Loss of H3K27 methylation identifies poor outcomes in adult-onset acute leukemia.

BACKGROUND: Acute leukemia is an epigenetically heterogeneous disease. The intensity of treatment is currently guided by cytogenetic and molecular genetic risk classifications; however these incompletely predict outcomes, requiring additional information for more accurate outcome predictions. We aimed to identify potential prognostic implications of epigenetic modification of histone proteins, with a focus on H3K4 and H3K27 methylation marks in relation to mutations in chromatin, splicing and transcriptional regulators in adult-onset acute lymphoblastic and myeloid leukemia. RESULTS: Histone 3 lysine 4 di- and trimethylation (H3K4me2, H3K4me3) and lysine 27 trimethylation (H3K27me3) mark expression was evaluated in 241 acute myeloid leukemia (AML), 114 B-cell acute lymphoblastic leukemia (B-ALL) and 14T-cell ALL (T-ALL) patient samples at time of diagnosis using reverse phase protein array. Expression levels of the marks were significantly lower in AML than in B and T-ALL in both bone marrow and peripheral blood, as well as compared to normal CD34+ cells. In AML, greater loss of H3K27me3 was associated with increased proliferative potential and shorter overall survival in the whole patient population, as well as in subsets with DNA methylation mutations. To study the prognostic impact of H3K27me3 in the context of cytogenetic aberrations and mutations, multivariate analysis was performed and identified lower H3K27me3 level as an independent unfavorable prognostic factor in all, as well as in TP53 mutated patients. AML with decreased H3K27me3 demonstrated an upregulated anti-apoptotic phenotype. In ALL, the relative quantity of histone methylation expression correlated with response to tyrosine kinase inhibitor in patients who carried the Philadelphia cytogenetic aberration and prior smoking behavior. CONCLUSION: This study shows that proteomic profiling of epigenetic modifications has clinical implications in acute leukemia and supports the idea that epigenetic patterns contribute to a more accurate picture of the leukemic state that complements cytogenetic and molecular genetic subgrouping. A combination of these variables may offer more accurate outcome prediction and we suggest that histone methylation mark measurement at time of diagnosis might be a suitable method to improve patient outcome prediction and subsequent treatment intensity stratification in selected subgroups.

Molecular patterns of response and treatment failure after frontline venetoclax combinations in older patients with AML.

The BCL-2 inhibitor venetoclax combined with hypomethylating agents or low-dose cytarabine represents an important new therapy for older or unfit patients with acute myeloid leukemia (AML). We analyzed 81 patients receiving these venetoclax-based combinations to identify molecular correlates of durable remission, response followed by relapse (adaptive resistance), or refractory disease (primary resistance). High response rates and durable remissions were typically associated with NPM1 or IDH2 mutations, with prolonged molecular remissions prevalent for NPM1 mutations. Primary and adaptive resistance to venetoclax-based combinations was most commonly characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS or biallelically perturbing TP53. Single-cell studies highlighted the polyclonal nature of intratumoral resistance mechanisms in some cases. Among cases that were primary refractory, we identified heterogeneous and sometimes divergent interval changes in leukemic clones within a single cycle of therapy, highlighting the dynamic and rapid occurrence of therapeutic selection in AML. In functional studies, FLT3 internal tandem duplication gain or TP53 loss conferred cross-resistance to both venetoclax and cytotoxic-based therapies. Collectively, we highlight molecular determinants of outcome with clinical relevance to patients with AML receiving venetoclax-based combination therapies.

A quantitative analysis of heterogeneities and hallmarks in acute myelogenous leukaemia.

Acute myelogenous leukaemia (AML) is associated with risk factors that are largely unknown and with a heterogeneous response to treatment. Here, we provide a comprehensive quantitative understanding of AML proteomic heterogeneities and hallmarks by using the AML Proteome Atlas, a proteomics database that we have newly derived from MetaGalaxy analyses, for the proteomic profiling of 205 patients with AML and 111 leukaemia cell lines. The analysis of the dataset revealed 154 functional patterns based on common molecular pathways, 11 constellations of correlated functional patterns and 13 signatures that stratify the outcomes of patients. We find limited overlap between proteomics data and both cytogenetics and genetic mutations. Moreover, leukaemia cell lines show limited proteomic similarities with cells from patients with AML, suggesting that a deeper focus on patient-derived samples is needed to gain disease-relevant insights. The AML Proteome Atlas provides a knowledge base for proteomic patterns in AML, a guide to leukaemia cell line selection, and a broadly applicable computational approach for quantifying the heterogeneities of protein expression and proteomic hallmarks in AML.

A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome.

Genetic changes are infrequent in acute myeloid leukemia (AML) compared with other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CGI methylator phenotype (CIMP) (A-CIMP+). A-CIMP was associated with longer overall survival (OS) in this data set (median OS, years: A-CIMP+=not reached, CIMP-=1.17; P=0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP+=2.34, A-CIMP-=1.00; P=0.01). Hypermethylation in A-CIMP+ favored CGIs (OR: CGI/non-CGI=5.21), and while A-CIMP+ was enriched in CEBPA (P=0.002) and WT1 mutations (P=0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP+ function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple data sets. We conclude that CIMP in AML cannot be explained solely by gene mutations (for example, IDH1/2, TET2), and that curability in A-CIMP+ AML should be validated prospectively.

Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD).

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

p53 pathway dysfunction is highly prevalent in acute myeloid leukemia independent of TP53 mutational status.

TP53 mutations are associated with the lowest survival rates in acute myeloid leukemia (AML). In addition to mutations, loss of p53 function can arise via aberrant expression of proteins that regulate p53 stability and function. We examined a large AML cohort using proteomics, mutational profiling and network analyses, and showed that (1) p53 stabilization is universal in mutant TP53 samples, it is frequent in samples with wild-type TP53, and in both cases portends an equally dismal prognosis; (2) the p53 negative regulator Mdm2 is frequently overexpressed in samples retaining wild-type TP53 alleles, coupled with absence of p21 expression and dismal prognosis similar to that of cases with p53 stabilization; (3) AML samples display unique patterns of p53 pathway protein expression, which segregate prognostic groups with distinct cure rates; (4) such patterns of protein activation unveil potential AML vulnerabilities that can be therapeutically exploited.

IDENTIFYING CANCER SPECIFIC METABOLIC SIGNATURES USING CONSTRAINT-BASED MODELS.

Cancer metabolism differs remarkably from the metabolism of healthy surrounding tissues, and it is extremely heterogeneous across cancer types. While these metabolic differences provide promising avenues for cancer treatments, much work remains to be done in understanding how metabolism is rewired in malignant tissues. To that end, constraint-based models provide a powerful computational tool for the study of metabolism at the genome scale. To generate meaningful predictions, however, these generalized human models must first be tailored for specific cell or tissue sub-types. Here we first present two improved algorithms for (1) the generation of these context-specific metabolic models based on omics data, and (2) Monte-Carlo sampling of the metabolic model ux space. By applying these methods to generate and analyze context-specific metabolic models of diverse solid cancer cell line data, and primary leukemia pediatric patient biopsies, we demonstrate how the methodology presented in this study can generate insights into the rewiring differences across solid tumors and blood cancers.

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

EphB1 Suppression in Acute Myelogenous Leukemia: Regulating the DNA Damage Control System.

UNLABELLED: Loss of ephrin receptor (EphB1) expression may associate with aggressive cancer phenotypes; however, the mechanism of action remains unclear. To gain detailed insight into EphB1 function in acute myelogenous leukemia (AML), comprehensive analysis of EphB1 transcriptional regulation was conducted. In AML cells, EphB1 transcript was inversely correlated with EphB1 promoter methylation. The presence of EphB1 allowed EfnB1 ligand-mediated p53 DNA binding, leading to restoration of the DNA damage response (DDR) cascade by the activation of ATR, Chk1, p53, p21, p38, CDK1(tyr15), and Bax, and downregulation of HSP27 and Bcl2. Comparatively, reintroduction of EphB1 expression in EphB1-methylated AML cells enhanced the same cascade of ATR, Chk1, p21, and CDK1(tyr15), which consequently enforced programmed cell death. Interestingly, in pediatric AML samples, EphB1 peptide phosphorylation and mRNA expression were actively suppressed as compared with normal bone marrow, and a significant percentage of the primary AML specimens had EphB1 promoter hypermethylation. Finally, EphB1 repression associated with a poor overall survival in pediatric AML. Combined, the contribution of EphB1 to the DDR system reveals a tumor-suppressor function for EphB1 in pediatric AML. IMPLICATIONS: The tumor-suppressor function of EphB1 is clinically relevant across many malignancies, suggesting that EphB1 is an important regulator of common cancer cell transforming pathways.

Are changes in HLA Ags responsible for leukemia relapse after HLA-matched allogeneic hematopoietic SCT?

Loss of heterozygosity (LOH) has been shown to be associated with leukemia relapse after haploidentical transplantation. Whether such changes are an important cause of relapse after HLA-matched transplantation remains unclear. We retrospectively HLA-typed leukemic blasts for 71 patients with AML/myelodysplastic syndrome obtained from stored samples, and the results were compared with those obtained at diagnosis and/or before the transplant. No LOH or any other changes in HLA Ag were found in any of the samples tested post transplant as compared with pretransplant specimens. One patient had LOH in HLA class I Ag (HLA-A,-B and -C); however, these changes were present in the pretransplant sample indicating that they occurred before the transplant. We concluded that, in contrast with haploidentical transplantation, HLA loss does not have a major role as a mechanism of relapse after allogeneic transplantation with a closely HLA-matched donor.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies.

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML.

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 vs 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

A phase II open-label study of the intravenous administration of homoharringtonine in the treatment of myelodysplastic syndrome.

Homoharringtonine is an alkaloid inhibitor of protein synthesis with activity in myeloid malignancies. We report a phase II pilot study of homoharringtonine in myelodysplastic syndrome (MDS). Induction consisted of homoharringtonine at 2.5 mg/m(2) via continuous infusion for 7 days. Maintenance was given every 4 weeks. Nine patients were enrolled: five with refractory anaemia with excess blasts, two with refractory anaemia with excess blasts in transformation, one each with refractory anaemia and chronic myelomonocytic leukaemia respectively. Median age was 70 years (55-84) and 6 (66%) were male. Per International Prognostic Scoring System (IPSS) two patients were intermediate-1, five intermediate-2 and two high-risk. Median chemotherapy courses were one (1-3). One patient (11%) responded with complete haematological and cytogenetic remission after one course. Eight patients did not respond (four had stable disease, two progressed to acute leukaemia and two died during induction - from aspergillus pneumonia and intracerebral haemorrhage respectively). Grade 3/4 myelosuppression seen in 56% (5/9). Serious non-haematological toxicities included one case of grade 4 left bundle branch heart block and one grade 3 nephrotoxicity. Median time between courses was 42 days (35-72 days). In conclusion homoharringtonine might have clinical activity in some patients with MDS.

Loss of SHIP-1 protein expression in high-risk myelodysplastic syndromes is associated with miR-210 and miR-155.

The myelodysplastic syndromes (MDSs) comprise a group of disorders characterized by multistage progression from cytopenias to acute myeloid leukemia (AML). They display exaggerated apoptosis in early stages, but lose this behavior during evolution to AML. The molecular basis for loss of apoptosis is unknown. To investigate this critical event, we analyzed phosphatidylinositol (PI) 3'kinase signaling, implicated as a critical pathway of cell survival control in epithelial and hematological malignancies. PI 3'kinase activates Akt through its production of 3' phosphoinositides. In turn, the phosphoinositides are dephosphorylated by two lipid phosphatases, PTEN and SHIP-1, in myeloid cells. We studied primary MDS-enriched bone marrow cells and bone marrow sections by western blotting, immunohistochemistry, immunocytochemistry and quantitative PCR for components of the SHIP/PTEN/PI 3'kinase signaling circuit. We reported constitutively activated Akt, variable levels of PTEN and uniformly decreased SHIP-1 expression in MDS progenitor cells. Overexpression of SHIP-1, but not the phosphatase-deficient form, inhibited myeloid leukemic growth. Levels of microRNA (miR)-210 and miR-155 transcripts, which target SHIP-1, were increased in CD34(+) MDS cells compared with their normal counterparts. Direct binding of miR-210 to the 3' untranslated region of SHIP-1 was confirmed by luciferase reporter assay. Transfection of a myeloid cell line with miR-210 resulted in loss of SHIP-1 protein expression. These data suggest that miR-155 and miR-210/SHIP-1/Akt pathways could serve as clinical biomarkers for disease progression, and that miR-155 and miR-210 might serve as novel therapeutic targets in MDS.

Low expression of PP2A regulatory subunit B55α is associated with T308 phosphorylation of AKT and shorter complete remission duration in acute myeloid leukemia patients.

The regulation of protein kinase B (AKT) is a dynamic process that depends on the balance between phosphorylation by upstream kinases for activation and inactivation by dephosphorylation by protein phosphatases. Phosphorylated AKT is commonly found in acute myeloid leukemia (AML) and confers an unfavorable prognosis. Understanding the relative importance of upstream kinases and AKT phosphatase in the activation of AKT is relevant for the therapeutic targeting of this signaling axis in AML. The B55α subunit of protein phosphatase 2A (PP2A) has been implicated in AKT dephosphorylation, but its role in regulating AKT in AML is unknown. We examined B55α protein expression in blast cells derived from 511 AML patients using reverse phase protein analysis. B55α protein expression was lower in AML cells compared with normal CD34+ cells. B55α protein levels negatively correlated with threonine 308 phosphorylation levels. Low levels of B55α were associated with shorter complete remission duration, demonstrating that decreased expression is an adverse prognostic factor in AML. These findings suggest that decreased B55α expression in AML is at least partially responsible for increased AKT signaling in AML and suggests that therapeutic targeting of PP2A could counteract this.

Evidence of a role for the novel zinc-finger transcription factor ZKSCAN3 in modulating Cyclin D2 expression in multiple myeloma.

Dysregulation of cyclin D2 contributes to the pathogenesis of multiple myeloma, and can occur through translocations that activate MAF/MAFB or MMSET/FGFR3. However, cyclin D2 induction can also be seen in the absence of such translocations, such as in patients with hyperdiploid disease, through unknown mechanisms. In UniGene cluster data-mining and ECgene analysis, we found that zinc-finger with KRAB and SCAN domains 3 (ZKSCAN3), a novel transcription factor, is overrepresented in this malignancy, and three consensus ZKSCAN3 binding sites were found in the cyclin D2 promoter. Analysis of a panel of myeloma cell lines, primary patient samples and datasets from Oncomine and the Multiple Myeloma Genomics Portal (MMGP) revealed expression of ZKSCAN3 messenger RNA (mRNA) in a majority of samples. Studies of cell lines by western blotting, and of primary tissue microarrays by immunohistochemistry, showed ZKSCAN3 protein expression in a majority, and in a manner that paralleled messenger levels in cell lines. ZKSCAN3 overexpression was associated with increased gene copy number or genomic DNA gain/amplification in a subset based on analysis of data from the MMGP, and from fluorescence in situ hybridization studies of cell lines and primary samples. Overexpression of ZKSCAN3 induced cyclin D2 promoter activity in a MAF/MAFB-independent manner, and to an extent that was influenced by the number of consensus ZKSCAN3 binding sites. Moreover, ZKSCAN3 protein expression correlated with cyclin D2 levels in cell lines and primary samples, and its overexpression induced cyclin D2. Conversely, ZKSCAN3 suppression using small hairpin RNAs (shRNAs) reduced cyclin D2 levels, and, importantly, inhibited myeloma cell line proliferation. Finally, ZKSCAN3 was noted to specifically bind to oligonucleotides representing sequences from the cyclin D2 promoter, and to the endogenous promoter itself in myeloma cells. Taken together, the data support the conclusion that ZKSCAN3 induction represents a mechanism by which myeloma cells can induce cyclin D2 dysregulation, and contribute to disease pathogenesis.