ALBERT

Description: 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.

Description: 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.

Description: 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

Description: Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .

Description: The inferior cure rate of T-cell acute lymphoblastic leukemia (T-ALL) is associated with inherent drug resistance. The activating NOTCH1 gene mutations have been reported to cause chemoresistance at the stem cell level1. Direct NOTCH1 inhibition has failed in clinical trials due to a narrow therapeutic window but targeting key oncogenic and metabolic pathways downstream of mutated NOTCH1 may offer novel approaches. We previously reported that rapid transformation of thymocytes at the DN3 differentiation stage into preleukemic stem cells (pre-LSC) requires elevated Notch1 in addition to the presence of Scl/Lmo11. Notably, we showed that cellular metabolism of NOTCH1-mutated T-ALLs depends on Oxidative Phosphorylation (OxPhos) and that OxPhos inhibition using the complex I inhibitor IACS-010759 (OxPhos-i) is efficacious in NOTCH1-mutated T-ALL patient derived xenografts (PDXs)2. Here, we investigated the link between NOTCH1-mutated chemoresistance and OxPhos in pre-leukemic and leukemic cells, utilizing comprehensive molecular and functional assays. We hypothesized that chemotherapy aided by OxPhos-i overcomes chemoresistance, depletes LSCs and combats T-ALL. First, we analyzed the role of OxPhos in downstream Notch1 targets at the pre- and leukemic stage considering four stages of thymocyte differentiation (D1-D4), in a mouse model of human T-ALL1. Gene set enrichment analysis (GSEA) implicated increased expression of Notch1 target genes starting at DN1, and OxPhos target genes were the highest-ranked gene set at DN3. Next, activation of Notch1 by its ligand DL4 and inhibition of OxPhos reduced viability of pre-LSCs, indicating that ligand-dependent activation of Notch1 signaling upregulates the OxPhos pathway and sensitizes pre-LSCs to OxPhos-i. To clarify the role of Notch1 signaling, we examined the effect of IACS-010759 on pre-leukemic thymocytes harboring LMO1, SCL-LMO1, NOTCH1, LMO1-NOTCH1 and SCL-LMO1-NOTCH1 with and without DL4 stimulation. We found that in the absence of DL4, only thymocytes harboring the Notch1 oncogene responded to OxPhos-i, whereas all DL4-stimulated thymocytes responded regardless of Notch1 status (Fig. 1a). In addition, at the leukemic stage, we found elevation of the OxPhos pathway driven by oncogenic Notch1 when we compared transcriptomes of SCL-LMO1 induced T-ALL in the presence or absence of the NOTCH1 oncogene. In line with the murine T-ALL NOTCH1 model, we performed transcriptome analysis of two independent T-ALL patient cohorts prior to chemotherapy, COG TARGET ALL (n=263) and AALL1231 (n=75), comparing transcriptomes of NOTCH1-mutated vs NOTCH1-wt T-ALLs. We found co-segregation of NOTCH1 mutations with significant upregulation of OxPhos and TCA cycle genes and downregulation of apoptosis signaling. Aiming to reverse the NOTCH1-controlled anti-apoptotic program and chemoresistance, we next tested the combination of Vincristine, Dexamethasone and L-Asparaginase (VXL) with IACS-010759. When compared to vehicle, OxPhos-i or VXL alone, only the VXL-OxPhos-i treatment caused an energetic crisis indicated by decreased OCR and ECAR (Seahorse), which translated to a profound reduction of viability (CTG, flow cytometry) in T-ALL cell lines (n=9) and primary T-ALL samples (n=5). Additionally, the IACS-VXL combination in vivo resulted in pan-metabolic blockade, which caused metabolic shut-down and triggered early induction of apoptosis in leukemic cells in peripheral blood, spleen and bone marrow (Fig. 1b). Single cell Proteomic analysis (CyTOF) of spleen showed reduced expression of cell proliferation marker -ki67, c-myc, ERK and p38 proteins, and reduction in number of leukemic cells. Finally, this combination therapy resulted in reduced leukemia burden and extension of overall survival across all three aggressive NOTCH1-mutated T-ALL PDX models (p