9 reduced leukemic growth and induced monocytic differentiation of t(8;21) AML cell lines in vitro and in vivo. The tumor-suppressive functions of miR-9 were specifically restricted to AML cell lines and primary leukemic blasts with t(8;21). On the other hand, these functions were not evident in AML blasts from patients with MLL rearrangements. We showed that miR-9 exerts its effects through the cooperation with let-7 to repress the oncogenic LIN28B/HMGA2 axis. Thus, miR-9 is a tumor suppressor-miR which acts in a stringent cell context-dependent manner.

Altered expression of miR-24, miR-126 and miR-365 does not affect viability of childhood TCF3-rearranged leukemia cells.

Among the microRNAs (miRNAs) that control different cellular processes, miR-24, miR-126 and miR-365 were shown to regulate cell cycle progression and apoptosis in various types of tumors. Interestingly, these three miRNAs were downregulated in pediatric TCF3-rearranged B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we showed that individual or combined overexpression of miR-24, miR-126 and miR-365 can neither alter the cell cycle progression nor the amount of apoptosis in 697, KASUMI-2 or MHH-CALL-3 TCF3-rearranged leukemic cells. We further integrated the miRNA-mRNA expression data of 37 children with BCP-ALL to identify candidate target genes for these three miRNAs. However, the expression levels of selected candidate target genes (ELL, EBF3 and IRF4 for miR-24, PITPNC1 for miR-126 and ZAP-70 for miR-365) did not reduce upon miRNAs overexpression in MHH-CALL-3 TCF3-rearranged leukemic cells. Although the expression level of AURKB-a validated target for miR-24-was reduced upon miR-24 overexpression in hepatocarcinoma HEP-G2 cells, overexpression of miR-24 cannot alter AURKB expression levels in MHH-CALL-3 TCF3-rearranged leukemic cells. Taken together, our data suggest that miRNAs' function is highly tissue-dependent and that a defined biological target gene or function of one miRNA in a specific tissue cannot be extended as a generalized target/function for that miRNA in all types of cells/tissues.

MiR-125b, miR-100 and miR-99a co-regulate vincristine resistance in childhood acute lymphoblastic leukemia.

MicroRNA-125b (miR-125b), miR-99a and miR-100 are overexpressed in vincristine-resistant acute lymphoblastic leukemia (ALL). Cellular viability of ETV6-RUNX1-positive Reh cells significantly increased in presence of 9 ng/mL vincristine upon co-expression of miR-125b/miR-99a (91 ± 4%), miR-125b/miR-100 (93 ± 5%) or miR-125b/miR-99a/miR-100 (82 ± 17%) compared with miR-125b-transduced cells (38 ± 13%, P<0.05). Co-expression of these miRNAs resulted in downregulation of DNTT, NUCKS1, MALAT1, SNRPE, PNO1, SET, KIF5B, PRPS2, RPS11, RPL38 and RPL23A (fold-change 1.3-1.9, p<0.05). Similarly, 7 out of these genes are lower expressed in vincristine-resistant ALL cells of children (p<0.05). The concerted function of miR-125b in combination with miR-99a and/or miR-100 illustrates the complexity of vincristine-resistant pediatric ALL.

Aurora kinases in childhood acute leukemia: the promise of aurora B as therapeutic target.

We investigated the effects of targeting the mitotic regulators aurora kinase A and B in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Aurora protein expression levels in pediatric ALL and AML patient samples were determined by western blot and reverse phase protein array. Both kinases were overexpressed in ALL and AML patients (P<0.0002), especially in E2A-PBX1-translocated ALL cases (P<0.002), compared with normal bone-marrow mononuclear cells. Aurora kinase expression was silenced in leukemic cell lines using short hairpin RNAs and locked nucleic acid-based mRNA antagonists. Aurora B knockdown resulted in proliferation arrest and apoptosis, whereas aurora A knockdown caused no or only minor growth delay. Most tested cell lines were highly sensitive to the AURKB-selective inhibitor barasertib-hydroxyquinazoline-pyrazol-anilide (AZD1152-HQPA) in the nanomolar range, as tested with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. But most importantly, primary ALL cells with a high aurora B protein expression, especially E2A-PBX1-positive cases, were sensitive as well. In adult AML early clinical trials, clear responses are observed with barasertib. Here we show that inhibition of aurora B, more than aurora A, has an antiproliferative and pro-apoptotic effect on acute leukemia cells, indicating that particularly targeting aurora B may offer a new strategy to treat pediatric ALL and AML.

The hunting of targets: challenge in miRNA research.

MicroRNAs (miRNAs) are small noncoding RNAs that control the expression of around 60% of the human protein-coding genes. In the past decade, deregulation of miRNAs (by expression and/or function) has been associated with the pathogenesis, progression and prognosis of different diseases, including leukemia. The number of discovered genes encoding miRNAs has risen exponentially in this period, but the numbers of miRNA-target genes discovered and validated lag far behind. Scientists have gained more in-depth knowledge of the basic mechanism of action of miRNAs, but the main challenge still remaining is the identification of direct targets of these important 'micro-players', to understand how they fine-tune so many biological processes in both healthy and diseased tissue. Many technologies have been developed in the past few years, some with more potential than others, but all with their own pros and cons. Here, we review the most common and most potent computational and experimental approaches for miRNA-target gene discovery and discuss how the hunting of targets is challenging but possible by taking the experimental limitations in consideration and choosing the correct cellular context for identifying relevant target genes.

Outcome in children with Down's syndrome and acute lymphoblastic leukemia: role of IKZF1 deletions and CRLF2 aberrations.

Children with Down's syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL) and have a low frequency of established genetic aberrations. We aimed to determine which genetic abnormalities are involved in DS ALL. We studied the frequency and prognostic value of deletions in B-cell development genes and aberrations of janus kinase 2 (JAK2) and cytokine receptor-like factor 2 (CRLF2) using array-comparative genomic hybridization, and multiplex ligation-dependent probe amplification in a population-based cohort of 34 Dutch Childhood Oncology Group DS ALL samples. A population-based cohort of 88 DS samples from the UK trials was used to validate survival estimates for IKZF1 and CRLF2 abnormalities. In total, 50% of DS ALL patients had ≥1 deletion in the B-cell development genes: PAX5 (12%), VPREB1 (18%) and IKZF1 (35%). JAK2 was mutated in 15% of patients, genomic CRLF2 rearrangements in 62%. Outcome was significantly worse in patients with IKZF1 deletions (6-year event-free survival (EFS) 45 ± 16% vs 95 ± 4%; P=0.002), which was confirmed in the validation cohort (6-year EFS 21 ± 12% vs 58 ± 11%; P=0.002). This IKZF1 deletion was a strong independent predictor for outcome (hazard ratio EFS 3.05; P=0.001). Neither CRLF2 nor JAK2 were predictors for worse prognosis. If confirmed in prospective series, IKZF1 deletions may be used for risk-group stratification in DS ALL.

The novel calicheamicin-conjugated CD22 antibody inotuzumab ozogamicin (CMC-544) effectively kills primary pediatric acute lymphoblastic leukemia cells.

We investigated whether the newly developed antibody (Ab) -targeted therapy inotuzumab ozogamicin (CMC-544), consisting of a humanized CD22 Ab linked to calicheamicin, is effective in pediatric primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells in vitro, and analyzed which parameters determine its efficacy. CMC-544 induced dose-dependent cell kill in the majority of BCP-ALL cells, although IC(50) values varied substantially (median 4.8 ng/ml, range 0.1-1000 ng/ml at 48 h). The efficacy of CMC-544 was highly dependent on calicheamicin sensitivity and CD22/CMC-544 internalization capacity of BCP-ALL cells, but hardly on basal and renewed CD22 expression. Although CD22 expression was essential for uptake of CMC-544, a repetitive loop of CD22 saturation, CD22/CMC-544 internalization and renewed CD22 expression was not required to achieve intracellular threshold levels of calicheamicin sufficient for efficient CMC-544-induced apoptosis in BCP-ALL cells. This is in contrast to studies with the comparable CD33 immunotoxin gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia (AML) patients, in which complete and prolonged CD33 saturation was required for apoptosis induction. These data suggest that CMC-544 treatment may result in higher response rates in ALL compared with response rates obtained in AML with Mylotarg, and that therefore clinical studies in ALL, preferably with multiple low CMC-544 dosages, are warranted.

MicroRNAs in acute leukemia: from biological players to clinical contributors.

MicroRNAs (miRNAs) are involved in the management of hematopoiesis. As a consequence, miRNA dysregulation causes disruption of the hematopoietic system and leukemia may arise. We here comprehensively discuss miRNAs found discriminative for cytogenetic and molecular subtypes of acute leukemia. These miRNAs are either known miRNAs involved in leukemogenesis with proven tumor suppressor or oncogenic activities or are newly identified by high-throughput sequencing with yet unknown function. Furthermore, forces are outlined that drive aberrant miRNA function, which include genetic abnormalities (for example, deletions, translocations and mutations) and epigenetic aberrations (for example, aberrant DNA methylation or histone modifications). Interestingly, leukemia-silenced miRNAs can be re-expressed upon treatment with de-methylating agents. Targeting miRNA expression may serve a therapeutical role, albeit at present this way of targeted therapy is in its infancy. However, emerging knowledge about the biology of miRNAs in leukemia may result into a role for these miRNAs in the diagnosis and treatment of acute leukemia.

Discovery of new microRNAs by small RNAome deep sequencing in childhood acute lymphoblastic leukemia.

MicroRNAs (miRNAs) relevant to acute lymphoblastic leukemia (ALL) in children are hypothesized to be largely unknown as most miRNAs have been identified in non-leukemic tissues. In order to discover these miRNAs, we applied high-throughput sequencing to pooled fractions of leukemic cells obtained from 89 pediatric cases covering seven well-defined genetic types of ALL and normal hematopoietic cells. This resulted into 78 million small RNA reads representing 554 known, 28 novel and 431 candidate novel miR genes. In all, 153 known, 16 novel and 170 candidate novel mature miRNAs and miRNA-star strands were only expressed in ALL, whereas 140 known, 2 novel and 82 candidate novel mature miRNAs and miRNA-star strands were unique to normal hematopoietic cells. Stem-loop reverse transcriptase (RT)-quantitative PCR analyses confirmed the differential expression of selected mature miRNAs in ALL types and normal cells. Expression of 14 new miRNAs inversely correlated with expression of predicted target genes (-0.49 ≤ Spearman's correlation coefficients (Rs)≤ -0.27, P ≤ 0.05); among others, low levels of novel sol-miR-23 associated with high levels of its predicted (antiapoptotic) target BCL2 (B-cell lymphoma 2) in precursor B-ALL (Rs -0.36, P = 0.007). The identification of >1000 miR genes expressed in different types of ALL forms a comprehensive repository for further functional studies that address the role of miRNAs in the biology of ALL.

Hypermethylation of specific microRNA genes in MLL-rearranged infant acute lymphoblastic leukemia: major matters at a micro scale.

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is the most aggressive type of childhood leukemia. To develop more suitable treatment strategies, a firm understanding of the biology underlying this disease is of utmost importance. MLL-rearranged ALL displays a unique gene expression profile, partly explained by erroneous histone modifications. We recently showed that t(4;11)-positive infant ALL is also characterized by pronounced promoter CpG hypermethylation. In this study, we investigated whether this widespread hypermethylation also affected microRNA (miRNA) expression. We identified 11 miRNAs that were downregulated in t(4;11)-positive infant ALL as a consequence of CpG hypermethylation. Seven of these miRNAs were re-activated after exposure to the de-methylating agent Zebularine. Interestingly, five of these miRNAs are associated either with MLL or MLL fusions, and for miR-152 we found both MLL and DNA methyltransferase 1 (DNMT1) as potential targeted genes. Finally, a high degree of methylation of the miR-152 CpG island was strongly correlated with a poor clinical outcome. Our data suggests that inhibitors of methylation have a potential beyond re-expression of hypermethylated protein-coding genes in t(4;11)-positive infant ALL. In this study, we provide additional evidence that they should be tested for their efficacy in MLL-rearranged infant ALL in in vivo models.

High BRE expression in pediatric MLL-rearranged AML is associated with favorable outcome.

Translocations involving the mixed lineage leukemia (MLL) gene, localized at 11q23, frequently occur in pediatric acute myeloid leukemia (AML). We recently reported differences in prognosis between the different translocation partners, suggesting differences in biological background. To unravel the latter, we used microarrays to generate gene expression profiles of 245 pediatric AML cases, including 53 MLL-rearranged cases. Thereby, we identified a specific gene expression signature for t(9;11)(p22;q23), and identified BRE (brain and reproductive organ expressed) to be discriminative for t(9;11)(p22;q23) (P<0.001) when compared with other MLL subtypes. Patients with high BRE expression showed a significantly better 3-year relapse-free survival (pRFS) (80±13 vs 30±10%, P=0.02) within MLL-rearranged AML cases. Moreover, multivariate analysis identified high BRE expression as an independent favorable prognostic factor within pediatric AML for RFS (HR=0.2, P=0.04). No significant differences were identified for 3-year event-free survival or for 3-year overall survival. Forced expression of BRE did not result in altered cell proliferation, apoptosis or drug sensitivity, which could explain the favorable outcome. In conclusion, overexpression of the BRE gene is predominantly found in MLL-rearranged AML with t(9;11)(p22;q23). Although further investigation for the role of BRE in leukemogenesis and outcome is warranted, high BRE expression is an independent prognostic factor for pRFS in pediatric AML.