ALBERT

Description: It is increasingly recognized that the tumor microenvironment plays a pivotal role in cancer initiation and progression. In mouse models it was shown that a genetically altered bone marrow (BM) micro milieu was sufficient to induce leukemia (Raaijmakers, Nature 2010); however, the pathogenic role and contribution of the BM stroma in leukemia initiation and during disease progression warrants further investigation. To address this, we have performed gene expression, methylation, RNAseq, whole exome sequencing (WES) in BM mesenchymal stroma cells (BM-MSC) and leukemic cells from AML patients (pts) to unravel underlying molecular alterations. We collected BM hematopoietic cells (BM-HC) as well as plastic-adherent BM-MSC from aspirates from AML pts and healthy donors (HD). BM-MSC were expanded to passage 4 and defined as CD73+/CD105+/CD271+/low/CD45-/CD33-. We investigated gene expression profiles (Affymetrix) of BM-MSC from newly diagnosed AML pts (n=20) and compared these to BM-MSC from HD (n=4). BM-MSC from AML pts displayed an altered expression signature with 191and 175genesbeingsignificantly 2-fold over- and under-expressed. KEGG analysis of differentially expressed genes in BM-MSC from AML pts exhibited enrichment for TGF-ß signalling, whereas downregulated genes were enriched for cytokine receptor interactions. Several of these candidates were validated in a larger set of BM-MSC samples by RT-PCR. One putative stroma-leukemia interaction molecule, lumican (LUM) was highly overexpressed in BM-MSC (n=60) from AML pts compared to HD (n=5; p value =0.019) indicating that LUM may affect the BM niche in AML. To explore the altered expression pattern in AML BM-MSC compared to HD BM-MSC, global methylation analyses (Illumina Infinium HumanMethylation 450 bead chip arrays) were performed in 5 AML pts where we had collected BM-HC and BM-MSC at 3 sequential time points [initial diagnosis (ID), remission (CR), relapse (REL); n=30] as well as in BM-HC and BM-MSC from HDs (n=6). A significantly different methylation profile was evident comparing AML BM-HC to the corresponding AML BM-MSC samples, the latter showing a homogenous pattern during the course of disease. When AML BM-MSC were compared to a set of HD BM-MSC, we identified 2416 differentially methylated CpG sites (p value

Description: Introduction: Despite the recent identification of the Ph-like subgroup of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), a large number of BCP-ALL patients lack cytogenetic and molecular defined lesions. To get a higher resolution and a broader molecular view of relapsed BCP-ALL, we designed a multi-omics study to reveal age-overriding relapse-driving alterations that may unravel novel molecular targets. Methods: We studied 150 paired samples (initial diagnosis: ID; relapse: REL; complete remission: CR) from 50 patients without known translocations. The cohort consisted of 24 adult and 26 pediatric patients with minimal residual disease 〈 0.05 % at CR. All patients were treated in population based German study trials (GMALL, BFM). We examined the mutational and copy number status via exome sequencing, obtained expression profiles and fusion-genes via RNA-sequencing and the methylation status via Illumina Methylation Array. Results: With a lenient approach detecting drivers and passengers, we identified significantly more mutations in REL compared to ID samples (adult median: 52 vs 38; pediatric median: 39 vs 27). In addition, we detected 4 hypermutators (more than 100 mutations per sample), 2 were pediatric and 2 were adult samples, 3 of which were REL samples. The most recurrently mutated genes were KRAS (n=15), NRAS (n=15), TP53 (n=13), CDC27 (n=13), KMT2D (n=11), IKZF1 (n=11), CREBBP (n=10) and FLT3 (n=6; Figure 1), with mutations present in both age cohorts. NT5C2, SYK and CHD1 were exclusively mutated in the pediatric cohort with at least 3 mutations. NT5C2 was also specific for early REL. Of all REL mutations, 225 mutations (14%, mean: 4 mutations/patient) were sub-clonal (under 〈 5% mutation frequency) at ID. Copy number alterations (CNA) varied greatly among pediatric and adult samples: 6% of pediatric and 18% of adult samples had aneuploidies and or copy neutral loss of heterozygosity of whole chromosomes. Chromosomal aberrations at ID persisted at relapse (100 %). Particular targets of CNA affected well-described genes like CDKN2A, CDKN2B, PAX5 on chr9p. Genes preferentially subjected to homozygous deletions were VPREB1 (n=6), SH2B3 (n=4), and ETV6 (n=2). All SH3B2 deletions were found in pediatric samples. On the epi-genomic level, the principal component analysis of the most variable CG-sites revealed a stable methylation profile during the course of the disease. However, we found a clear separation into a smaller pediatric-dominated cluster (n=24; 20 pediatric, 4 adult) and a larger mixed-age cluster (n=76; Fig. 1, Cluster A). Differentially methylated regions, affecting a total of 269 genes, characterized the separation of the smaller cluster, henceforth called Methylation Deregulated (MDR) cluster. The samples of the MDR cluster showed also a distinct gene expression profile by RNA-seq supporting a tight connection between the methylation status and its transcriptional program. A subset of 97 genes was differentially expressed including MAPK and PDGFR genes as most prominently deregulated. Additionally we defined a MDR expression classifier comprising 30 genes (Fig. 1). On the mutational level, the MDR samples had 20 % fewer mutations (mean: 25.3) compared to the remaining samples (mean: 31.3) and fewer CNVs for the most frequently affected genes. Characterising the non-MDR samples, a third of those were categorized as Ph-like ALL using the 15 gene classifier in an unsupervised clustering; this signature also coincided with the presence of well-known fusion-genes (Fig. 1, Cluster B). The remaining samples were defined by chromosomal instability (CI; Fig. 1, Cluster C). In the CI cluster, mutations in epigenetic regulators were twice as frequent when compared to the remaining samples. Conclusions: We describe three distinct clusters in relapsed BCP-ALL, which are characterized by a different genetic alterations: a novel MDR cluster by distinct methylation changes, the Ph-like cluster by gene fusions and the CI cluster by chromosomal instability. The cluster assignment was stable over the course of the disease. All clusters occurred in pediatric and adult patients, with the methylation-driven cluster predominantly in pediatrics. The MDR cluster showed significantly fewer mutations and CNVs compared to the other two clusters. The MDR samples showed activation of the MAPK signaling pathway pointing to actionable therapeutic targets. Figure 1 Figure 1. Disclosures Gökbuget: Pfizer: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.

Description: Abstract 1389 Overexpression of the ETS transcription factor ERG in subtypes of acute lymphoblastic and myeloid leukemias has been correlated with a poor prognosis. The underlying ERG mediated chemotherapy resistance of the leukemic cells may explain the link of poor outcome to ERG expression. We recently observed that ERG overexpression in K562 cells induced adhesion and morphological changes with elongated with bi-directional protrusions. This morphological transformation was mediated by WNT11, a direct target of ERG in acute leukemia. Herein, we determined the ERG transcriptional program responsible for cell adhesion and drug resistance potential. A genome wide transcriptional profiling was performed to identify candidate genes responsible for the observed cell shape changes. mRNA from doxycycline induced K562 cells harbouring tet-on inducible ERG expression constructs (pTRE-Tight-BI-DsRed-ERG) were analyzed with Affymetrix GeneChip (U133 2.0 plus). Non induced clones were used as a control. Genes significantly upregulated after ERG induction included WNT11 and genes associated with biological adhesion included CD44, ITGA10, FLT4, SELP, CD24, TYROBP and SHANK3. The upregulation (≥ 2-fold) of these potentially novel targets of ERG were also validated by RT-PCR. Interestingly, cell cultures of ERG induced cells incubated with a combination of both WNT11 and CD44 antibodies to block cell adhesion showed inhibition of the ERG induced morphological transformation. Furthermore, FACS analysis of ERG induced cells stained for Annexin V showed an increase in apoptosis (24%) by the addition of CD44 and WNT11 antibodies (dilutions 1: 250) whereas the no antibody control was measured at 60%) with the addition of CD44 and WNT11 (dilutions 1: 250) blocking antibodies to co-culture assays. Cell culture of ERG induced K562 cells with a stromal monolayer bed resulted in resistance to cytarabine (Ara-C) and to midostaurin (PKC412). Apoptosis evaluation of DsReD ERG/Annexin V double positive cells treated with either Ara-C (10–30μg/ml) or PKC412 (10–30μM) were measured by FACS at only 14% and 20%, respectively. In contrast, non induced K562 cultures (absent of ERG) had a significantly higher apoptosis induction range: 46% to 55% after exposure to Ara-C and 56% to 65% after exposure to PKC412. We conclude that direct contact and cell adhesion with stromal cells promote an ERG-dependent survival mechanism and mediate resistance to Ara-C or PKC412. In addition, the observed cell death effects by WNT11 and CD44 antibodies in cultures with ERG induced cells strongly indicate their involvement in ERG-dependent cellular adhesion. We propose that, in acute leukemia patients with aberrant ERG overexpression, ERG may mediate genetic and morphological transformation (i.e. cell adhesion) of leukemic cells resulting in an escape mechanism and the development of resistance to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3306 Introduction: Heat shock protein (HSP) 70 is aberrantly expressed in acute leukemias and other hematologic and solid malignancies, promoting tumor cell survival and therapy resistance. Recently, the small molecule pifithrin-μ (2-phenylethynesulfonamide) has been identified as a direct inhibitor of inducible HSP70, showing antiproliferative activity in different cell lines of solid tumors. Here, we analysed the in vitro antileukemic effect of pifithrin-μ in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines, as well as in primary AML blasts. In addition, incubations of pifithrin-μ with cytarabine, the histone deacetylase inhibitor SAHA, the HSP90 inhibitor 17-AAG, and the multikinase inhibitor sorafenib were performed to evaluate the potential use of combination therapies with pifithrin-μ in acute leukemias. Methods: Leukemic cell lines KG-1a (AML), K562 (CML in blast crisis), K562r (cytarabine-resistant K562), NALM-6 (B-lineage ALL), TOM-1 (B-lineage ALL, BCR-ABL pos.), Jurkat (T-lineage ALL), BE-13 (T-lineage ALL) and 9 bone marrow cell samples from newly diagnosed or relapsed AML patients were exposed to pifithrin-μ. Cell viability of all cell lines listed above was quantified by WST-1 assay. Subsequent functional analyses were performed on KG-1a and NALM-6 cells. Apoptosis was determined by annexin-V/7-AAD staining and subsequent flow cytometric analysis. Activated caspase-3 was detected by flow cytometry. Levels of the cell signaling kinase Akt were measured by intracellular staining and FACS analysis. Coincubations of pifithrin-μ with cytarabine, SAHA, 17-AAG or sorafenib were performed in KG-1a, NALM-6 and TOM-1, using WST-1 assays to analyse cytotoxic effects of combination therapies. Results: Pifithrin-μ at low micromolar concentrantions significantly inhibited viability of all acute leukemia cell lines tested, with IC50 values ranging from 2.5 to 12.7 μM independent of the differentiation lineage. Importantly, viability of both cytarabine-sensitive and -resistant K562 cells was effectively inhibited by pifithrin-μ. The median IC50 of primary AML blasts was 8.8 μM, ranging from 5.7 to 11.8 μM with no obvious differences regarding patients' clinical or genetic characteristics. Apoptosis was induced in a time- and dose-dependent fashion with a rate of specific apoptosis of 46% at 4 μM pifithrin-μ for NALM-6 and 36% at 40 μM pifithrin-μ for KG1a. In NALM-6, treatment with 3 μM pifithrin-μ for 24 hours resulted in a significant increase in the cleaved, active form of caspase-3, whereas in KG1a no increase in active caspase-3 was detected. Intracellular concentrations of Akt were markedly reduced after 12 hours incubation of NALM-6 with pifithrin-μ. In NALM-6, KG-1a, and TOM-1 combination treatment of pifithrin-μ at concentrations below the IC50 with either SAHA, 17-AAG or sorafenib resulted in a significant decrease of cell viability compared to corresponding monotherapy. Thus in NALM-6 combination of 2 μM pifithrin-μ with 0.6 μM SAHA inhibited viability by 73%, compared to 22% and 0% inhibition for either drug alone (p

Description: AML in the elderly has a very poor outcome. Despite advances in the characterization of molecular alterations in younger AML patients, comprehensive studies in elderly AML are still lacking to uncover their distinct underlying molecular alterations. In this project, we investigated genetic and epigenetic modifications to unravel the molecular background of this unfavorable disease. To capture a broad spectrum of relevant alterations in elderly AML, we performed genomic profiling by target enrichment of 555 candidate genes (mutated in cancer), followed by next generation sequencing on an Illumina HiSeq 1500 platform. Reads were mapped to NCBI hg19 RefSeq and SNV or INDELs were called if they were non-silent coding variations with a coverage of at least 〉 30x and a variant allele frequency higher than 20%. The mean coverage of the target sequence was 382±160x and 98±2% of the target sequence had a read depth of at least 30x. We applied this sequencing strategy on diagnostic bone marrow samples of 100 AML patients enrolled on the Studienallianz Leukämie (SAL) registry. This cohort was composed of patients 65 to 90 years old (median 72 years), 76% were classified as de novo AML, 19% as secondary AML, 5% as therapy-related AML. In addition, we investigated the DNA methylation profile for this cohort with an Illumina 450k methylation array to further characterize the heterogeneity of elderly AML. Overall, 817 mutations were detected in 292 of the 555 candidate genes, 80 of these genes were mutated in more than two patients. A median number of 7 genes were mutated per patient (range: 1 to 23). Several known mutations were identified with a particular high frequency: DNMT3A 33%, TET2 24%, SRSF2 23%, ASXL1 21%, RUNX1 18%, IDH1 17%, NPM1 15%, IDH2 and BCOR 10% each. We also identified novel aberrations (not previously reported in AML) that provide new insights into the specificity of this disease, including mutations in the PI3K/mTOR pathway (PIK3C2B, MTOR), DNA damage proteins (BRCA2, ERCC2, FANCC, PMS1) and histone modifiers (EP300, JARID2, NSD1, MYST3). When compared to younger AML (less than 65 years, TCGA cohort NEJM 2013), elderly AML showed significantly higher mutation rates in ASXL1 (21% vs 1%), TET2 (24% vs 7%), RUNX1 (18% vs 7%), BCOR (10% vs 1%) and BRCA2 (8% vs 0%). In addition, we found a high rate of mutations in splicing regulators affecting 38% of elderly AML patients (SRSF2 23%, U2AF1 6%, SF3B1 5%, DDX5 3%, ZRSR2 2%), similarly distributed between de novo and secondary AML. Notably, 15% of elderly AML patients had mutations in the DNA repair genes TP53, NBN, ATM, FANCA, FANCC, likely responsible for drug resistance and unfavorable outcome: patients with DNA repair mutations had a median survival of only 4 months, compared to 16 months for patients without these mutations (p=5.99e-5); variations in these DNA repair proteins predicted poor overall survival independently of the TP53 mutational status (p=0.004). To describe the molecular heterogeneity of the disease we included 152 genes (mutated in more than 1 patient) to build a reactome functional interaction network and identified 9 different network modules. The first most prominent module comprised 15 mutual exclusively mutated genes including DNMT3A and genes of the DNA repair pathway (p=0.013; purple network in figure). The second most distinct module included 12 genes also being altered in a mutually exclusive manner (p=4.9e-4): NPM1, RNA splicing and transport genes (green network in figure). Together the alterations from these two modules affected 88% of the patients and showed no significant overlap (mutual exclusivity test p=7.5e-4). These findings indicate that elderly AML is characterized by two distinct molecular patterns, with patients frequently having one of the two modules altered (mutual-exclusive mutation plot of the two modules in figure). In conclusion, elderly AML harbors a high frequency of molecular alterations in spliceosome components, epigenetic regulators and in DNA repair factors, the latter being associated with poor prognosis. The characterization of recurrent mutations may guide the development of new strategies to adapt treatment for older AML patients. In this regard, the molecular categorization of elderly AML into two groups (DNA repair or RNA processing deficient) underscores the distinct biology and the need for molecularly driven therapeutic approaches. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.