SUMMARY Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R) and 20 older children (MLL-R cases) with leukemia. Our data demonstrated infant MLL-R ALL to have one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite the paucity of mutations, activating mutations in kinase/PI3K/RAS signaling pathways were detected in 47%. Surprisingly, however, these mutations were often sub-clonal and frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (a mean of 6.5/case versus 1.3/case, P=7.15×10−5) and contained frequent mutations (45%) in epigenetic regulators, a category of genes that with the exception of MLL was rarely mutated in infant MLL-R ALL.

SUMMARY To define the mutation spectrum in non-Down syndrome acute megkaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL leukemia samples. Our analysis identified a cryptic chromosome 16 inversion [inv(16)(p13.3q24.3)] in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling, and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Here we sequence 633 genes, encoding the majority of known epigenetic regulatory proteins, in over 1000 pediatric tumors to define the landscape of somatic mutations in epigenetic regulators in pediatric cancer. Our results demonstrate a marked variation in the frequency of gene mutations across 21 different pediatric cancer subtypes, with the highest frequency of mutations detected in high-grade gliomas, T-lineage acute lymphoblastic leukemia, medulloblastoma, and a paucity of mutations in low-grade glioma, and retinoblastoma. The most frequently mutated genes are H3F3A, PHF6, ATRX, KDM6A, SMARCA4, ASXL2, CREBBP, EZH2, MLL2, USP7, ASXL1, NSD2, SETD2, SMC1A, and ZMYM3. Importantly, we identify novel loss-of-function mutations in the ubiquitin-specific-processing protease 7 (USP7) in pediatric leukemia, which result in a decrease in deubiquitination activity. Collectively, our results help to define the landscape of mutations in epigenetic regulatory genes in pediatric cancer and yield a valuable new database for investigating the role of epigenetic dysregulations in cancer.

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving subunits of the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n=87) and adult (n=78) samples, including cases with RUNX1-RUNX1T1 (n=85) or CBFB-MYH11 (n=80) rearrangements, by whole-genome or whole-exome sequencing. In addition to previously reported somatic mutations in the Ras signaling pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a recurrent and previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated a remarkably different spectrum of cooperating mutations as RUNX1-RUNX1T1 cases harbored recurrent somatic mutations in DHX15 and ZBTB7A, as well as an enrichment of somatic mutations in epigenetic regulators, including ASXL2, and in components of the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscape of cooperating mutations between these related AML subtypes.

We explored the impact of mutations in the NOTCH1, FBW7 and PTEN genes on prognosis and downstream signaling in a well-defined cohort of 47 pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. In T-ALL lymphoblasts, we identified high frequency mutations in NOTCH1 (n=16), FBW7 (n=5) and PTEN (n=26). NOTCH1 mutations resulted in 1.3-3.3-fold increased transactivation of a HES1 reporter construct over wild-type NOTCH1; mutant FBW7 resulted in further augmentation of reporter gene activity. NOTCH1 and FBW7 mutations were accompanied by increased median transcripts for NOTCH1 target genes (HES1, DELTEX1, cMYC). However, none of these mutations were associated with treatment outcome. Elevated HES1, DELTEX1 and cMYC transcripts were associated with significant increases in transcript levels of several chemotherapy relevant genes, including MDR1, ABCC5, reduced folate carrier, asparagine synthetase, thiopurine methyltranserase, Bcl-2 and dihydrofolate reductase. PTEN transcripts positively correlated with HES1 and cMYC transcript levels. Our results suggest that (1) multiple factors should be considered with attempting to identify molecular-based prognostic factors for pediatric T-ALL, and (2) depending on the NOTCH1 signaling status, modifications in the types or dosing of standard chemotherapy drugs for T-ALL, or combinations of agents capable of targeting NOTCH1, AKT and/or mTOR with standard chemotherapy agents may be warranted.

NOTCH1 is activated by mutation in more than 50% of human T-cell acute lymphoblastic leukemias (T-ALLs) and inhibition of Notch signaling causes cell-cycle/ growth arrest, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor phosphatase and tensin homolog (PTEN) is also mutated or lost in up to 20% of cases. It was recently observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition, raising concern that patients with PTEN-negative disease may fail Notch inhibitor therapy. As these studies were limited to established cell lines, we addressed this issue using a genetically defined mouse retroviral transduction/bone marrow transplantation model and observed primary murine leukemias to remain dependent on NOTCH1 signaling despite Pten loss, with or without additional deletion of p16 Ink4a /p19 Arf IntroductionThe 4 mammalian Notch genes (NOTCH1-4) encode a family of highly conserved type I transmembrane receptors that are normally activated by ligands of the Delta/Serrate/Lag-2 family expressed on the surface of neighboring cells. Once activated by ligand, the Notch receptors undergo proteolytic cleavage first by a disintegrin and metalloprotease, then by a ␥-secretase, which releases the intracellular domain (ICN) from the plasma membrane to translocate to the nucleus to stimulate transcription of downstream target genes in complex with the DNA-binding factor CBF1(RBPJ)/ suppressor of hairless/Lag-1 and coactivators of the Mastermind family. Although regulated NOTCH1 signaling is important for normal T-cell development, 1 it is frequently activated by mutation in the human cancer T-cell acute lymphoblastic leukemia (T-ALL). 2 The potent oncogenicity of activated NOTCH1 has been demonstrated in murine bone marrow transduction/transplantation models and several transgenic mouse lines. 3 Activating NOTCH1 mutations occur in more than 50% of primary human T-ALLs and cluster in the heterodimerization (HD) and C-terminal proline-, glutamic acid-, serine-, and threonine-rich (PEST) domains. 4 HD mutations result in weakened association or complete dissociation of the receptor subunits, and thus lead to heightened/constitutive activation of the receptor. 5 PEST domain mutations often generate premature stop codons that delete the PEST degron, and thus enhance signaling by reducing turnover/ prolonging half-life of activated ICN. 6 When present together, the HD and PEST mutations occur in cis, and stimulate signaling in a synergistic fashion. 4 Interestingly, a similar overall frequency of Notch1 mutations (mostly PEST, but some HD) has been observed in various mouse models of T-ALL, underscoring the importance of Notch1 signaling in T-cell leukemogenesis. In addition, both human and murine T-ALL cells bearing NOTCH1 mutations are frequently sensitive to treatment with inhibitors of Notch signaling including ␥-secretase inhibitors (GSIs) that induce G1 cell-cycle/ growth arrest and in some cases apoptosis. 4,7-10 Based on these findings, GSIs and other inhi...

Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts that are predicted to be pathologic including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI, and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2 which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain of function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor (MEP) gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathologic drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.