Amanda Larson Gedman

Data-driven phenotypic dissection of AML reveals progenitor-like cells that correlate with prognosis

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.

The landscape of somatic mutations in epigenetic regulators across 1,000 paediatric cancer genomes

Studies of paediatric cancers have shown a high frequency of mutation across epigenetic regulators. Here we sequence 633 genes, encoding the majority of known epigenetic regulatory proteins, in over 1,000 paediatric tumours to define the landscape of somatic mutations in epigenetic regulators in paediatric cancer. Our results demonstrate a marked variation in the frequency of gene mutations across 21 different paediatric cancer subtypes, with the highest frequency of mutations detected in high-grade gliomas, T-lineage acute lymphoblastic leukaemia and 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. We identify novel loss-of-function mutations in the ubiquitin-specific processing protease 7 (USP7) in paediatric leukaemia, which result in decreased deubiquitination activity. Collectively, our results help to define the landscape of mutations in epigenetic regulatory genes in paediatric cancer and yield a valuable new database for investigating the role of epigenetic dysregulations in cancer.

The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.

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 cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has 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 this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10−5) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

An Inv(16)(p13.3q24.3)-Encoded CBFA2T3-GLIS2 Fusion Protein Defines an Aggressive Subtype of Pediatric Acute Megakaryoblastic Leukemia

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic 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 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.

The genomic landscape of core-binding factor acute myeloid leukemias

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving 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 known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.

Abstract A15: The landscape of somatic mutations in the core-binding factor acute myeloid leukemias

Acute myeloid leukemia (AML) results from mutations that promote aberrant self-renewal, proliferation and differentiation. The specific combination of mutations not only influences the phenotype of the leukemia but also its response to therapy. The core-binding factor (CBF) leukemias represent one of the most common forms of AML and are defined by genetic lesions that target the RUNX1(AML1)/CBFβ transcription factor complex. These include t(8;21)[RUNX1-ETO], inv(16) and t(16;16)[CBFβ-MYH11], point mutations in RUNX1, and several other rarer translocations that target RUNX1. The AML1/CBFβ transcription factor complex functions as a master transcriptional regulator that is essential for the generation of definitive hematopoietic stem cells during development, and plays an important role in the differentiation of a number of hematopoietic lineages including T cells and megakaryocytes. Using a conditional Runx1-Eto murine knock-in model, we previously demonstrated that the expression of Runx1-Eto was insufficient to induce leukemia. To define the landscape of mutation that cooperate with RUNX1-ETO in leukemogenesis, the St. Jude Children9s Research Hospital - Washington University Pediatric Cancer Genome Project performed whole genome DNA Sequencing (WGS) on a cohort of 17 pediatric CBF leukemia [7 t(8;21) and 10 inv(16) cases. Somatic mutations were identified using the PCGP analytical pipeline and all identified mutations were experimentally validated. The frequency of the identified mutations were then assessed by performing exome sequencing on a validation cohort consisting of 151 pediatric and adult CBF leukemias [79 t(8;21) and 72 inv(16)]. The combined analyses identified very few copy number alterations (CNAs) or structural alterations per case, and an average of only 12.5 somatic non-synonymous single nucleotide variations or insertions/deletions per case. Despite the low burden of mutations, recurrent activating mutations in the Kinase/RAS signaling pathway (NRAS, KIT, FLT3, KRAS, and PTPN11, and inactivating mutations in NF1) were collectively identified in >67% of cases An even more striking result was the identification of a high frequency of loss-of-function mutations in genes that encode key epigenetic regulatory proteins (28% including ASXL2, EZH2, UTX, SETD2, MLL2/3), and missense and loss of function mutations in genes that encode components of the cohesin complex (10% including SMC1A, SMC2, RAD21). Moreover, the frequency of both classes of mutations were significantly higher in the t(8;21) versus inv(16) containing cases (epigenetic regulatory mutations 48% vs 6.25% and cohesin mutations 20% vs 0%). No significant difference was noted in the frequency of cohesin mutations between pediatric and adult cases. The identified mutations targeted genes encoding core components of the cohesion complex required to generate the ring shaped DNA binding structure, SMC1A, SMC3, and RAD21, as well as a single case with a mutation in the SMC3 acetyl transferase ESCO2. In addition, loss of the X chromosome is a frequent somatic event in female patients with t(8;21) leukemia and SMC1A is located on Chr. X and is not subjected to X inactivation, suggesting that loss of Chr. X leads to haploinsufficiency. The majority of leukemias with cohesion mutations also contain activating mutation in the RAS/Kinase signaling pathway, suggesting that these mutations cooperate with AML1-ETO to induce leukemia. Collectively, these data provide an unprecedented view of the range of cooperating mutations that occur in the CBF leukemias and highlight fundamental differences between t(8;21) and inv(16) containing cases. Citation Format: Amanda Larson Gedman, Jinjun Cheng, Xiang Chen, Ina Radtke, Anna Andersson, Heather Mulder, Kristy Boggs, Bhavin Vadodaria, Donald Yergeau, Joy Nakitandwe, Lars Bullinger, Michael Kuehn, Hartmut Doehner, Konstanze Doehner, Sheila Shurtleff, John Easton, Richard Wilson, Jinghui Zhang, James R. Downing. The landscape of somatic mutations in the core-binding factor acute myeloid leukemias. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A15.

Abstract 4116: Notch1 regulates AKT-T308 dephosphorylation through modulation of the PP2A phosphatase in GSI-resistant T-cell acute lymphoblastic leukemia (T-ALL) cells.

Pediatric T-ALL typically has a poorer prognosis than B-precursor ALL, as T-ALL patients exhibit 5-event free survival rates approximating 70-75% compared to > 90% for B-precursor-ALL. Notch1 activating mutations occur in more than 50% of T-ALL cases. Notch1 mutations cluster within the heterodimerization (HD) and/or PEST domains and liberate activated intracellular Notch 1 (ICN1) by increasing susceptibility to HD domain cleavage by gamma-secretase (GS) and/or by increasing the half-life of ICN1, respectively. This results in increased expression of ICN1 target genes including hes1, deltex1, cmyc, igf1r, calcineurin, e2a, and il7r, which affect cell proliferation and survival at least in part by activating AKT. Notch1 increases Hes1 expression and transcriptional silencing of PTEN which antagonizes AKT activation. Mutational loss of PTEN, which occurs frequently in T-ALL, renders cells resistant to Notch1 inhibition with GS inhibitors (GSIs). This suggests that Notch1 favors AKT activation. However, Notch1 can exert inhibitory control over AKT signaling even in GSI-resistant, PTEN-null Jurkat cells, which have mutant activated Notch1. We found that both GSI treatment and Notch1 knockdown (N1KD) in Jurkat cells increased AKT activation loop (T308) phosphorylation and signaling, and protected cells from induction of apoptosis. This was not due to increased AKT phosphorylation by PI3K. Rather this was due to decreased dephosphorylation of AKT-T308 in the N1KD cells. PP2A is the major Ser/Thr phosphatase that dephosphorylates AKT at T308. Treatment of cells with the PP2A inhibitor okadaic acid increased AKT-T308 phosphorylation in non-targeted control (NTC) cells but had no overt effect in the N1KD cells, suggesting decreased PP2A activity. Notch1 primarily functions as a transcriptional regulator and could affect genes encoding the PP2A catalytic or regulatory subunits. However, neither the levels of the individual subunits nor PP2A catalytic activity were changed in N1KD cells. By immunoprecipitation, N1KD cells showed a decreased interaction between PP2A and AKT. This was accompanied by increased phosphorylation of AKT-T308 but also of other PP2A targets including AMPK and p70S6K. Increased phosphorylation of these targets also resulted from transient transfection with a dominant-negative MAML which interferes with ICN1 transcriptional effects. This was not mediated by cMyc, as established by cMyc transfections and treatment with a cMyc inhibitor. Conversely, transfection with Hes1 decreased phosphorylation of these PP2A targets in the N1KD cells. This suggests a causal role for Hes1, at least in part, in the Notch1 effects on PP2A and AKT-T308 phosphorylation. To our knowledge, these effects of Notch1 and Hes1 on PP2A and their impact on AKT and AMPK signaling have not been previously described. Citation Format: Eric Christopher Hales, Steven M. Orr, Amanda Larson Gedman, Jeffrey W. Taub, Larry H. Matherly. Notch1 regulates AKT-T308 dephosphorylation through modulation of the PP2A phosphatase in GSI-resistant T-cell acute lymphoblastic leukemia (T-ALL) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4116. doi:10.1158/1538-7445.AM2013-4116

Abstract 2226: Mechanisms for cross-talk between Notch1 and AKT signaling in T-cell acute lymphoblastic leukemia

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) has a variable prognosis, with many patients exhibiting high-risk features at diagnosis, including older age and high presenting white blood cell counts. Activating mutations involving the Notch1 heterodimerizeration and PEST domains occur in more than 50% of T-ALL cases. Increased Notch1 activity promotes survival and proliferation of leukemic cells, in part by transcriptionally repressing PTEN (via HES1). This provides compelling rationale for the use of gamma secretase inhibitors (GSIs) to block Notch1 activation and to restore PTEN levels, which antagonize AKT signaling. Further, PTEN mutations are implicated in GSI resistance. PTEN activity is also modulated by an assortment of posttranslational modifications and Notch1 may regulate PTEN at the posttranslational level. To investigate the impact of Notch1 on AKT signaling, independent of its transcriptional regulation of PTEN, we used PTEN-null Jurkat cells with constitutively active Notch1 and conditional ectopic PTEN expression under control of a tetracycline (tet)-inducible promoter. Induction of PTEN was accompanied by increased phosphorylation of AKTT308, increased insulin-like growth factor 1 receptor (IGF1R) protein and transcripts, and increased Notch1. Increased AKTT308 phosphorylation was blocked with an IGF1R inhibitor (GSK1904259A) and was abolished in Jurkat cells expressing a tet-inducible G129R PTEN phosphatase-inactive mutant. This suggests that PTEN can activate AKT signaling by modulating IGF1R levels. In Jurkat cells, inhibition of Notch1, either through GSI treatment or shRNA knockdown (KD), resulted in increased phosphorylation of AKTT308. Although this result could involve Notch1-dependent decreased cMyc, reflecting indirect effects of cMyc on mTOR1 and negative feedback on AKT, this was not impacted by treatment with a cMyc inhibitor (10058-F4) or by ectopic overexpression of cMyc, suggesting that other effectors and mechanisms are involved. Microarray analysis was performed on wild-type and KD Jurkat cells. Using a 1.5-fold cutoff, 1208 differentially regulated genes were identified. These include PH domain leucine-rich repeat protein phosphatase-1 (PHLPP1) and prokineticin-2 (PROK2), both of which could significantly impact AKT phosphorylation in response to Notch1. By real-time RT-PCR, PHLPP1 was significantly decreased (∼4-fold), and PROK2 was significantly increased (∼6-fold) in Notch1 KD cells compared to wild-type cells. Studies are underway to characterize the potential roles of PHLPP1 and PROK2 in mediating the inhibitory effects of Notch1 on AKT activation in T-ALL. Better understanding of the cross-talk between Notch1 and AKT in T-ALL may identify rational therapeutic strategies for combining GSIs with inhibitors of AKT, mTOR, or IGF1R, and/or for designing therapies based on capacities for constitutive Notch1 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2226. doi:1538-7445.AM2012-2226

Abstract 4869: Whole genome sequence analysis of MLL rearranged infant acute lymphoblastic leukemias reveals remarkably few somatic mutations: A Report From the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project

Infant acute lymphoblastic leukemia (ALL) is characterized by MLL rearrangements (MLLr) and poor prognosis. To determine the complement of somatic mutations in this high risk leukemia, we performed whole genome sequencing (WGS) on 22 infants with MLL rearranged ALL. An analysis of the structure of the MLLr revealed that over half had complex rearrangements involving either three or more chromosomes, carried cryptic rearrangements, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In three cases, genetic rearrangements were predicted to generate in addition to the MLL-partner gene fusion, novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B, AFF1-RAD51B-MLL; MLLT10-ATP5L-YPEL4 / ATP5L-YPEL4. An analysis of the number of non-silent mutations revealed infant ALL to have the lowest frequency of somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLLr, a mean of only 3.5 SVs and 2.2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case. Despite the paucity of mutations several pathways were recurrently targeted including PI3K/RAS pathway in 45% (KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and ARHGAP32 (p200Rho/GAP)), B cell differentiation in 23% as a result of mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2. WGS of two infant ALL relapse samples and comparison with their matched diagnostic samples revealed a marked increase in the number of mutations at relapse. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone. Because of the exceedingly low number of mutations detected in infant ALL, we used exome sequencing to determine the frequency of non-silent SNVs in 20 MLLr leukemias (9 ALLs, 10 AMLs and 1 AUL) in older children (7-19 yrs). This analysis revealed that non-infant pediatric MLL leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 7.4/case in older patients vs 2.2/case in infants, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4869. doi:1538-7445.AM2012-4869

Abstract 4867: Identification of an inv(16)-encoded CBFA2T3-GLIS2 fusion protein in 34% of non-infant acute megkaryoblastic leukemias: A report from the Pediatric Cancer Genome Project

Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with Down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4867. doi:1538-7445.AM2012-4867