ALBERT

Beschreibung: Bortezomib (BTZ) was recently evaluated in a randomized Phase 3 clinical trial which compared standard chemotherapy (cytarabine, daunorubicin, etoposide; ADE) to standard therapy with BTZ (ADEB) for de novo pediatric acute myeloid leukemia. While the study concluded that BTZ did not improve outcome overall, we examined patient subgroups benefitting from BTZ-containing chemotherapy using proteomic analyses. The proteasome inhibitor BTZ disrupts protein homeostasis and activates cytoprotective heat shock responses. We measured total heat shock factor 1 (HSF1) and phosphorylated HSF1 (HSF1-pSer326) in leukemic cells from 483 pediatric patients using Reverse Phase Protein Arrays. HSF1-pSer326 phosphorylation was significantly lower in pediatric AML compared to CD34+ non-malignant cells. We identified a strong correlation between HSF1-pSer326 expression and BTZ sensitivity. BTZ significantly improved outcome of patients with low-HSF1-pSer326 with a 5-year event-free survival of 44% (ADE) vs. 67% for low-HSF1-pSer326 treated with ADEB (P=0.019). To determine the effect of HSF1 expression on BTZ potency in vitro, cell viability with HSF1 gene variants that mimicked phosphorylated (S326A) and non-phosphorylated (S326E) HSF1-pSer326 were examined. Those with increased HSF1 phosphorylation showed clear resistance to BTZ vs. those with wild type or reduced HSF1-phosphorylation. We hypothesize that HSF1-pSer326 expression could identify patients that benefit from BTZ-containing chemotherapy.

Beschreibung: The proteasome degrades unneeded and damaged proteins. Tumor cells highly depend on increased protein production and their degradation suggesting that malignant cells with a high proliferation index will be more sensitive to proteasome inhibition. The addition of the proteasome inhibitor Bortezomib (Velcade, 'BTZ') to standard pediatric AML chemotherapy (cytarabine, daunorubicin and etoposide, 'ADE') depleted leukemia-initiating cells in a phase 2 clinical trial in pediatric AML (pedi-AML) patients. A randomized phase 3 clinical trial was then conducted by the Children's Oncology Group (COG) comparing ADE and ADE+BTZ treatment regimes in pedi-AML (AAML1031). To determine if there were specific protein expression profiles that correlated with response to BTZ-containing chemotherapy, we analyzed key components of the proteome of pedi-AML that participated in the trial using reverse phase protein arrays (RPPA). RPPA-based expression of 293 validated antibodies was tested in 500 leukemia samples and compared to expression in CD34+ samples from healthy individuals (n=30). Among all proteins, FOXO3 expression was identified as the protein with the highest influence on outcome in the ADE group. The expression of FOXO3 was prognostic for event-free survival (EFS) in both univariate (HR = 0.56, 95% CI = 0.34-0.90, P = 0.017) and multivariate (HR = 0.55, 95% CI = 0.34-0.88, P = 0.013) analysis. All patients were divided into two clusters (low and high) based on their FOXO3 expression level using median FOXO3 expression in normal CD34+. Kaplan-Meier survival analysis showed poor OS (3 year OS 65.3% vs. 73.9%, P = 0.03) and EFS (3 year EFS, 42.8% vs. 55%, P = 0.01) in low FOXO3 expressors (n=119) compared to patients with high FOXO3 expression (n=291) (fig. 1A). Notably, the poor prognostic effect of low FOXO3 for OS was seen in ADE (3 year OS 60% vs. 72.3%, P = 0.03), but not in ADE-BTZ (3 year OS 70.3% vs. 75.3%, P = 0.23) (fig. 1B). This suggests that in particular patients with low FOXO3 may be eligible candidates for BTZ-addition. To validate our findings, we performed knockdown (KD) of FOXO3 using a short hairpin approach in OCI-AML3 (p53WT) and THP-1 (p53null) cell lines. KD FOXO3 in OCI-AML3 had a short-term growth advantage compared to controls (Day 4, P = 0.004), but not KD FOXO3 THP-1 cells suggesting a role for p53 in the FOXO3 functional pathway. KD FOXO3 cells were more resistant to doxorubicin and etoposide combination therapy than controls (P = 0.04), confirming our clinical observations. Since therapeutic regimes in AML are currently shifting towards Bcl-2 inhibition by Venetoclax (ABT-199, 'ABT'), we were eager to test whether BTZ and ABT could act in synergy, and if this is related to FOXO3 expression. Single low dose BTZ and ABT did not reduce cell numbers after 3 days, but were effective when used in combination (

Beschreibung: Background: Lineage-specific gene transcription signatures between AML and ALL are recognized, but post-translational phenotype-specific protein expression profiles remain undefined. We hypothesized that functional proteomic patterns vary between AML and ALL and that the activity state of cells correlates with response to therapy within subgroups, complementing cytogenetic and molecular data. Methods: Reverse phase protein arrays (RPPA) were generated using bone marrow (BM) and peripheral blood (PB) samples from newly diagnosed B-ALL (n=114), T-ALL (n=14), and AML (n=241) adult patients admitted at the MD Anderson Cancer Center. RPPA allowed simultaneous expression measurement of 229 highly validated protein antibodies including 3 Histone 3 (H3) post-translational methylation regulatory modifications; H3K4Me2, H3K4Me3 and H3K27Me3. Results: Unsupervised clustering of histone modification protein expressions distinguished AML from ALL in freshly prepared lysates from BM (n=241) and PB (n=127) as well as when BM and PB samples were combined (fig. 1A). The ALL-enriched cluster was dominated by high H3K27Me3. Elevated H3K27Me3 levels were found in the BM derived leukemic blasts compared to PB blasts in ALL (P 〈 0.001), but not AML (P = 0.35). Trimethylation of the repressive mark H3K27 is catalyzed by the polycomb group protein Ezh2. Oncogenic gain-of-functions of Ezh2 are seen in patients with lymphoid malignancies and others have shown that mutated Ezh2 increased H3K27Me3 in B-cells which associated with tumorigenesis. H3K27Me3 and Ezh2 antibody expressions were highly correlated in another RPPA of ALL and AML we created (R2=0.49, P 〈 0.001). Profiling of methylation marks using unsupervised clustering in ALL divided patients in 2 clusters that correlated with survival (fig. 1B-C, P = 0.02). Cluster 1 (C1) with higher H3K27Me3, H3K4Me2 and H3K4Me3 was associated with better outcome. In ALL, Ph+ historically associated with poor prognosis but outcomes have improved substantially with the use of tyrosine kinase inhibitors (TKI). In our cohort, 11/26 Ph+ ALL patients were treated with TKIs and it is notable that sensitivity to TKIs correlated with cluster membership; all C1 patients (high degree of methylation) were alive after 7 years of follow-up in contrast to none of the TKI-treated Ph+ ALL patients in cluster 2 (C2, low degree of methylation) (fig. 1D, P = 0.01). Recently, TKI resistance in Ph+ ALL has been proposed to associate with smoking due to altered DNA methylation patterns caused by chemical components of cigarette smoke. Retrospectively, we identified that 2 of 11 TKI treated patients were smokers. Both had membership in C2, were resistant against TKIs and died after 1 year. Thus, 2 out of 3 resistant TKI treated Ph+ ALL were smokers compared to none of the 8 responders. We then aimed to identify proteins that are potentially downregulated by increased expression of the repressive mark H3K27Me3. Pathway enrichment analysis of 59 significant negatively correlated proteins with H3K27Me3 revealed that these are involved in tyrosine kinase activity and resistance, including Jak/STAT and PI3K/Akt signaling pathways. If these pathways are less activated in patients with high H3K27Me3, then this can partially explain the increased sensitivity to TKIs in this subgroup. Clinically, no differences were found in age, BM and PB blast counts between TKI-treated C1 and C2 patients to provide an explanation for the higher death rate in C2. Conclusion: ALL and AML share some pathophysiology and the identification of differences in the functional activity of cells may contribute to a better understanding of the etiology of both diseases. Here we report that high H3K27Me3 protein levels in BM and PB distinguish ALL from AML and are related to TKI sensitivity in Ph+ ALL. Histone methylation status defines a group of Ph+ ALL patients that does not benefit from the addition of TKI therapy. The idea that smoking alters the epigenetic machinery in TKI resistant Ph+ ALL has been proposed and warrants further investigation. Fig. 1 A) Heatmap showing histone methylation levels in BM and PB from AML and ALL patients. B) Heatmap showing histone methylation levels in ALL BM and PB. Unsupervised clustering divided samples into 2 clusters. C) ALL patients in C1 survived longer than patients in C2 (P = 0.02). D) Increased long-term sensitivity for TKI therapy in C1 Ph+ ALL patients compared to C2 (100 vs. 0%, P = 0.01). Figure.1 Disclosures Jabbour: AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Adaptive: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Amgen: Consultancy, Research Funding.

Beschreibung: Background: Dysregulation of histone modifying marks and their modulators leads to aberrant gene expression and could contribute to leukemogenesis via misregulation of gene transcription of tumor suppressor genes and oncogenes. Although understanding of the role of the epigenome in cancer has expanded greatly, until recently there has not been a comprehensive characterization of protein expression patterns for multiple histone modifications, histone modification-related proteins (HistModProt), or their association with clinical characteristics in AML. We recently demonstrated that adult AML HistModProt form recurrent patterns of expression that predict prognosis (van Dijk et al. 2018). We now seek to determine whether HistModProt expression can predict prognosis in pediatric AML. Methods: We simultaneously analyzed expression of 7 histone modification marks (H3 core protein, H3K4me2 (2 antibodies), H3K4me3, H3K9me2, H3K27me3, H3K36me3) and 17 HistModProt (ASH2L, BMI1, BRD4, NCL, CLPP, HDAC1, HDAC2, HDAC3, HDAC6, hnRNPK, JMJD6, KDM1A, NPM1 (2 antibodies), SIRT1, SIRT6, WTAP) in 505 de novo pediatric AML patients by the reverse phase protein array (RPPA) methodology. Expression was compared to 20 non-malignant CD34+ bone marrow derived samples. Patients were clustered by the progeny clustering algorithm coupled with k-means, which computationally calculated the optimal number of clusters. Patients were treated according the COG Phase 3 AAML1031 trial with a 1:1 randomization to cytarabine (ara-C), daunorubicin, etoposide (ADE) ± the proteasome inhibitor bortezomib. Results: Cluster analysis identified 4 groups of correlated Protein Groups (PrG) that defined 5 recurrent Patient Clusters (PC, figure 1). The first PrG consisting of HDAC's, SIRT's and BRD4 showed homogeneous normal range expression across all patients. PrG2 contains most varying expression of HistModProt and was therefore termed as modulators. PrG3, miscellaneous defined (misc) group, had universally low expression across all patients. PrG4 consists of the histone marks including total H3. Interestingly, all variation between the PC was due to modulation within PrG2 modulators and PrG4 histone marks. Patient protein patterns in PC1 were identified as closest to that of the normal bone marrow derived CD34+ cells by performing linear discriminant analysis and were therefore defined as most normal-like cluster. PC2 had very low expression of both PrG2 and PrG4, representing a more deactivated/off cluster. PC3 had somewhat low histone marks, with higher modulators relative to the CD34+ normal-like PC1. PC4 showed relative normal levels of histone marks but had the highest levels of modulators (i.e. NPM1, NCL and hnRNPK). PC5 had elevated levels of both modulators and histone marks and therefore PC4 and PC5 were considered to represent a more activated/on protein signature that was associated with a higher proliferative potential, as manifested by higher WBC, and percent peripheral blasts and absolute blood count (all p 〈 0.001). These findings correlate with clinical features of adult AML patients with upregulated HistModProt. However, in adults, different proteins (e.g. high BRD4 and KDM1) were more prominent in the upregulated HistModProt signature. HistModProt PC membership did not correlate with outcome overall (OS) or event free survival (EFS). However, pediatric patients with the on signature (PC4 and PC5) that received the ADE + bortezomib regimen had a trend towards longer EFS compared to those treated with ADE (P = 0.055), whereas PC2 patients did not benefit from the addition of bortezomib (p = 0.43). Conclusion: Similar to adult AML patients, recurrent patterns of HistModProt were observed in pediatric AML and associates with outcome. Relative low expression of histone marks and HistModProt correlated with favorable outcomes in both adults and pediatrics suggesting that these patients contain a more open chromatin state with potential for diagnostic and prognostic implications in AML at all age. Figure legend: RPPA-based heatmap of 505 de novo pediatric AML samples containing 7 histone marks and 17 HistModProt. The five cluster colors along the top bar delineate the five identified protein clusters (PC). Protein expression is normalized for each protein to range from the lowest (blue) to the highest (red) compared to non-malignant CD34+ cells. Identification of protein groups (PrG's) is shown. Disclosures No relevant conflicts of interest to declare.

Beschreibung: INTRODUCTION: Pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are heterogeneous diseases mediated by changes in protein expression. As most chemotherapeutic agents target proteins, and because overall survival of pediatric AML is far inferior to both pre-B and T-ALL, we aimed to compare the proteomic landscape of pediatric T-ALL and AML, with the goal of determining common AML-T-ALL pathways that are potentially targetable with novel agents. METHODS: Reverse phase protein arrays (RPPA) analysis was used to measure protein expression in 858 acute leukemia samples (358 T-ALL and 500 AML, 723 pediatric (〈 18 yrs.), 135 adults (≥18 yrs.)) and 61 normal CD34+ samples using 270 validated antibodies. Expression levels were normalized against CD34+ cells. Proteins were allocated into 30 functionally related subgroups (Protein Functional Group (PFG)). A progeny clustering algorithm was applied to each PFG to search for strong correlations between proteins and to identify an optimal number of Protein Clusters (PC). Block clustering identified PC that recurrently co-occurred together (Protein Constellation (CON)) and patients that expressed similar combination of CON were defined as Protein Signature (SIG). Proteins that were differentially expressed were identified using the Student's t-test or ANOVA, with a Bonferroni adjusted p-value (0.05/ 270 = 0.00019)). RESULTS: Of the 270 analyzed proteins, 131 proteins (49%) were differentially expressed between T-ALL and AML; 60 were higher in T-ALL, 71 in AML. Similar to our previous analysis in adult AML and ALL, cell cycle regulators (CDKN1A, CDKN1B) and 2 of the 5 histone marks (H3K36Me3 & H3K4Me3) were higher expressed in T-ALL compared to AML. Heat shock proteins (HSP90AA1_B1, HSPA1A_L, HSPB1 and HSPB1-pSer82) were higher in AML as well as translation proteins EIF2S1, EIF4E and EIF4EBP1 and ribosomal proteins RPS6-pSer235_236 and RPS6KB1, while expression of the translation inhibitory proteins EIF2S1-pSer51 and EIF2AK2-pThr451 was lower in AML compared to T-ALL. Next, cluster analysis in the context of 30 PFG resulted in 133 PC. The majority (n=102) of PC were expressed in both diseases, 30 PC (22.6%) were AML-specific, and only one PC was specific to T-ALL (characterized by high CDKN1A, CDKN1B and CCND1, but low WEE1, CCNB1 and RB1-pSer). Co-clustering of the 133 PC identified 14 CON that formed 17 SIG. Three CON (5, 9, 10) were associated with AML, 2 with T-ALL (2, 13) and 8 CON were observed in both diseases. In contrast, 15 of SIG were associated with either T-ALL or AML, and two SIG (9, 10) included a mixture of both diseases (P 〈 0.001, annotation bar Figure 1 "Disease")). SIG were associated with gender (P 〈 0.001), but not with CNS-status and ethnicity (Hispanic vs. non-Hispanic). No age-specific (kids vs. adults) signatures were observed. For each SIG and CON, proteins significantly higher or lower expressed compared to the normal CD34+ cells were identified. CONCLUSIONS: This study provides support for our previous hypothesis that pediatric T-ALL and AML can be characterized by recurrent protein expression patterns. While most PC and CON were found in both diseases, SIG (i.e. combinations of protein expression patterns) were specific to either T-ALL or AML. We found similar results when comparing B-ALL to AML in adults. Shared CON indicate that there are common protein expression patterns between pediatric T-ALL and AML. Proteins or pathways with similar utilization (e.g. CON3, 5) in both diseases may allow for information on clinical utility from one disease to be applicable to the other. Those with differential utilization are likely to be uninformative with respect to clinical utility in the other disease. Figure. "MetaGalaxy" analysis for pediatric AML and T-ALL. Each row represents one protein clusters (n = 133), each column represents one patient (n = 858). Blue indicates membership for that particular protein cluster. Annotation bar shows strong correlation with disease (yellow = T-ALL, blue = AML). No associations were seen for age (blue = adult, pink = pediatric) or Ethnicity (blue = Hispanic, yellow = non-Hispanic). Figure 1 Disclosures No relevant conflicts of interest to declare.

Beschreibung: Background: Acute myeloid leukemia (AML) 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 predictions. We aimed to identify potential prognostic implications of epigenetic modification of histone proteins, with a focus of H3K27 methylation in relation to mutations in chromatin, splicing and transcriptional regulators. Material and methods: Histone methylation mark expressions were evaluated in a cohort of 241 AML bone marrow (BM) and peripheral blood (PB) samples from patients admitted at the MD Anderson Cancer Center relative to their expression in CD34+ BM derived samples from healthy donors. Simultaneous analysis of 230 proteins was performed using the reverse phase protein array - a high-throughput, quantitative proteomic platform that enables identification of aberrant expressed proteins and the pathways they act in. Additional mutational analysis was performed on 65 BM samples. Results:H3K27Me3 was significantly lower in both BM and PB leukemic-derived samples compared to their expression in normal BM (figure 1A). A greater loss of H3K27Me3 associated with increased proliferative potential and shorter overall survival (OS) in the whole patient population (n=241, HR=0.64, 95% CI=0.47-0.87, p