Key Points• CN/AML patients have a high frequency of CSF3R and RUNX1 mutations. • CSF3R and RUNX1 mutations induce elevated proliferation of CD34 1 cells.Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a 20% risk of evolving into leukemia or myelodysplastic syndrome (MDS). Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R mutations. In contrast to their high frequency in CN patients who developed leukemia or MDS, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric acute myeloid leukemia. A sequential analysis at stages prior to overt leukemia revealed RUNX1 mutations to be late events in leukemic transformation. Single-cell analyses in 2 patients showed that RUNX1 and CSF3R mutations were present in the same malignant clone. Functional studies demonstrated elevated granulocyte colony-stimulating factor (G-CSF)-induced proliferation with diminished myeloid differentiation of hematopoietic CD34 1 cells coexpressing mutated forms of RUNX1 and CSF3R. The high frequency of cooperating RUNX1 and CSF3R mutations in CN patients suggests a novel molecular pathway of leukemogenesis: mutations in the hematopoietic cytokine receptor (G-CSFR) in combination with the second mutations in the downstream hematopoietic transcription fator (RUNX1). The detection of both RUNX1 and CSF3R mutations could be used as a marker for identifying CN patients with a high risk of progressing to leukemia or MDS. (Blood. 2014;123(14):2229-2237

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.

MicroRNAs (miRNAs) are crucial components of homeostatic and developmental gene regulation. In turn, dysregulation of miRNA expression is a common feature of different types of cancer, which can be harnessed therapeutically. Here we identify miR-139-5p suppression across several cytogenetically defined acute myeloid leukemia (AML) subgroups. The promoter of mir-139 was transcriptionally silenced and could be reactivated by histone deacetylase inhibitors in a dose-dependent manner. Restoration of mir-139 expression in cell lines representing the major AML subgroups (t[8;21], inv[16], mixed lineage leukemia-rearranged and complex karyotype AML) caused cell cycle arrest and apoptosis in vitro and in xenograft mouse models in vivo. During normal hematopoiesis, mir-139 is exclusively expressed in terminally differentiated neutrophils and macrophages. Ectopic expression of mir-139 repressed proliferation of normal CD34(+)-hematopoietic stem and progenitor cells and perturbed myelomonocytic in vitro differentiation. Mechanistically, mir-139 exerts its effects by repressing the translation initiation factor EIF4G2, thereby reducing overall protein synthesis while specifically inducing the translation of cell cycle inhibitor p27(Kip1). Knockdown of EIF4G2 recapitulated the effects of mir-139, whereas restoring EIF4G2 expression rescued the mir-139 phenotype. Moreover, elevated miR-139-5p expression is associated with a favorable outcome in a cohort of 165 pediatric patients with AML. Thus, mir-139 acts as a global tumor suppressor-miR in AML by controlling protein translation. As AML cells are dependent on high protein synthesis rates controlling the expression of mir-139 constitutes a novel path for the treatment of AML.

The most important reason for therapy failure in pediatric acute myeloid leukemia (AML) is relapse. In order to identify miRNAs that contribute to the clonal evolution towards relapse in pediatric AML, miRNA expression profiling of 127 de novo pediatric AML cases were used. In the diagnostic phase, no miRNA signatures could be identified that were predictive for relapse occurrence, in a large pediatric cohort, nor in a nested mixed lineage leukemia (MLL)-rearranged pediatric cohort. AML with MLL- rearrangements are found in 15-20% of all pediatric AML samples, and reveal a relapse rate up to 50% for certain translocation partner subgroups. Therefore, microRNA expression profiling of six paired initial diagnosis-relapse MLL-rearranged pediatric AML samples (test cohort) and additional eight paired initial diagnosis-relapse samples with MLL-rearrangements (validation cohort) was performed. A list of 53 differentially expressed miRNAs was identified of which the miR-106b~25 cluster, located in intron 13 of MCM7, was the most prominent. These differentially expressed miRNAs however could not predict a relapse in de novo AML samples with MLL-rearrangements at diagnosis. Furthermore, higher mRNA expression of both MCM7 and its upstream regulator E2F1 was found in relapse samples with MLL-rearrangements. In conclusion, we identified the miR-106b~25 cluster to be upregulated in relapse pediatric AML with MLL-rearrangements.

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease with respect to biology as well as outcome. In this study, we investigated whether known biological subgroups of pediatric AML are reflected by a common microRNA (miRNA) expression pattern. We assayed 665 miRNAs on 165 pediatric AML samples. First, unsupervised clustering was performed to identify patient clusters with common miRNA expression profiles. Our analysis unraveled 14 clusters, seven of which had a known (cyto-)genetic denominator. Finally, a robust classifier was constructed to discriminate six molecular aberration groups: 11q23-rearrangements, t(8;21)(q22;q22), inv(16)(p13q22), t(15;17) (q21;q22), NPM1 and CEBPA mutations. The classifier achieved accuracies of 89%, 95%, 95%, 98%, 91% and 96%, respectively. Although lower sensitivities were obtained for the NPM1 and CEBPA (32% and 66%), relatively high sensitivities (84%−94%) were attained for the rest. Specificity was high in all groups (87%−100%). Due to a robust double-loop cross validation procedure employed, the classifier only employed 47 miRNAs to achieve the aforementioned accuracies. To validate the 47 miRNA signatures, we applied them to a publicly available adult AML dataset. Albeit partial overlap of the array platforms and molecular differences between pediatric and adult AML, the signatures performed reasonably well. This corroborates our claim that the identified miRNA signatures are not dominated by sample size bias in the pediatric AML dataset. In conclusion, cytogenetic subtypes of pediatric AML have distinct miRNA expression patterns. Reproducibility of the miRNA signatures in adult dataset suggests that the respective aberrations have a similar biology both in pediatric and adult AML.

Acute myeloid leukemia (AML) accounts for approximately 15-20% of all childhood leukemias, and despite dramatic improvements in treatment outcome, only approximately 70% of children with AML are cured.

Congenital neutropenia (CN) is a rare inherited disorder of hematopoiesis with a 20% risk of evolving into acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Using next-generation sequencing in 31 CN patients who developed leukemia we found that 20 of the 31 patients (64.5%) had mutations in RUNX1 (runt-related transcription factor 1). Of these 20 patients, 19 had inherited mutations associated with CN. Intriguingly, the majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R (colony stimulating factor 3 receptor) mutations. Other leukemia-associated mutations (EP300, FLT3-ITD, CBL, and SUZ12) were less frequent. In eight patients, we detected two distinct heterozygous RUNX1 mutations. These mutations were localized to the splice-acceptor site of intron 4, affecting splicing of exons 3 and 4, which encode the Runt homology/DNA binding domain (RHD) of RUNX1, or solely in the RHD or were present in both RHD and trans-activation domain (TAD). In two patients, we were able to perform allele-specific analysis of RUNX1 mutations. Patient #10 had an Phe13TrpfsX14 deletion on one allele of RUNX1 and an Arg139ProfsX47 deletion on the other allele. In Patient #14, two RUNX1 mutations were on the same allele; one of the mutations (Met240Ile) was inherited from the mother and was localized two amino acids before the TAD, and the second acquired mutation (Arg139Gly) was in the RHD of RUNX1. Ten patients with RUNX1 mutations developed monosomy 7 and six patients developed trisomy 21 at diagnosis of leukemia. In contrast to their high frequency in CN evolving into AML, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric AML. RUNX1 mutations were mainly found in pediatric AML patients with an adverse prognosis. A sequential analysis at stages prior to overt leukemia in ten CN/AML patients showed that RUNX1 mutation is a late event in leukemogenic transformation. In 6 of 10 patients, a CSF3R mutation occurred prior to RUNX1 mutations (24-192 months prior to CN/AML for CSF3R mutations vs. 1-36 months prior to CN/AML for RUNX1 mutations). Interestingly, monosomy 7 or trisomy 21 appeared after acquisition of RUNX1 mutations and no additional chromosomal aberrations were detected by array-CGH. Single-cell analyses in two patients revealed that RUNX1 and CSF3R mutations were segregated in the same malignant clone. Moreover, functional studies demonstrated elevated G-CSF-induced proliferation with diminished myeloid differentiation of hematopoietic CD34+ cells after co-transduction with mutated RUNX1 and CSF3R, in comparison to cells transduced with mutated RUNX1 or mutated CSF3R only. The importance of RUNX1 mutations in leukemogenic transformation was substantially strengthened by the analysis of a unique family with two siblings suffering from CN that subsequently transformed to AML. In both children, cooperating RUNX1 and CSF3Rmutations were detected that were not present in healthy family members. Taken together, the high frequency and the time course of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia suggests a unique molecular pathway of leukemogenesis similar as has been reported in the Gilliland-Griffin two-hit hypothesis for AML development. The concomitant detection of RUNX1 and CSF3Rmutations represents a useful biomarker for identifying CN patients with a high risk of progressing to leukemia or MDS. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

S.E. and J.K.K. as well as J.H.K. and M.M.H.E. contributed equally to this study. MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and in the development of leukemia. In addition, modulation of miRNA expression can be exploited therapeutically. To identify tumor suppressive miRNAs in pediatric acute myeloid leukemia (AML), we performed a large-scale miRNA expression profiling in 90 cytogenetically characterized, de novo AML cases using a RT-qPCR platform. In total, 253 miRNAs were significantly differentially expressed between patients with MLL rearrangements, t(8;21), inv(16), t(7;12), and t(15;17). Hierarchical clustering of patient samples using these sets of miRNA values showed that t(15;17) samples clearly cluster away from the other pediatric AML samples while t(7;12) patients cluster closely to core binding factor AMLs, (t(8;21) and inv(16). These three groups largely cluster away from the majority of MLL rearranged samples. Eight miRNAs specifically downregulated in MLL rearranged, t(8;21) and inv(16) AMLs were functionally evaluated in vitro using three cell lines representing those cytogenetic groups: THP-1 (MLL rearranged), KASUMI-1 (t[8;21]) and ME-1 (inv[16]). Two of two miRNAs tested in KASUMI-1 cells (miR-9 and miR-582), two of three miRNAs tested in ME-1 (miR-192/194 bicistron and miR-660) and one of three miRNAs tested in THP-1 (miR-181a/b bicistron) reduced cell growth and colony-forming capacity upon ectopic expression. In KASUMI-1 cells, one miRNA was identified, miR-9, that not only reduced cell growth and colony forming capacity but also strongly induced monocytic differentiation in concert with calcitriol without affecting apoptosis. During normal hematopoiesis miR-9 is only expressed in macrophages.The effects on cell growth, colony-forming capacity and differentiation were confirmed in a second AML cell line with t(8;21), SKNO-1. The differentiation induction was restricted to t(8;21) leukemic cell lines, while its growth inhibitory function was also evident in normal CD34+ hematopoietic stem and progenitor cells. Most strikingly, miR-9 exerted a tumor suppressive function in primary leukemic blasts from patients with t(8;21) (n=2), but not in patients with MLL rearrangements (n=3). Using global gene expression studies upon ectopic miR-9 expression, we identified and validated LIN28B and HMGA2 as high fidelity target genes of miR-9 by RT-qPCR, western blotting and luciferase reporter assays. LIN28B is known to suppress let-7 processing. Indeed, miR-9 overexpresion increased the levels of mature let-7 family members, also leading to HMGA2 downregulation. ShRNA-mediated downregulation of LIN28B or HMGA2 partially recapitulated the effects of miR-9 on proliferation and differentiation of t(8;21) cell lines. Thus, miR-9 is a tumor suppressor-miR in t(8;21) de novo pediatric AML, that acts in a stringent cell context dependent manner in concert with let-7 family members by repressing the oncogenic LIN28B/HMGA2 axis. This work was supported by grants to J.H.K. from the German National Academic Foundation (KL-2374/2-1) and to J.K.K., L.V., A.D.vO, C.M.Z. and M.M.H.-E. from the Children Cancer Free Foundation (KIKA, project 49). Disclosures: No relevant conflicts of interest to declare.