Chromatin Profiles Are Prognostic of Clinical Response to Bortezomib-Containing Chemotherapy in Pediatric Acute Myeloid Leukemia: Results from the COG AAML1031 Trial.

The addition of the proteasome inhibitor bortezomib to standard chemotherapy did not improve survival in pediatric acute myeloid leukemia (AML) when all patients were analyzed as a group in the Children's Oncology Group phase 3 trial AAML1031 (NCT01371981). Proteasome inhibition influences the chromatin landscape and proteostasis, and we hypothesized that baseline proteomic analysis of histone- and chromatin-modifying enzymes (HMEs) would identify AML subgroups that benefitted from bortezomib addition. A proteomic profile of 483 patients treated with AAML1031 chemotherapy was generated using a reverse-phase protein array. A relatively high expression of 16 HME was associated with lower EFS and higher 3-year relapse risk after AML standard treatment compared to low expressions (52% vs. 29%, p = 0.005). The high-HME profile correlated with more transposase-accessible chromatin, as demonstrated via ATAC-sequencing, and the bortezomib addition improved the 3-year overall survival compared with standard therapy (62% vs. 75%, p = 0.033). These data suggest that there are pediatric AML populations that respond well to bortezomib-containing chemotherapy.

Clinical relevance of proteomic profiling in de novo pediatric acute myeloid leukemia: a Children's Oncology Group study.

Pediatric acute myeloid leukemia (AML) remains a fatal disease for at least 30% of patients, stressing the need for improved therapies and better risk stratification. As proteins are the unifying feature of (epi)genetic and environmental alterations, and are often targeted by novel chemotherapeutic agents, we studied the proteomic landscape of pediatric AML. Protein expression and activation levels were measured in 500 bulk leukemic patients' samples and 30 control CD34+ cell samples, using reverse phase protein arrays with 296 strictly validated antibodies. The multistep MetaGalaxy analysis methodology was applied and identified nine protein expression signatures (PrSIG), based on strong recurrent protein expression patterns. PrSIG were associated with cytogenetics and mutational state, and with favorable or unfavorable prognosis. Analysis based on treatment (i.e., ADE vs. ADE plus bortezomib) identified three PrSIG that did better with ADE plus bortezomib than with ADE alone. When PrSIG were studied in the context of cytogenetic risk groups, PrSIG were independently prognostic after multivariate analysis, suggesting a potential value for proteomics in combination with current classification systems. Proteins with universally increased (n=7) or decreased (n=17) expression were observed across PrSIG. Certain proteins significantly differentially expressed from normal could be identified, forming a hypothetical platform for personalized medicine.

RPPA-based proteomics recognizes distinct epigenetic signatures in chronic lymphocytic leukemia with clinical consequences.

The chronic lymphocytic leukemia (CLL) armamentarium has evolved significantly, with novel therapies that inhibit Bruton Tyrosine Kinase, PI3K delta and/or the BCL2 protein improving outcomes. Still, the clinical course of CLL patients is highly variable and most previously recognized prognostic features lack the capacity to predict response to modern treatments indicating the need for new prognostic markers. In this study, we identified four epigenetically distinct proteomic signatures of a large cohort of CLL and related diseases derived samples (n = 871) using reverse phase protein array technology. These signatures are associated with clinical features including age, cytogenetic abnormalities [trisomy 12, del(13q) and del(17p)], immunoglobulin heavy-chain locus (IGHV) mutational load, ZAP-70 status, Binet and Rai staging as well as with the outcome measures of time to treatment and overall survival. Protein signature membership was identified as predictive marker for overall survival regardless of other clinical features. Among the analyzed epigenetic proteins, EZH2, HDAC6, and loss of H3K27me3 levels were the most independently associated with poor survival. These findings demonstrate that proteomic based epigenetic biomarkers can be used to better classify CLL patients and provide therapeutic guidance.

Bortezomib is significantly beneficial for de novo pediatric AML patients with low phosphorylation of the NF-κB subunit RelA.

PURPOSE: The addition of the proteasome inhibitor (PI) bortezomib to standard chemotherapy (ADE: cytarabine [Ara-C], daunorubicin, and etoposide) did not improve overall outcome of pediatric AML patients in the Children's Oncology Group AAML1031 phase 3 randomized clinical trial (AAML1031) . Bortezomib prevents protein degradation, including RelA via the intracellular NF-kB pathway. In this study, we hypothesized that subgroups of pediatric AML patients benefitting from standard therapy plus bortezomib (ADEB) could be identified based on pre-treatment RelA expression and phosphorylation status. EXPERIMENTAL DESIGN: RelA-total and phosphorylation at serine 536 (RelA-pSer536 ) were measured in 483 patient samples using reverse phase protein array technology. RESULTS: In ADEB-treated patients, low-RelA-pSer536 was favorably prognostic when compared to high-RelA-pSer536 (3-yr overall survival (OS): 81% vs. 68%, p = 0.032; relapse risk (RR): 30% vs. 49%, p = 0.004). Among low-RelA-pSer536 patients, RR significantly decreased with ADEB compared to ADE (RR: 30% vs. 44%, p = 0.035). Correlation between RelA-pSer536 and 295 other assayed proteins identified a strong correlation with HSF1-pSer326 , another protein previously identified as modifying ADEB response. The combination of low-RelA-pSer536 and low-HSF1-pSer326 was a significant predictor of ADEB response (3-yr OS: 86% vs. 67%, p = 0.013). CONCLUSION AND CLINICAL RELEVANCE: Bortezomib may improve clinical outcome in a subgroup of AML patients identified by low-RelA-pSer536 and low-HSF1-pSer326 .

Heat shock factor 1 (HSF1-pSer326) predicts response to bortezomib-containing chemotherapy in pediatric AML: a COG report.

Bortezomib (BTZ) was recently evaluated in a randomized phase 3 clinical trial by the Children's Oncology Group (COG) that compared standard chemotherapy (cytarabine, daunorubicin, and etoposide [ADE]) vs standard therapy with BTZ (ADEB) for de novo pediatric acute myeloid leukemia (AML). Although the study concluded that BTZ did not improve outcome overall, we examined patient subgroups benefiting from BTZ-containing chemotherapy using proteomic analyses. The proteasome inhibitor BTZ disrupts protein homeostasis and activates cytoprotective heat shock responses. Total heat shock factor 1 (HSF1) and phosphorylated HSF1 (HSF1-pSer326) were measured in leukemic cells from 483 pediatric patients using reverse phase protein arrays. HSF1-pSer326 phosphorylation was significantly lower in pediatric AML compared with CD34+ nonmalignant 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 = .019). To determine the effect of HSF1 expression on BTZ potency in vitro, cell viability with HSF1 gene variants that mimicked phosphorylated (S326A) and nonphosphorylated (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 who benefit from BTZ-containing chemotherapy.

Targeted therapy in acute myeloid leukemia: current status and new insights from a proteomic perspective.

Introduction: The biological heterogeneity of acute myeloid leukemia (AML) complicates personalized medicine. Individual prognosis is typically based on the presence of chromosomal and genetic lesions. Nevertheless, these classifications often lack a priori information about response to therapy. Since the protein expression landscape reflects the functional activity state of cells, we hypothesize that analyzing this can be used for the identification of protein activity markers to provide better risk stratification as well as may provide targeted therapeutic guidance in AML.Areas covered: Herein, we review recently new adopted drugs in the treatment for AML and discuss how quantitative proteomic techniques may contribute to better therapeutic selection in AML.Expert commentary: The net functional state of the cell is defined by the activity of protein within all the pathways that are active in the cell. Recognition of the proteomic profile of the leukemic blast could, therefore, complement current classification systems by providing a better a priori description of what pathways are important within a cell as a guide to the selection of therapy for the patient.

Histone Modification Patterns Using RPPA-Based Profiling Predict Outcome in Acute Myeloid Leukemia Patients.

Posttranslational histone tail modifications are known to play a role in leukemogenesis and are therapeutic targets. A global analysis of the level and patterns of expression of multiple histone-modifying proteins (HMP) in acute myeloid leukemia (AML) and the effect of different patterns of expression on outcome and prognosis has not been investigated in AML patients. Here we analyzed 20 HMP by reverse phase protein array (RPPA) in a cohort of 205 newly diagnosed AML patients. Protein levels were correlated with patient and disease characteristics, including survival and mutational state. We identified different protein clusters characterized by higher (more on) or lower (more off) expression of HMP, relative to normal CD34+ cells. On state of HMP was associated with poorer outcome compared to normal-like and a more off state. FLT3 mutated AML patients were significantly overrepresented in the more on state. DNA methylation related mutations showed no correlation with the different HMP states. In this study, we demonstrate for the first time that HMP form recurrent patterns of expression and that these significantly correlate with survival in newly diagnosed AML patients.