ALBERT

Description: Introduction: Given the dismal outcome of relapsed pediatric ALL, there is an urgent need to identify underlying drug resistance mechanisms. We have previously discovered that chemosensitivity can be restored by epigenetic reprogramming (Bhatla et al, Blood 2012). Based on our prior work, we hypothesize that epigenetic changes play a major role in mediating chemoresistance and relapse in pediatric ALL. To develop a comprehensive map of relapse specific epigenetic alterations and to understand the impact of epigenetic alterations on the relapse specific gene expression signature, we have embarked on an unbiased genome-wide approach to map the location of key histone marks by chromatin immunoprecipitation sequencing (ChIP-seq) in diagnosis-relapse patient pairs with B-lymphoblastic leukemia. Methods: To date, we have performed ChIP-seq on 13 matched diagnosis/relapse cryopreserved bone marrow samples from patients enrolled on Children’s Oncology Group protocols. We assessed histone marks associated with promoters (H3K4me3, H3K9ac), enhancers (H3K27Ac) and those which are rather widely distributed in euchromatin and heterochromatin (H3K9me3, H3K27me3). 51-cycle single-end sequencing was performed using the Illumina HiSeq2000 Analyzer. Reads were aligned to the Human reference genome (assembly hg19) using the Burrows-Wheeler Alignment tool (BWA, v0.7.7) and post-preocessed using Samtools (v0.1.18). Enriched binding sites (“peaks”) were determined by the peak-calling algorithm, MACS2 (v2.0.10.20131216) using a q-value of 0.01 to define significance. Histone peak deposition on the promoters and enhancer regions were correlated with gene expression data from microarrays obtained from NCI’s TARGET initiative (Therapeutically Applicable Research to Generate Effective Treatment) on the same patients. Promoter regions were defined as 3 kb upstream and downstream of Transcription Start Site (TSS). Super-enhancers were identified by executing the ROSE algorithm (Hnisz et al, Cell 2013). Results: Promoter and enhancer region analysis was carried out only on activating histone marks (H3K4me3, H3K9ac and H3K27Ac) due to their expected uniform and enriched deposition in these regions. We observed that approximately 50% of the genes exhibited transcriptional activation or repression with respective concordant gain or loss of activating histone marks in the majority of patients, while the regulation of rest of the genes seemed independent of histone modification. Next, we sought to determine the impact of histone modification from diagnosis to relapse on the gene expression signature previously established in a cohort of 49 diagnosis-relapse patient pairs (Hogan et al, Blood 2011). Of 60 genes, 46 genes had one or more activating histone marks differentially deposited in the 6 kb promoter region in one or more of the patient samples analyzed and showed concordant expression. Furthermore, differentially up-regulated relapse specific genes such as FOXM1, FANCD2, PRMT7, CENPM and PTBP1 showed concordant deposition of activating histone marks in approximately 50% of relapse samples. Likewise, 5 down-regulated genes including SMEK2 and FOXP1 had concordant loss of these marks in approximately 50% of relapse samples. In order to identify the compendium of distal regulatory enhancers that may govern transcription, we generated chromatin state maps based on the histone modification H3K27ac, which depicts active enhancers. This analysis suggested that the super-enhancers deposited adjacent to genes having higher expression at diagnosis relative to relapse (eg. JARID2, TLE4, ETS1, EBF1 and CIITA), are implicated in transcriptional regulation. Likewise, genes involved in DNA replication and repair such as PHB and TOP3B and those involved in immune regulation such as CD34, IGLL1 and LMO2 were up-regulated with concordant gain of super-enhancers at the time of relapse. Conclusions: In a pilot ChIP-seq analysis of 13 ALL diagnosis/relapse pairs, we have identified several candidate genes, whose transcription appear to be epigenetically regulated and are markers of aggressive disease. Our study further implicates a potential use for epigenetic therapy for the treatment of relapsed ALL. Disclosures No relevant conflicts of interest to declare.

Description: Background: Survival rates for acute lymphoblastic leukemia (ALL) have risen dramatically but up to 20% of patients relapse and their prognoses are dismal. Resistance to glucocorticoid (GC) agonists is a hallmark of relapsed ALL and a strong predictor of outcome at diagnosis (Dördelmann M et. al, Blood 1999, Schmiegelow K et. al, Leukemia 2001, Tissing WJ et. al, Leukemia 2003). However, the underlying biological pathways that mediate sensitivity to GCs remain to be determined. In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cells. Methods: Genome-wide screening was performed using pooled shRNA libraries coupled with next generation sequencing to identify genes that when depleted promote GC sensitivity. Statistical analysis using Bioinformatics for Next Generation Sequencing (BiNGS) and Redundancy and Fold Change (RFC) were employed to identify candidate genes that mediate prednisolone sensitivity (Porter et. al, Leukemia 2012). Validation of hits from the primary screen were performed in Reh and RS4;11 cells. Knockdown of candidate genes MEK2 and MEK4 was determined by western blot. Changes in chemosensitivity upon MEK2 and MEK4 knockdown were determined by Cell Titer-Glo assay (Promega). The levels of apoptotic cells upon chemotherapy treatment in control and knockdown cell lines was determined by Annexin V-PE and 7-Amino-actinomycin D (7AAD) staining (Annexin V-PE Apoptosis Detection Kit, BD Pharmingen, San Diego, CA, USA), followed by flow cytometry using the FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). The levels of downstream GC target genes including NR3C1, GILZ, and BIM were determined by RT-PCR. The levels of target proteins including GR, pERK, ERK, MEK1, and p53 were determined by western blot. pERK levels in primary matched pairs were determined by multiparameter phosphoflow cytometry. Results: In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of this data with integrated analysis of relapse-specific genetic and epigenetic changes (Hogan et. al, Blood 2012) allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. Interestingly, depletion of MAPK pathway members, MEK2 and MEK4, increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to GCs by increasing the mRNA and protein levels of the glucocorticoid receptor (GR). This resulted in greater induction of the GR target genes GILZ and BIM upon prednisolone exposure over time. Importantly, depletion of MEK4 did not affect sensitivity of ALL cells to other chemotherapy agents (doxorubicin, etoposide, and 6-thioguanine). By contrast, MEK2 knockdown increased the sensitivity of cells to each of the chemotherapy agents tested including prednisolone, doxorubicin, etoposide, and 6-thioguanine. Depleting MEK2 decreased activated pERK and increased levels of p53. Over expression of a dominant negative p53 in MEK2 deficient cells reversed sensitivity to doxorubicin and prednisolone, indicating that MEK2 expression mediates chemosenstivity in a p53 dependent manner. Furthermore, inhibition of MEK1/2 pharmacologically with trametinib increased sensitivity of ALL cells to chemotherapy. Trametinib treatment also resulted in increased levels of p53. To determine if activation of the MAPK pathway in patients is associated with recurrent disease we examined seven matched diagnosis and relapse primary samples for MAPK activation as determined by pERK staining, and observed increased pERK levels at relapse in all samples tested. Conclusion: Our data indicate that activation of the MAPK pathway promotes chemoresistance and may drive the development of recurrent disease in pediatric ALL.Asdisrupting MEK2 and MEK4 sensitizes cells to chemotherapy, this makes the MAPK pathway an attractive target for therapeutic intervention in relapsed ALL. Disclosures No relevant conflicts of interest to declare.

Description: Introduction While childhood acute lymphoblastic leukemia (ALL) is highly curable, up to 20% of children will relapse, with dismal prognosis, warranting the need for novel therapies. Previously, using an integrated genomic approach on matched diagnosis-relapse samples, we identified overactivation of the Wnt pathway as a mechanism of disease recurrence at relapse (Hogan et al, Blood 2011). Aberrant Wnt signaling has been linked to cancers of the liver, colon, breast, skin and more recently hematologic malignancies. To validate our findings and determine if Wnt inhibition could restore chemosensitivity in relapsed ALL, we sought to examine directly whether Wnt is activated at relapse in paired samples (examining expression of activated b-catenin and its downstream target Survivin (BIRC5) using multiparameter phosphoflow cytometry) and tested the efficacy of a recently developed small molecule Wnt inhibitor, iCRT14, that specifically interferes with the b-catenin-TCF interaction (Gonzalves et al, PNAS 2011), in ALL cell lines and patient samples. Methods B and T-ALL cell lines were treated with iCRT14 and the expression of target genes were determined by quantitative RT-PCR.10 paired diagnosis-relapse patient samples obtained from the Children’s Oncology Group were washed, fixed and stained simultaneously with caspase 3, CD10, activated b-catenin and survivin and the change in expression of activated b-catenin and survivin from diagnosis to relapse was measured by multiparameter phosphoflow cytometry in each patient by gating on the caspase 3 negative, CD10 positive leukemic blasts. To test the effect of Wnt inhibition on chemosensitivity, B-ALL cell lines were pretreated with iCRT14 for 48 hours prior to incubation with traditional chemotherapy for an additional 24 hours. The response to increasing doses of iCRT14 and chemo, alone and in combination, was assessed by cell viability (Cell Titer-Glo Luminescent Assay (Promega)) and apoptosis (FACS analysis with AnnexinV-PE/7AAD staining (BD Bioscience)). Protein levels of apoptotic markers were assessed. Also, 4 newly diagnosed and 4 relapsed patient samples were treated ex vivo with iCRT14 (20 and 30 uM) and prednisolone, alone and in combination. Drug combination results were analyzed using the Calcusyn program which calculates a Combination Index (CI): CI〉1.1=antagonism, 0.9-1.1=additive and 80% apoptosis by hour 72 with the maximal chemotherapy dose in all cell lines. Change in the protein levels of cleaved PARP and cleaved caspase 3 was seen. The 4 diagnosis patients were very sensitive to prednisolone as expected, precluding synergism with iCRT14. The relapsed patient samples were much less sensitive to prednisolone alone (40% decrease in viability in relapsed patients vs 80% in new diagnoses). Interestingly, all the relapsed patients showed enhanced chemosensitivity with Wnt inhibition. 3 out of 4 relapsed patients showed strong synergism (CI=0.03-0.6) with both doses of iCRT14 and 1 patient showed additive to synergistic effects (CI=0.7 and 1). Conclusion Overactivation of the Wnt pathway may lead to chemoresistance in relapsed ALL. Wnt Inhibition restores chemosensitivity and induces apoptosis in ALL cell lines and primary patient samples making it a potential therapeutic approach. Disclosures: No relevant conflicts of interest to declare.

Description: The goal of this project was to identify and target metabolic vulnerabilities of leukemia stem cells (LSCs) to improve therapeutic outcomes for patients with AML. We have previously shown that primary human LSCs reside in a unique metabolic condition characterized by a relatively low oxidative state (termed "ROS-low") and increased levels of glutathione (Lagadinou et al. Cell Stem Cell, 2013). Cells in this condition are highly dependent on oxidative phosphorylation for survival, in striking contrast to many tumor cells which often rely on glycolysis; indicating that LSCs are governed by distinct metabolic properties. To further elucidate key metabolic properties of LSCs, we measured differences in the global metabolome of ROS-Low LSCs in comparison to ROS-high AML blasts. Our preliminary data demonstrated that ROS-low LSCs have higher levels of amino acids and require amino acid catabolism for survival. We hypothesized that certain individual amino acids may be more important for LSC survival. If true, then targeting specific amino acids may be an avenue towards improved AML therapy. To determine if any individual amino acid is essential for LSC survival, we analyzed AML cells from five patients that were systematically cultured in media lacking one of the twenty amino acids. Cysteine depletion was consistently the most cytotoxic, showing decreased cell viability and colony forming potential of LSCs. We next determined the effect of an engineered human enzyme that selectively degrades cysteine and cystine (AEB3103, Aeglea BioTherapeutics, Inc.) on LSC viability and colony forming potential. We found that AEB3103 treatment decreased viability of LSCs in all AML specimens tested and significantly decreased colony formation (p

Description: Effective targeting of the acute myeloid leukemia (AML) leukemia stem cell (LSC) population may allow for deep, durable remissions and curative potential. In older, newly diagnosed AML patients who are not candidates for induction, venetoclax + azacitidine (aza) targets specific metabolic vulnerabilities of LSCs, resulting in very promising clinical outcomes. In our single institution experience treating 45 previously untreated AML patients with venetoclax + aza both in the context of the multi-institutional study NCT02203773 (N=33) and with off-label use (N=12), 36/45 (80%) achieved a complete remission (CR) or CR with incomplete count recovery (CRi). In the relapsed/refractory (R/R) setting, the efficacy of venetoclax + aza has been reported to be significantly worse. In our single-institution off-label experience (N=7), only 1/7 (14%) R/R patients had a CR/CRi (p=0.005 compared to the untreated group). R/R and untreated patients had similar baseline characteristics, although more R/R patients had an antecedent hematological disorder (Table 1). Multivariate analysis showed cytogenetic risk and R/R disease as the sole predictors of response to venetoclax + aza (Table 2). In light of existing data regarding biological changes that occur in LSCs after treatment and subsequent relapse, we aimed to determine whether laboratory analysis of LSCs from patients treated with venetoclax + aza would show differential sensitivity to this therapy in the up-front vs R/R setting that could help to explain the different clinical activity. We have previously shown that LSCs from untreated patients are uniquely reliant on oxidative phosphorylation (OXPHOS), and that venetoclax + aza targets LSCs by decreasing OXPHOS. Therefore, we tested the hypothesis that inferior responses of R/R patients to venetoclax + aza are due to changes in OXPHOS regulation in relapsed LSCs. LSCs were defined as cells bearing relatively low levels of reactive oxygen species (ROS-low), an effective means of enriching primary human LSCs. We found that in contrast with untreated patients, venetoclax + aza does not decrease viability or OXPHOS in LSCs from R/R patients (Fig1). Furthermore, R/R LSCs had altered fatty acid metabolism that contributed to these OXPHOS differences, with increased flux of fatty acids into the TCA cycle (Fig 2). In addition, R/R samples compensated for the metabolic perturbations that occurred upon exposure to venetoclax + aza through upregulation of fatty acid uptake and metabolism into the TCA cycle (Fig 3). Fatty acid metabolism is controlled by multiple genes and pathways. Integral to its activity is the gene Carnitine Palmitolytransferase 1 (CPT1), due to its pivotal role in the beta-oxidation of long chain fatty acids. Investigation of the Cancer Genome Atlas AML dataset reveals higher expression of CPT1 leads to significantly worse overall survival, suggesting increased fatty acid metabolism may drive a more resistant LSC population in R/R AML patients. We also found elevated baseline levels of CPT1 in patients who progressed on venetoclax + aza compared to those that had long term remissions (not shown). Therefore we utilized the CPT1 inhibitor etomoxir to block fatty acid metabolism. We found addition of etomoxir to cultures of R/R LSCs rescued the ability of venetoclax + aza to decrease OXPHOS and re-sensitized R/R LSCs to venetoclax + aza (Fig 4). To prove that this novel regimen targets functionally-defined R/R LSCs we performed ex vivo treatment followed by xenotransplantation of R/R patient specimens, which showed that upon etomoxir addition, engraftment potential is significantly decreased over venetoclax + aza alone (not shown). Therefore we propose a novel mechanism for the increased resistance of R/R AML patients to venetoclax + aza involving altered energy metabolism. We find increased fatty acid metabolism in R/R patient specimens, and targeting this pathway using the CPT1 inhibitor etomoxir leads to sensitization to venetoclax + aza and rescued targeting of OXPHOS, allowing for LSC eradication. Disclosures Pollyea: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Curis: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees.

Description: Previous studies have demonstrated the importance of energy metabolism as it relates to numerous aspects of leukemia stem cell (LSC) biology. Specifically, in acute myelogenous leukemia (AML), it has been shown that LSCs have a unique reliance on oxidative phosphorylation (OXPHOS), and that inhibition of B-cell lymphoma 2 (BCL-2) acts to down-regulate OXPHOS and eradicate LSCs in pre-clinical models. In the clinical setting, when BCL-2 inhibitor venetoclax is combined with azacitidine, high response rates (~80%) in elderly de novo AML patients have been observed (PMID: 29339097). Nonetheless, a significant portion of these patients ultimately experience disease progression. The mechanism of resistance and characteristics of these patients is poorly understood. Our preliminary data shows that the venetoclax + azacitidine (ven/aza) regimen targets LSCs through alteration of energy metabolism. Specifically, the regimen disrupts the TCA cycle leading to decreases in ATP production and inhibition of OXPHOS. This metabolic targeting is central to ven/aza efficacy and we hypothesize that resistance and progression of AML patients is due to compensatory mechanisms that restore sufficient levels of OXPHOS (PMID:3333149). Investigation of such mechanisms led us to explore the potential activity of MCL1. Previous studies have shown MCL1 can influence venetoclax resistance, however little is known about MCL1's role in metabolism, although a report in breast cancer cells suggests MCL1 modulates OXPHOS (PMID: 28978427). In leukemia, MCL1 expression has been shown to be partially upregulated through mutations in protein tyrosine phosphatase non- receptor type 11 (PTPN11), and PTPN11 mutations have been shown to increase LSC frequency. Thus, we hypothesized that PTPN11 mutations may confer resistance to venetoclax-based regimens at least partially by up-regulation of MCL1. To test this hypothesis, we investigated the relationship between PTPN11 mutations, MCL1, and the metabolic phenotype. In comparison to specimens with a wild type allele, LSCs isolated from PTPN11 mutant patient specimens showed increased levels of OXPHOS as well as glycolysis, amino acids, and fatty acids, suggesting an ability to utilize multiple energy sources for survival. PTPN11 mutant specimens also show decreased sensitivity to venetoclax, suggesting OXPHOS is not affected by venetoclax to the same degree as PTPN11 wild type specimens (fig. 1). Furthermore, when we introduced a mutated allele of PTPN11 into a primary AML specimen we observed increased oxidative phosphorylation and glycolysis, which correlated with decreased in vitro sensitivity to venetoclax (fig. 2). To test the potential role of MCL1 in PTPN11 mutant specimens, we employed a small molecule MCL-1 inhibitor. Metabolic analysis of specimens treated with the MCL-1 inhibitor showed decreased OXPHOS in PTPN11 mutant specimens (fig 3). Further, PTPN11 mutant specimens exhibit increased sensitivity to the MCL-1 inhibitor (fig. 4). To investigate a potential mechanistic link to clinical observations, we next examined 45 older, previously untreated AML patients from our institution who received ven/aza, both in the context of the multi-institutional study NCT02203773 (N=33) and with off-label use (N=12). Of 12 variables examined, only the presence of PTPN11 predicted shorter response duration (table 1). In addition, of the 9 patients who progressed ven/aza, 2 (22%) acquired PTPN11 mutations upon progression, further suggesting PTPN11 may represent a resistance mechanism to this regimen. Notably, PTPN11 is not preferentially detected in patients who progress after regimens other than ven/aza. In conclusion, AML containing PTPN11 mutations exhibit a unique energy metabolism profile. These specimens also appear to have increased sensitivity to MCL-1 inhibitors. The presence of PTPN11 mutations represents both a novel method for predicting response to ven/aza and a potential strategy for targeting patients who progress. We propose that addition of an MCL-1 inhibitor for treatment of AML patients bearing PTPN11 or related mutations may increase therapeutic responses. Disclosures Savona: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Consultancy. Fesik:Boehringer Ingelheim: Consultancy. Pollyea:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Curis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy.

Description: Recent studies from our group and others have revealed a role for ETV6 germline mutations in the predisposition to ALL. Although ETV6 is among the most commonly mutated genes in ALL, its mechanistic role in leukemogenesis remains unclear. ETV6 is an ETS family transcription factor. ETV6 regulates gene transcription through homo- and hetero- oligomerization with other ETS family members and transcriptional repressors. The germline mutation (P214L amino acid change) identified by our group and others impairs the transcriptional activity and nuclear localization of ETV6 in a dominant negative fashion. The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia. To identify functions of ETV6 in B cell development, we queried the gene expression commons database for evidence of Etv6 expression during B cell development. Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (P