Metabolomic Profiling Demonstrates Postprandial Changes in Saturated Fatty Acids and Glycerophospholipids Are Associated With Fasting Inflammation

Objectives Metabolic flexibility is the responsiveness to heterogeneous physiologic conditions, such as food ingestion. A key unresolved question is how inflammation affects metabolic flexibility. Our study objective was to compare metabolic flexibility, specifically the metabolomic response to a standardized meal, by fasting inflammation status, among Guatemalan adults. Methods Participants (n = 302, median age 44 years, 43.7% men) received a standardized, mixed-macronutrient liquid meal. Plasma samples (fasting, 2 hr post-meal) were assayed by dual column liquid chromatography (reverse phase [C18] and hydrophilic interaction liquid chromatography [HILIC]) with ultra-high resolution mass spectrometry, and for concentrations of 6 inflammation biomarkers (hsCRP, leptin, resistin, IL-10, adiponectin, TNFsR). We summed the 6 fasting inflammation biomarker Z-scores, after reverse-coding of anti-inflammation biomarkers. For feature selection, we identified features with peak areas differing between fasting and post-meal (FDR-adjusted q < 0.05) and compared median log2 postprandial/fasting peak area ratios by inflammation indicators. Results 1,397 C18 and 967 HILIC features had significant log2 postprandial/fasting peak area ratios (q < 0.05). These log2 feature peak area ratios clustered differentially between people in the lowest tertile vs higher 2 tertiles of the inflammation index. The inflammation index was directly associated with log2 ratios of arachidic acid, and inversely associated with log2 ratios of caproic acid and lysophosphatidic acid, adjusting for age and sex (all P < 0.05). The log2 ratio of arachidic acid was negatively correlated with resistin, IL-10, adiponectin, and TNFsR concentrations (all P < 0.05). The log2 ratio of caproic acid was positively correlated with adiponectin and IL-10 concentrations, and negatively correlated with hsCRP concentration (all P < 0.05). The log2 ratio of lysophosphatidic acid was positively correlated with IL-10 and adiponectin, and log2 ratio of phosphatidylinositol was positively correlated with hsCRP (all P < 0.05). Conclusions Our findings showed that postprandial responses of saturated fatty acids and glycerophospholipids were associated with fasting inflammation. Funding Sources National Institutes of Health (HD075784, HL007745).

Single Nucleotide Polymorphism Array-Based Signature of Genetic Ploidy Groups in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is characterised by a number of recurrent chromosomal abnormalities which inform prognosis. Low hypodiploidy (HoTr) and high hyperdiploidy (HeH) are genetic subgroups associated with large non-random ploidy shifts, specifically 30-39 chromosomes and 51-65 chromosomes respectively. HoTr ALL often presents with a near triploid karyotype of 60-78 chromosomes through chromosomal endoreduplication without cytokinesis. This presents a diagnostic challenge in distinguishing this poor risk entity from good risk HeH ALL. To date, classification of such challenging cases has been based on the modal chromosome number, the pattern of specific gains, and identification of loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) arrays where possible. However, loss of cellular context and mixed cell populations (normal diploid, low hypodiploid and near-triploid) when analysing SNP arrays can pose additional analytical difficulty. The aim of this SNP array study was to (1) determine the level of inaccurate genetic subgrouping when cytogenetics only was used to distinguish HeH from doubled up HoTr and (2) develop a diagnostic algorithm to reliably call HeH and HoTr without supporting cytogenetic analysis. SNP arrays were performed on diagnostic ALL samples of 85 patients (46 HoTr, 39 HeH) using Illumina CytoSNP 850k (n=80) or Affymetrix Cytoscan HD (n=5) arrays. Probe level data were uploaded to Nexus Copy Number 10 (BioDiscovery) and manual segments spanning the length of each chromosome were created. No further data pre-processing was carried out and log2 ratio of 0 was automatically assigned to the median log2 ratio of the sample. Chromosomal log2 ratios were normalized within each sample and a variety of machine-learning techniques used to cluster the samples independently of assigned diagnosis. Cases residing in the incorrect cluster based on initial diagnosis were examined in detail using information from cytogenetics and SNP array interpretation. SNP arrays were analysed from 46 HoTr (median age 50.5 years (range 7-87), 43% male) and 39 HeH (median age 7 years (range 1-58), 56% male) patients. Unsupervised clustering of log2 ratios showed a clear distinction between HeH and HoTr patients (figure (A)). Six cases clustered incorrectly based on cytogenetic diagnosis. After detailed interpretation of all cases, including identifying LOH affecting chromosomes 3, 7, 15, 16 and 17, 3/6 cases initially classified as HeH were highly suggestive of HoTr ALL despite having <60 chromosomes. Similarly, 1/6 cases was cytogenetically diagnosed with HoTr but had a SNP array pattern typical of HeH ALL. We identified chromosomes 1, 4, 11, 17, 19, and 21 as those contributing most to the distinction in the HoTr and HeH signatures with the log2 ratio of chromosome 1 the most highly discriminatory in this cohort. Using whole chromosome log2 ratios, HoTr and HeH ALL have distinct profiles. SNP array analysis highlighted at least 4 patients whose ploidy subgroups appeared incorrectly called by cytogenetics, which can affect risk stratification. Crucially, these data call into question the accepted modal chromosome numbers for HeH and HoTr in the near triploid phase and suggest this alone cannot be used to classify patients into these ploidy groups. All samples incorrectly classified as HeH ALL were from adults aged >40 years, suggesting this good risk subgroup is even rarer than previously thought in older adults. After re-classification, our cohort only contained 5/41 adults >40 years with HeH ALL, signifying that HoTr ALL is the commonest genetic ploidy group in older adults with ALL and must still be considered in cases with 50-60 chromosomes. Copy number analysis from SNP arrays is challenging in samples with marked ploidy shift and mixed cell populations. Importantly, a number of our samples had significant contaminating normal DNA and LOH could not be confirmed visually (figure (B)), underlying the need for additional factors to aid classification. Our analysis only takes into account log2 ratio of entire chromosomes, thus permitting a measure of the relative over and under-representation of specific chromosomes within the sample. This method accurately clusters patients even when LOH cannot be clearly visualized from B-allele frequency due to contaminating non-leukemic DNA and supports the development of a diagnostic classifier based on chromosomal log2 ratios. Disclosures Fielding: Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Incyte: Consultancy.

Integrated Multi-Level Omics to Characterize Bortezomib Resistance in Multiple Myeloma

Introduction The use of proteasome inhibitors (PIs), such as bortezomib (BTZ), in multiple myeloma (MM) has markedly increased the survival of newly diagnosed patients. Although advancements in therapeutic regimens in the past decade have improved prognosis, we lack knowledge of the mechanisms that lead to drug resistance. To assess the contributors to BTZ-resistance, we integrated steady-state metabolomics, proteomics and gene expression from two naïve and BTZ-resistant cell line models. In addition, gene expression associated with ex vivo PI resistance has been analyzed. Potential predictive biomarkers of PI-resistance and novel targets for combination therapy will be investigated. Methods Parental cell lines, RPMI 8226 and U266, were acquired from ATCC. 8226-B25 and U266-PSR (kind gift from Dr. S. Grant) BTZ-resistant derivatives were selected from their respective parental naïve cell lines by chronic drug exposure. Untargeted metabolomics, activity-based protein profiling (ABPP), and expression proteomics data were acquired using liquid chromatography-mass spectrometry. Gene expression profiles of both cell lines and ex vivo patient specimens were derived from RNAseq. Metabolomics and proteomics data were normalized with iterative rank order normalization. Significantly different genes, proteins, and metabolites were integrated for pathway mapping and identification of biomarkers for PI resistance. Results Consistent with previous findings, kynurenine, a product of tryptophan catabolism, is significantly altered in both of our cell line models. In the 8226 and 8226-B25 pair, PI resistance was associated with increased kynurenine and positively correlated with TDO2 and IDO1 overexpression consistent with published literature (Li et al. Nature Medicine, 2019, 25, 850-60). As expected, PSMB2, a subunit of the proteasome, is overexpressed and has a higher activity in both 8226-B25 and U266-PSR in the ABPP and expression proteomics, and higher expression in 8226-B25 RNAseq data. PSMB2 is also overexpressed and significant in the RNAseq patient data, increasingly from newly diagnosed/pre-treatment to early relapse (p-value 2E-4) and late relapse (p-value 0.0052). In addition, CD38 is an enzyme responsible for conversion of NAD+ to nicotinamide and ADP-ribose. It has increased expression in MM cells and is significantly downregulated in ABPP (log2 ratio -4.25, p-value 2E-13), expression proteomics (log2 ratio -2.5), and RNAseq (log2 ratio -2.6, p-value 5E-6) in the 8226-B25 BTZ-resistant cells. In the steady-state metabolomics of the 8226-B25 cells, ADP-ribose (log2 ratio 4.11, p-value 2E-5) is the most upregulated known metabolite. This change suggests a downstream result of resistance within this interaction and a potential biomarker of PI resistance. However, gene expression of CD38 in patient samples was relatively unchanged. CD38 was not detected in the U266-PSR proteomics or RNAseq data and ADP-ribose (log2 ratio -0.63, p-value 0.06) was not significantly altered, suggesting a different mechanism of resistance in this cell line. Conclusions Though common mechanisms of PI resistance were identified, our data clearly show that BTZ-resistance arises by heterogeneous means in the two cell line models, promoting the need for biomarkers that can determine resistance and predict response in individual patients (or cohorts). Decreased expression of CD38 in 8226-B25 could elucidate mechanisms of PI resistance and immune response evasion strategies of MM cells. Further investigation of CD38 expression as a BTZ-resistance biomarker could lead to improving combination therapies with monoclonal antibodies, such as daratumumab, and PIs in newly diagnosed MM patients by predicting response prior to treatment. Further examination of ADP-ribose metabolism may lead to the mechanism of synergy between PARP inhibitors and proteasome inhibitors. Ultimately, we plan to integrate and utilize these multi-omics approaches in patient specimens and improve MM patient care by identifying PI resistance biomarkers to predict patient response. Disclosures Shain: Adaptive Biotechnologies: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

TP53 Alterations Are Frequent in Patients over 60 Years with B-Precursor Acute Lymphoblastic Leukemia (ALL) and Low Hypodiploid/Near Triploid (HoTr) Karyotype; They Correlate with RB1 Deletion and Leukemic Telomere Gain

Introduction TP53 is the most frequently altered gene in cancer. Until recently data on its frequency and prognostic impact in ALL has been scant, particularly in the elderly. TP53 alteration in the context of telomere shortening (the hallmark of aging) results in telomere attrition and genomic instability with resultant overexpression of telomerase to overcome the genomic crisis. Objective To determine TP53 alteration frequency and its association with cytogenetic subgroups, key B cell differentiation/cell cycle genes abnormalities and induction therapy response in ALL patients aged ≥60 and its impact on leukemic telomere state Methods Patients enrolled in the UK NCRI UKALL14 and UKALL60+ trials aged ≥60 years were assessed at diagnosis for 17p deletions by cytogenetics, TP53 alterations by FISH and/or direct sequencing of hot spots exons 5-8. Data were correlated with IKZF1, CDKN2A/B, PAR1, BTG, RB1, ETV6, EBF and PAX5 gene copy number status, assessed by MLPA (P335-B1). MYC-rearrangement was an exclusion criterion. Minimal residual disease (MRD) was assessed by quantitative PCR (qPCR) for Ig/TCR rearrangements or BCR-ABL1, where applicable. Leukemic DNA telomere length relative to that of remission DNA was assessed by monochrome multiplex (MM) qPCR and expressed as a log2 ratio. Using expectation maximization mixture model a log2 ratio >3.18 defined gain (reflecting telomerase overexpression) and values below this were defined as loss. Results The cohort included 63 patients; characteristics are presented in table 1. Median age was 63 years (range 60–83). TP53 state by cytogenetics and/or sequencing was available in all. Eleven had TP53 alteration (17%); TP53 mutation only, n=3; TP53 deletion only, n=6; TP53 deletion and mutation, n=2. Cytogenetics was available in 60 patients. Nine patients had HoTr (14%). Seven of 9 patients with HoTr had TP53 alteration (78%) versus 4/51 without HoTr (4%); p<0.001. MLPA was done in 57 patients. Those with TP53 alteration had more copy number alterations (CNA)(median 4 versus 1; p<0.01). Twelve (21%) had RB1 deletion; 3 were due to cytogenetically visible 13q abnormalities rather than ‘true' intragenic aberration. Four of 9 patients with TP53 alteration had RB1 deletion versus 8/51 without TP53 alteration, p=0.07. All other microaberrations were not significantly associated with TP53 alteration. Sixty-two achieved complete remission after 2 cycles of induction. MRD state (n=32) was not impacted by TP53 (2/5 with TP53 alteration and 13/27 without were MRD positive after 2 cycles of induction; p=0.563). Fourteen of 59 (24%) patients relapsed; median of four months from diagnosis (range 2–21), three had TP53 alteration versus 11 without TP53 alteration; p=0.65. Relapse occurred significantly earlier in patients with TP53 alteration (median two months versus eight months; p=0.03). Leukemic telomere state was assessed in 28 patients. Three of 4 patients with telomere gain had TP53 alteration versus 3/24 without; p=0.02 Conclusions This is one of the largest reports of TP53 frequency in older patients with non-MYC-rearranged ALL. TP53 was deleted and/or mutated in 17%, with highest frequency in those with HoTr (7/9). RB1 deletion was the commonest associated microaberration, noted in over a third of patients. TP53 alteration was associated with earlier relapse but had no impact on induction response. TP53 alteration was notably associated with frequent CNA and telomere gain suggesting a putative potential role of TP53 in genomic instability though sample size for the latter limits assertion of this hypothesis. Mechanistic work to shed insight into this model is currently underway. Table 1: Patient characteristics TP53 deleted and/or mutated n=11 TP53 wild type n=52 Age at Diagnosis, median(range) 63(60-75) 63(60-83) Gender; M:F 06:05 23:29 Phenotype, n (%) · Precursor B cell 11 (100) 51 (98) · Precursor T cell 0 (0) 1 (2) Cytogenetics (risk stratification as per Moorman et al. Blood 2007; 109:3189–97), n (%) Standard Risk 0 (0) 23 (44) t(9;22) 1 (9) 21 (40) t(4;11) 0 (0) 2(4) Ho/Tri 7 (64) 2(4) Complex 2 (18) 2 (4) Unknown 1 (9) 2 (4) Morphological Remission after Cycle 2 Induction, n (%) 11 (100) 51(98) Induction Deaths, n (%) 2 (18) 15 (29) CNA by MLPA, median (range) 4 (2-7) 1 (0-2) RB1 deletion, n (%) · Present 4 (36) 8 (15) · Absent 5 (45) 43 (83) · Unknown 2 (18) 1 (2) Leukemic Telomere State · Gain 3 (27) 3 (6) · Loss 1 (9) 21 (40) · Unknown 7 (64) 28 (54) Disclosures No relevant conflicts of interest to declare.

Gistic Evaluation in Sezary Syndrome

Abstract 4814 Sezary Syndrome (SS) is characterized by specific chromosomal abnormalities, however is not completely clarified yet which are the genetics hits associated to the initial phase or associated to disease progression. In this study, employing both high density Comparative Genomic Hybridization array (aCGH) and Single Nucleotide Polymorphism (SNP) array technologies, we elucidated the most frequent and significant chromosomal gain and loss regions, and new potentially relevant aberration for the disease progression onset. A total of 30 samples derived from 18 SS patients were analyzed on the Gene Chip Human Mapping 10K Array (Affymetrix). Genotype call and signal information performed by dChip SNP Software (http://www.dchip.org) provided us normalization and simultaneous measurement of Loss of Heterozygosity (LOH) and DNA Copy Number (CN) changes. We have further refined our analysis with a systematic method, Genomic Identification of Significant Targets in Cancer (GISTIC), able to identify regions that were significantly amplified o deleted, assigning a G-score that considers the amplitude of the aberration as well as the frequency of its occurrence across samples (http://genepattern.broadinstitute.org). Our data, generating by the integration of this two methods (see Table 1), revealed 19 significant focal event, including 10 amplification (10p, 17q, 8q, 6p, 4p, 1q, 18q, 21q, 3q, 1p) and 9 deletions (14q, 17p, 10p, 9p, 8p, 13q, 12p, 6q, 2p). In addition, new significant aberrant regions were identified, such as losses of 9p21.3, locus of an important tumor-suppressor gene CDKN2A (p16INK4a/p14ARF) and CDKN2B (p15INK4b). Recently, several study have reported the great influence of alteration in p16INK4a/p14ARF for prognosis of Diffuse large B-cell lymphoma (DLBL), primary cutaneous DLBCL leg-type, Blastic plasmacytoid dendritic cell neoplasm (BPDCN). Across our set of samples, 9p21.3 deletion occur in 8/30 samples from 6 patient, prevalently in heterozygosity state (Log2 ratio from −0.3484 to –0.7691). Two cases presented homozygous deletion (Log2 ratio from −1.2157 to –1.5342), and in four patients losses appeared only in a following phase of observation, suggesting a tumor progression event. Table 1 Significant regions of chromosomal gain Genes in Region Significant regions of chromosomal loss Genes in Region Cytoband Q value GISTIC peak Cytoband Q value GISTIC peak 10p12.33 8.07E-13 17765313-19264851 8 14q11.2 1.85E-40 21395737-22130391 1 17q11.1 3.54E-09 20147238-62034035 563 17p13.1 2.15E-10 1-15053486 234 8q22.3 2.93E-07 70887466-146274826 314 10p11.22 1.98E-07 30280444-32664641 8 6p25.3 0.0020062 1-1263549 6 9p21.3 0.00074825 21283632-22089567 11 4p15.32 0.0071803 17395812-20340589 4 8p23.3 0.00076308 1-32177352 179 1q23.3 0.039674 158849705-159641626 8 13q21.31 0.0028295 63681448-65120691 0 18q22.3 0.1913 1-76117153 251 12p13.1 0.025412 9304151-17414118 85 21q22.3 0.1913 1-46944323 223 6q24.1 0.029124 139319577-141635965 4 3q29 0.2191 169050880-199505740 163 2p23.3 0.043403 24636437-43910832 126 1p36.31 0.11633 1-20338260 239 Disclosures: No relevant conflicts of interest to declare.

Identification of Multiple Subclones in Peripheral T-Cell Lymphoma, Not Otherwise Specified with Genomic Aberrations

Abstract 298 Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) is a heterogeneous group of nodal and extra-nodal mature T-cell lymphomas that do not belong to any of the hitherto characterized T cell lymphoma subtypes. Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). PTCL, NOS with genomic aberrations has been shown to resemble lymphoma-type ATLL in terms of genomic aberration patterns, histopathology and prognosis (Clin Cancer Res., 15:30–38). We have recently shown that a majority of patients with acute-type ATLL have multiple subclones that were likely produced in lymph nodes (Blood, 117:5473–5478). In this study, high-resolution oligo-array comparative genomic hybridization (CGH) was employed to determine whether PTCL, NOS with genomic aberrations also possesses multiple subclones as found in ATLL. Thirteen cases of PTCL, NOS were available for 44K high-resolution array CGH analysis. These samples analyzed in this study were shown to be monoclonal by Southern blot analysis. We obtained average log2 ratios of regions where aberrations continued over 400 probes and showed a linear average ratio of >0.1 or <-0.1. The log2 ratio imbalance was defined when a patient had a different average log2 ratio for gain or loss regions. The results showed that 11 of the 13 cases (84.6%) had a log2 ratio imbalance, suggesting that multiple subclones exist in PTCL, NOS with genomic aberrations. As previously demonstrated, the average log2 ratio of aberrant regions reflected the ratio of tumors (Blood, 117:5473–5478). In one case, as represented in Figure 1, all tumor cells had regions of loss on chromosomes 3 and 13. Fifty-four percent of tumor cells had a region of loss on chromosome 16, and 83% of tumor cells had loss on chromosome 17. It is therefore speculated that clonal evolution took place as shown in Figure 2. In an effort to examine the association between multiple subclones and prognosis, we used our previous BAC array data for 24 cases and found that the presence of multiple subclones was associated with a poor prognosis (p=0.0279) (Figure 3). Our finding concerning the presence of multiple subclones in PTCL, NOS and ATLL should assist greatly in revealing the mechanism of lymphoma development, progression and drug-resistance associated with these diseases. Disclosures: No relevant conflicts of interest to declare.

Array Comparative Genomic Hybridization Revealed Polyclonality In Acute Type Adult T-Cell Leukemia/Lymphoma and PTCL NOS

Abstract 3096 Introduction: Adult T-cell leukemia/lymphoma (ATLL) is the neoplasia caused by Human T-cell leukemia virus type 1 (HTLV-1). HTLV-1-infected CD4+ T lymphocytes accumulate genetic and epigenetic changes during an extensive latency period, which can lead to neoplastic growth. Some studies have suggested that a rapidly growing clone in HTLV-1 provirus integrated T cells is selected, thus resulting in monoclonal expansion of more malignant tumor cells. In the present study, we analyzed ATLL samples using oligoarray comparative genomic hybridization (oligo-aCGH) and revealed the presence of multiple clones in the majority of ATLL patients investigated. Our data suggested that PTCL NOS is similar to ATLL with regard to polyclonal tumor cell expansion. Methods: We performed high-resolution oligo-aCGH (Agilent Technologies Santa Clara, CA, USA) containing a 44,000 probe set against 26 paired samples comprising peripheral blood and lymph node from 13 patients with acute type ATLL. Sixteen lymph node samples from PTCL NOS patients were also examined. Results: All of ATLL and PTCL NOS had genomic aberrations as determined by oligo-aCGH. 1) Polyclonality of ATLL oligo-aCGH analysis showed log2 ratio average imbalances among some chromosomes in 13 of the 26 ATLL samples. For example, in the case 1 lymph node sample (Figure 1), regions of gain were detected on chromosomes 2, 3, 7, 8 and 9 as shown by the log 2 ratio in parentheses: chromosome 2 (0.10), chromosomes 3 and 7 (0.41), chromosome 8 (0.25), and chromosome 9 (0.15). These results indicate that chromosome imbalance is present in one patient sample (Figure1). Importantly, in the case 1 peripheral blood sample, regions of gain were only detected on chromosomes 3, 7 and 8, with an associated log2 ratio of almost 0.53, suggesting the absence of no chromosome imbalance. These differential genomic aberration patterns between peripheral blood and lymph node samples were found in 9 out of 13 ATLL cases (Table 1). These cases strongly indicated that the majority of ATLL patients possess polyclonal tumors. 2) Clones were selected in the peripheral blood All cases examined had common genomic abnormalities between blood and lymph node samples from the same patient. However, cases 1, 2, 4, 8 and 12 indicated that selected clones from multiple clones of lymph node tumor cells had expanded in peripheral blood. A schematic representation of case 1 is shown in Figure 2. 3) Polyclonality of PTCL NOS Eight out of 12 PTCL NOS cases showed a log2 ratio imbalance, suggesting that similar polyclonal expansion is present in PTCL NOS. An evaluation of the four remaining cases was not possible since the log2 ratio imbalance was too low. Conclusions: Our results suggested that selected clones derived from a common origin may be present at the same time in acute type ATLL based on genomic analysis. The multi-clonal growth found in ATLL lymph node is also suggested to be present in PTCL NOS. Disclosures: No relevant conflicts of interest to declare.

Synthetic Lethal RNAi Screen of the Human Kinome with Cytarabine in Myeloid Leukemias.

Abstract 590 Background: To improve response and increase the number of patients responding to Ara-C, new genes/mechanisms/pathways need to be discovered that underlie and determine the sensitivity to Ara-C. We hypothesized that applying synthetic lethal RNAi screens of >570 kinases of the human kinome +/- Ara-C would be a fast and unbiased way to identify kinases that modulate sensitivity to Ara-C in myeloid cells. Methods: First, we developed a High-Throughput RNAi (HT-RNAi) platform for transient siRNA gene silencing in myeloid cells. Using lipid-based reverse transfection, TF1 and THP-1 myeloid cells were incubated with 2 different siRNA sequences per gene targeting 572 kinases (Tibes, EHA 2009). At 48 hrs, Ara-C at an IC30 dose was added and proliferation/viability was measured using a luminescence-based assay 48 hours after Ara-C treatment. Additionally, to assess high- vs. low dose Ara-C, a screen at an IC70 dose of Ara-C was performed in TF1 cells. Screens were performed in duplicate. The log2 ratio of each individual treatment of si+Ara-C over si alone was calculated [siRNA+drug : siRNA]. Hits were defined as the log2 ratio of individual siRNA >2 standard deviation (STD) below the median of all siRNA treatments (log2 ratios) on an individual plate or for the entire screen, respectively. Results: Transfection efficacy, determined by a universal lethal siRNA vs. non-silencing and buffer control, was between 75-80% for TF1 and >85% for THP-l. Correlation was high, i.e. for THP-1 (same Ara-C dose) with R2=0.67 for duplicate screens. Data was first analyzed to identify reproducible hits across independent duplicate RNAi screens for each cell line. Hits were defined of at least one same sequence siRNA reducing viability in combination with Ara-C in both (duplicate) screens to greater than 2 STD compared to siRNA or Ara-C alone (see also Methods). For TF1, 9 genes (1.6%) met these criteria. The top hit in TF1 was ATR for which all 4/4 siRNA sensitized to Ara-C; 4 genes each were positive for 3/4 and 2/4 siRNA. For THP-1, 8 genes were hits (7 genes with 2/4 siRNA and 1 gene with 3/4 siRNA) representing 1.4% of the entire screen. Strong hits in TF1 included ABL-1 and CHEK1 and for THP-1, ABL-2 and CHEK1. Apart from the top hit CHEK1, only one other gene reduced proliferation/viability in combination with Ara-C across both cell lines and thus, may be a novel universal sensitizer to Ara-C. This gene is centrally involved in regulating trafficking of membrane and receptor kinases of the MAPK family/pathways. Comparing high- vs. low-dose Ara-C, 4 genes exhibited sensitization to Ara-C at low concentrations only, and 10 genes at high Ara-C concentrations (each 2/2 si). Genes sensitizing at low Ara-C are involved in cell-cycle (3 of 4 genes); at high Ara-C, KEGG pathway analysis showed enrichment for purine, pyrimidine, IGF and MAPK pathways. In secondary functional validation screens using 4 siRNA sequences, CHEK1 has validated, whereas cell-cycle regulatory genes (i.e. AURKB) have not to date. In preliminary experiments, treatment of myeloid cells with CHEK1 inhibitors sensitized to Ara-C. A list of high-priority candidate targets is assembled and targets are being validated. Conclusions: Herein, we present the first large-scale kinase RNAi synthetic lethal screen in myeloid cells in combination with Ara-C. Only few kinases, 1-2% of the kinome, were identified whose silencing sensitizes to Ara-C. Two genes were potential universal sensitizers in AML: CHEK1, for which inhibitors are in early clinical development including in AML. We are currently conducting clinical trials with a novel, specific well tolerated CHEK inhibitor plus chemotherapy in solid tumors. Supported by our data we are encouraged to translating this concept into design of combination trials of CHEK1 inhibitor with Ara-C in AML. A potential novel sensitizer gene to Ara-C was identified that regulates kinase membrane trafficking and can activate the MAPK pathway. Validation of this gene is currently ongoing. The initial finding that distinct genes may modulate response at different Ara-C doses is of importance and could have clinical implications in the future. Functional genomics provide a fast and attractive approach to identify molecular targets and vulnerabilities in AML. RNAi synthetic lethal screens in leukemias hold promise for design of rational combination therapies that can be rapidly translated into the clinic. Disclosures: Tibes: Arizona Biomedical Research Commission: Peer reviewed grant funding for the presented project.

Identification of SGK1 as a novel predictive marker for treatment response in medulloblastoma

10006 Background: We have recently demonstrated that both gains (5-year OS 16%) and losses (5-year OS 100%) of chromosome 6q define distinct clinical and biological subgroups of medulloblastoma. However, the underlying molecular mechanisms accounting for the prognostic impact of these aberrations so far remained unclear. Methods: To identify potential candidate genes on chromosome 6q targeted by these genomic aberrations, a genome wide mRNA expression profiling study was performed for 47 well-characterized medulloblastoma samples and correlated to chromosome 6q DNA copy-number status. Results were validated by real-time quantitative PCR (RTQ-PCR). SGK1 protein expression was examined by immunohistochemistry on a tissue-microarray which represents a large independent cohort of uniformly treated medulloblastoma patients (n = 260). Results of both studies were correlated with survival data available for all investigated samples. Results: Expression profiling revealed the AKT homologue SGK1 as an interesting candidate gene on chromosome 6q showing very close correlation to 6q copy-number status. Direct correlation could be confirmed by RTQ-PCR showing that SGK1 mRNA levels were significantly upregulated in tumors with gain of chromosome 6q (median log2-ratio when normalized against normal cerebellum = 3.88) and, in contrast, downregulated in cases carrying deletions of this region (median log2-ratio = -1.97). On protein level, we found high SGK1 protein levels to be highly associated with poor overall survival as assessed by immunohistochemistry (Kaplan-Meier method; p < 0.00001). Conclusions: We identified SGK1 as an interesting novel candidate gene on chromosome 6q contributing to the striking differences in overall survival of cases with chromosome 6q gain or loss, who are known to have a very poor or especially good prognosis, respectively. The fact that SGK1 encodes for a serum- and glucocorticoid inducible kinase which is both structurally and functionally homologous to the antiapoptotic kinase AKT further underlines the potential role of this gene. Based on the reported results, we propose SGK1 protein expression as a novel prognostic marker in medulloblastoma. No significant financial relationships to disclose.