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To study the prognostic relevance of rare genetic aberrations in acute myeloid leukemia (AML), such as t(16;21), international collaboration is required. Two different types of t(16;21) translocations can be distinguished: t(16;21)(p11;q22), resulting in the FUS-ERG fusion gene; and t(16;21)(q24;q22), resulting in RUNX1-core binding factor (CBFA2T3). We collected data on clinical and biological characteristics of 54 pediatric AML cases with t(16;21) rearrangements from 14 international collaborative study groups participating in the international Berlin-Frankfurt-Münster (I-BFM) AML study group. The AML-BFM cohort diagnosed between 1997 and 2013 was used as a reference cohort. RUNX1-CBFA2T3 (n = 23) had significantly lower median white blood cell count (12.5 × 109/L, P = .03) compared with the reference cohort. FUS-ERG rearranged AML (n = 31) had no predominant French-American-British (FAB) type, whereas 76% of RUNX1-CBFA2T3 had an M1/M2 FAB type (M1, M2), significantly different from the reference cohort (P = .004). Four-year event-free survival (EFS) of patients with FUS-ERG was 7% (standard error [SE] = 5%), significantly lower compared with the reference cohort (51%, SE = 1%, P < .001). Four-year EFS of RUNX1-CBFA2T3 was 77% (SE = 8%, P = .06), significantly higher compared with the reference cohort. Cumulative incidence of relapse was 74% (SE = 8%) in FUS-ERG, 0% (SE = 0%) in RUNX1-CBFA2T3, compared with 32% (SE = 1%) in the reference cohort (P < .001). Multivariate analysis identified both FUS-ERG and RUNX1-CBFA2T3 as independent risk factors with hazard ratios of 1.9 (P < .0001) and 0.3 (P = .025), respectively. These results describe 2 clinically relevant distinct subtypes of pediatric AML. Similarly to other core-binding factor AMLs, patients with RUNX1-CBFA2T3 rearranged AML may benefit from stratification in the standard risk treatment, whereas patients with FUS-ERG rearranged AML should be considered high-risk.

Pediatric Acute Myeloid Leukemia (AML) is a rare, but deadly cancer. Outcomes over the last 20 years have remained stagnant with an overall 5-year survival rate < 70% and relapse rates around 50%. Further, few new therapies have been successfully introduced to improve these outcomes. Here we report that exploiting deficiencies in DNA damage repair (DDR) is a potential therapeutic strategy for AML. Poly-ADP Ribose Polymerase (PARP) inhibitors were initially developed to target deficient homologous recombination (HR) in BRCA1/2 mutated cancers by blocking single stranded base repair following DNA damage, leading to an accumulation of double stranded DNA breaks, thereby inducing apoptosis. To evaluate the activity of PARP inhibition in pediatric AML, talazoparib was tested as a single agent and in combination with standard chemotherapeutic agents in human AML cell lines representing low (Kasumi-1 and ME-1), intermediate (AML193), and high-risk (CTS, CMS, MOLM-13, and CHRF288-11) disease based on their genomic mutations. Talazoparib showed the highest efficacy as a single agent in all four cell lines with genomic lesions found in high-risk AML subtypes. After combination drug screens, topotecan (synergistic) and gemcitabine (additive) were chosen to move forward to in vivo testing. Our investigational combination was tested in vivo in four murine models representing pediatric AML subtypes harboring AML1-ETO9a (low risk), MLL-AF6 (high risk), CBAF2T3-GLIS2/JAK2 V617F (high risk) and NUP98-KDM5A (high risk) oncogenes. Mice received a backbone of either current standard of care chemotherapy (SOC; anthracycline plus cytarabine) or topotecan plus gemcitabine. NUP98-KDM5A and MLL-AF6 positive mice receiving single agent talazoparib were found to have prolonged survival compared to vehicle alone (p=0.019 and p Synergy experiments with ATM inhibitor AZD0156 demonstrated tremendous synergy with talazoparib in sensitive cell lines with almost no synergy in those that were resistant, suggesting that sensitive cell lines are unable to efficiently activate the HR pathway to repair double stranded breaks induced by PARP inhibition whereas resistant cells can overcome inhibition. To determine the HR response to DNA damage in our cell lines, we exposed them to 1uM topotecan for 2 hours and then measured γH2AX response at 0, 4 and 24 hours. γH2AX is a sensor of DNA damage and therefore increases with DNA damage and decreases with repair. PARP inhibitor sensitive cell lines had persistence of gamma H2AX at 24hrs while resistant cell lines had at least partial resolution of damage, confirming that PARP inhibitor sensitive cell lines have aberrant DNA damage response through HR. RNA sequencing of our cell lines revealed a correlation between Phosphatase and tensin homolog (PTEN) transcript levels and PARP sensitivity. Western blotting confirmed that PTEN was downregulated or absent in both cell lines and murine leukemias that were sensitive to PARP inhibitors. In contrast to the CMS cell line that carries the CBFA2T3-GLIS2 fusion, murine leukemias with CBAF2T3-GLIS2/JAK2 V617F had high levels of PTEN, supporting the hypothesis that sensitivity to PARP inhibitors is due to loss of PTEN. In conclusion, we report that a subset of pediatric AML with high- risk features are sensitive to PARP inhibition due to deficient DDR through HR. Downregulation of PTEN is a candidate biomarker of response to PARP inhibitors in these patients. This data illuminates a promising therapeutic vulnerability in a patient population where new targeted treatments are vital to improve outcomes. Disclosures No relevant conflicts of interest to declare.

Rearrangements in KMT2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis, with an event-free-survival (EFS) of 33.6-36.9%. In the context of the remarkable improvements in the treatment of childhood ALL, the dismal outcome of infantile KMT2Ar ALL and the lack of any significant progress for more than two decades are striking . The St. Jude Total Therapy 16 study (the most recently reported study of a program for childhood ALL that begun in 1962) yielded a 5-year EFS of 88.2% overall. Total 16 enrolled all subtypes of newly diagnosed pediatric ALL patients including infants, with intensity of treatment adapted to presenting clinical and genomic features, and early treatment response as determined by minimal/measurable residual disease (MRD). KMT2Ar infants were treated on an intensified high-risk arm and received clofarabine in combination with cyclophosphamide and etoposide (CCE) at two points during treatment: Induction days 22-25 and Reinduction I. Infants who lacked KMT2Ar and KMT2Ar patients who were one year of age or older received the same risk-directed treatment plan given to all other patients enrolled on study. A total of 28 patients with KMT2Ar were enrolled on Total 16; the 19 patients > 1 year of age received standard-risk therapy, and the 9 patients < 1 year of age received high-risk therapy on the infant arm with CCE. The probabilities of 5-year EFS and overall survival in KMT2Ar patients > 1 year of age and those < 1 year of age were 73.3% vs. 44.4% (p=0.071) and 84.2% vs. 55.6% (p=0.060), respectively. Six of the nine infants were MRD-positive on Induction day 15 prior to CCE (MRD-positive range, 0.012% to 13.7%; median, 2.13%) with MRD negative status ( 1 year of age and 12.5% for those < 1 year of age (p=0.454). Five infants remain alive (four in CR1, one in CR2), while four expired in CR1. Three deaths were secondary to infection, including a multi-drug resistant soft tissue bacterial infection during Induction days 1-21, a respiratory syncytial virus pneumonia during Reinduction II, and a chronic parainfluenza 3 infection during Continuation weeks 70-101 that led to chronic pneumonitis and interstitial fibrosis. The fourth patient developed grade 5 pulmonary hypertension following induction, a complication potentially compounded by their presenting WBC count of 905 x 10 9/L and pulmonary leukostasis. A comparison of 3-year cumulative risk of selected major toxic effects of treatment revealed that high-risk infants had a lower incidence of asparaginase allergic reactions, osteonecrosis, hyperglycemia, and pancreatitis; in contrast, the incidence of fever and neutropenia, hepatic toxicity and seizures, was similar in high-risk patients regardless of age. Infants had a higher risk of thrombosis (46.7% vs. 23.1%, p 1 year of age that received one or more clofarabine-containing Reintensification chemotherapy cycles prior to hematopoietic stem cell transplant in first remission (CR1). This revealed a higher frequency of infections in infants, suggesting a greater susceptibility to this complication independent from clofarabine exposure (mean number of episodes, 2.39 vs. 1, p Disclosures Gruber: Kura Oncology: Consultancy. Coustan-Smith: Juno Therapeutics: Patents & Royalties; Nkarta Therapeutics: Current holder of individual stocks in a privately-held company; Medisix Therapeutics: Current holder of individual stocks in a privately-held company. Campana: Nkarta Therapeutics: Current holder of stock options in a privately-held company; Medisix Therapeutics: Current holder of stock options in a privately-held company; Juno: Other: patent licensing payments; Juno Therapeutics (a Bristol-Myers Squibb company),: Other: patents on methods for minimal residual disease detection.. Evans: St. Jude Children's Research Hospital, Emeritus Member (began Jan 2021): Ended employment in the past 24 months; BioSkryb, Inc.: Membership on an entity's Board of Directors or advisory committees; Princess Máxima Center for Pediatric Oncology, Scientific Advisory Board, Chair: Membership on an entity's Board of Directors or advisory committees. Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee.

CBFA2T3-GLIS2 is an oncogenic fusion gene that acts as the driver mutation in ~18% of pediatric patients with acute megakaryoblastic leukemia (AMKL) that do not have Down syndrome. CBFA2T3-GLIS2 driven leukemia carries the worst prognosis among all pediatric acute myeloid (AML) subtypes, with event free and overall survival probabilities of 8% and 14% respectively. Chemotherapy and stem cell transplantation fail to cure this disease, thus innovative treatment approaches are needed. While monoclonal antibody and chimeric antigen receptor T-cell (CAR-T) therapies have proven successful in acute lymphoblastic leukemia (ALL), similar successes haven't been realized in AML due to the lack of targetable antigens that eradicate leukemia cells while minimizing off target toxicities such as depletion of normal myeloid cells which can lead to prolonged neutropenia. An alternative is T cell receptor engineered T cell (TCR-T) immunotherapy which use heterodimers consisting of alpha and beta peptide chains to recognize polypeptide fragments presented by MHC molecules on the tumor cells. An advantage of this approach is the ability to recognize intracellular tumor specific and tumor associated antigen fragments in addition to extracellular proteins which results in a wider range of targets. To investigate the immunogenicity of CBFA2T3-GLIS2 positive AMKL cells, we established a humanized patient derived xenograft (PDX) murine model by conditioning 15 immunodeficient NSG-SGM3 mice with 200 rads 24 hours prior to transplantation of 3.5 million CBFA2T3-GLIS2 positive PDX cells derived from an individual female patient. After 20 days of engraftment, 2.5 million HLA class I matched PBMCs from an unrelated female donor were introduced. Experimental mice (engrafted with both PDX cells and PBMCs), leukemia only control mice, and PBMC only control mice were sacrificed at days 30, 35, and 40. Flow cytometry analysis at day 40 showed dramatic reduction of leukemic blasts (huCD45lo, huCD34+) when exposed to PBMCs compared to the leukemia only control (1.48% and 93.7%, respectively). Circulating human Granzyme A was elevated at day 40 in the PDX mice with PBMCs compared to the PBMC only control (9665.4 pg/ml vs. 789.7 pg/ml, p Disclosures Gruber: Kura Oncology: Consultancy.

CBFA2T3-GLIS2 is the most prevalent fusion oncogene in pediatric acute megakaryoblastic leukemia patients without Down syndrome and is associated with an event free survival of only 8%. A cryptic inversion event on chromosome 16 joins the three nervy homology regions (NHR) of CBFA2T3 to the five zinc fingers of GLIS2. This configuration enables the encoded chimeric transcription factor to bind GLIS2 consensus sequences throughout the genome and recruit transcriptional activators and repressors to alter gene expression and enhance self-renewal capability. Few cooperating mutations have been identified in patients harboring this fusion which suggests it is the sole oncogenic driver. The molecular mechanism by which CBFA2T3-GLIS2 drives leukemogenesis is not fully understood. Identification of components critical to the transcriptional complex and their role in gene regulation may reveal novel therapeutic targets to improve patient outcomes. Studies on the wild type CBFA2T3 and GLIS2 proteins have demonstrated interactions with the transcriptional regulators ETO and CtBP1 respectively. Further p300 has been shown to play a role in transcriptional regulation imparted by both transcription factors. We therefore hypothesize the fusion promotes transcriptional activation when the histone acetyltransferase p300 and the transcription factor ETO are recruited through NHR1 and NHR2, respectively. When the co-repressor CtBP1 is recruited through the PXDLS motif, located in the GLIS2 portion of the fusion, transcriptional repression predominates. Association of these co-factors with the fusion was confirmed through co-immunoprecipitations. Site-directed mutagenesis was then used to systematically delete NHR1 and NHR2 and mutate the PXDLS motif to evaluate the resultant effects on transcriptional regulation, self-renewal, and leukemogenesis imparted by the fusion. A luciferase reporter assay was used to assess transcriptional activation of the BMP2 promoter, a gene which is known to be upregulated by the CBFA2T3-GLIS2 fusion. Loss of NHR1, NHR2, or NHR3 did not alter the ability of the fusion to activate transcription. In contrast, loss of NHR1 and NHR2 in combination (NHR1-2Δ) and mutation of the PXDLS domain decreased transcriptional activation compared to the wild type fusion. The effect of the mutations on self-renewal capability was then evaluated through colony formation assays. Consistent with the luciferase reporter assay, NHR1-2Δ and PXDLS mutants decreased the number of colonies at week six compared to the unmanipulated fusion. Next, we investigated the effect of these mutations on leukemogenesis. Murine models harboring the CBFA2T3-GLIS2 fusion without cooperating mutations have been unsuccessful and patient-derived xenograft models are limited and difficult to manipulate. Therefore, we developed a novel in vivo model of CBFA2T3-GLIS2 driven leukemia. CD34+ stem cells were isolated from human cord blood and transduced with a lentivirus construct encoding the fusion and a GFP reporter. The cells were then differentiated to megakaryoblasts using human TPO and IL1-beta and sorted for purity prior to injection into immunodeficient NSG-SGM3 recipient mice. The fusion positive human primary megakaryoblasts induced a serially transplantable leukemia within 180 days that recapitulates CBFA2T3-GLIS2 positive patient specimens on a transcriptional and protein level. In contrast to our in vitro studies where NHR2 deletion alone did not alter transcriptional activation and self-renewal, the loss of this domain abrogated leukemogenesis in vivo, suggesting a dependency on the association of ETO with the fusion. Mice that received PXDLS mutant cells, however, developed leukemia at a normal latency suggesting that CtBP1 is not required. This study confirms the CBFA2T3-GLIS2 fusion is sufficient for oncogenic transformation of human CD34+ stem cells. We demonstrate that disruption of ETO, p300, and CtBP1 recruitment to the transcriptional complex decreases transcriptional regulation and self-renewal imparted by the fusion. The loss of ETO was the most detrimental to leukemogenesis in our murine model, uncovering a potential new pathway for the development of targeted therapies. Ongoing studies include CUT&RUN sequencing for the fusion, ETO, and CtBP1 to determine co-occupancy of target genes to further understand those that are critical in transformation. Disclosures Gruber: Kura Oncology: Consultancy.

Myeloid sarcoma is a tumor mass consisting of blasts at extramedullary sites, most frequently the skin and soft tissues. Skin involvement, also termed leukemia cutis, is a well-described entity in congenital leukemia and is present in approximately 50% of patients diagnosed within the first 4 weeks of life. Most cases of congenital leukemia cutis harbor KMT2A rearrangements, as do up to 80% of cases of infantile leukemia, and are typically accompanied by bone marrow involvement. Here we describe a rare CIC-rearrangement as the driver mutation for a case of congenital cutaneous myeloid sarcoma in a newborn female who was noted at birth to have a violaceous, nodular rash. Skin biopsy of the nodular rash shortly after birth with immunohistochemical staining was consistent with myeloid sarcoma. Bilateral bone marrow aspirates and biopsies were also performed, but failed to reveal an aberrant blast population by morphology and flow cytometry. Cytogenetics of the cutaneous myeloid sarcoma was significant for t(10;19)(q23;q13.2) and fluorescence in situ hybridization performed on the tumor confirmed a CIC-rearrangement. CIC-rearranged sarcomas are a new entity of undifferentiated small round cell sarcoma characterized by fusion events involving the CIC gene but are not a known to be driver mutations in myeloid neoplasms. Despite the absence of a blast population in the bone marrow, 75% of analyzed metaphases carried the t(10;19)(q23;q13.2). To further explore the underlying genomic events, whole genome, exome, and transcriptome sequencing was performed on both sarcoma and bone marrow specimens. Sequencing revealed an in-frame CIC-NUTM2A fusion gene present in both the skin and the bone marrow which has been previously described in a single case of undifferentiated soft tissue sarcoma. There were no large-scale chromosomal losses or gains and no copy neutral loss of heterozygosity events. Two exonic single nucleotide variations (SNVs) were detected, both of which were limited to the skin sarcoma and not predicted to be pathogenic. In vitro and in vivo modeling demonstrated that the CIC-NUTM2A fusion protein was highly leukemogenic. Colony forming assays performed using transduced murine bone marrow revealed that the CIC-NUTM2A fusion conferred self-renewal in contrast to CIC, NUTM2A, and the reciprocal NUTM2A-CIC product which failed to serially replate (p To further understand the acquisition of a malignant phenotype by morphology between the bone marrow and the sarcoma, targeted deep sequencing was performed for all tier 1, 2 and 3 mutations identified by whole genome and exome sequencing. A shared ancestral clone was identified in both specimens along with three subclones specific to the sarcoma. Four SNVs acquired in the sarcoma were found to be present in regulatory regions of genes that were also differentially expressed between the sarcoma and the bone marrow including C6orf120, SMURF1, TJAP1, and PID1. PID1 was found to be downregulated in the malignant sarcoma specimen and has been previously shown to be a regulator of the AKT/PI3K pathway. Low PID1 expression has been associated with poor outcomes in other malignancies, including pediatric glioblastoma. A genome wide CRISPR screen of our CIC-NUTM2A positive murine leukemia cells revealed an enrichment for PID1 deficient cells as well as PTEN confirming the cooperativity between CIC-NUTM2A and the AKT/PI3K pathway (p=0.009 and p=0.005 respectively). In conclusion, we describe a rare fusion gene, CIC-NUTM2A, which leads to an aggressive myeloid malignancy in both humans and mice. Targeted deep sequencing demonstrated the clonal evolution from the bone marrow and acquisition of a cooperating mutation targeting the AKT/PI3K pathway in a subset of extramedullary cells that led to morphologic transformation. A thorough interrogation of the bone marrow in patients with myeloid sarcoma is warranted even in the absence of morphologic and immunophenotypic blasts by flow cytometry to optimally track residual disease during treatment. Disclosures No relevant conflicts of interest to declare.

Introduction: Cytarabine, daunorubicin and etoposide (ADE) are commonly used for remission and intensification of pediatric acute myeloid leukemia (AML). However, development of drug resistance is a major cause of treatment failure. In this study, we performed a comprehensive evaluation of expression levels of genes of pharmacological significance (pharmacokinetic /pharmacodynamic) to ADE and derived a drug response score predictive of treatment outcomes in pediatric AML patients. Methods: This study included 163 cases (median age=8.79 year, range= (0.013-21.1)) with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression and clinical data available. We used a penalized LASSO regression algorithm (glmnet R-package) to fit a cox regression model on diagnostic leukemic cell gene expression levels of 66 genes of pharmacological significance to ADE. We performed 1000 bootstraps of LASSO regression with event free survival (EFS) as the outcome variable and the five genes represented in at least 95% of the models were included to build an ADE-Response Score (ADE-RS) equation. Patients were classified into low or high score groups using recursive portioning implemented in Rpart-R package and evaluated for association with minimal residual disease after induction I (MRD1), EFS and overall survival (OS). ADE response score equation was further validated using RNA-Seq gene-expression data obtained from diagnostic samples of 432 pediatric AML patients enrolled in Children's Oncology Group (COG) AAML0531 and AAML03P1 treatment protocols. Results: After applying LASSO regression, we defined the equation: ADE-RS = (0.128 x DCTD) - (0.0993 x TOP2A) + (0.212 x ABCC1) - (0.113 x MPO) - (0.126 x CBR1) to develop ADE-response score (ADE-RS), followed by classifying patients into low (60%; 98 patients) or high (40%; 65 patients) score groups. Patients in the high ADE-RS group had significantly worse EFS (HR=4.07(2.43-6.84), P < 0.0001; Figure 1A) and OS (HR= 4.54(2.42-8.49), P In a multivariable cox-regression models that included pLSC6-ADE response score groups, MRD1 status, risk groups, WBC at diagnosis and age in AML02 cohort, high pLSC6-ADE score group was found significantly associated with poor EFS (HR=6.02(2.71-13.2), P Discussion: In this study, we defined a pharmacological response score focused on key genes of PK/PD significance to ADE. We further integrated LSC score with the ADE response score to improve our ability to predict treatment outcome in AML patients across different clinical trials. ADE-RS was composed of five genes: DCTD, which is a deaminase, involved in ara-C inactivation; CBR1, a carbonyl reductase involved in inactivation of daunorubicin (DNR); MPO, myeloperoxidase, an etoposide activator; ABCC1, an efflux transporter of DNR and etoposide; and TOP2A, DNA topoisomerase II alpha, which is a target for DNR and etoposide. Integrated pLSC6 and ADE-RS has a potential to predict treatment outcomes using diagnostic gene expression levels and accordingly develop treatment strategies to improve treatment outcome. Figure 1 Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding.

Acute myeloid leukemia (AML) comprises a heterogeneous group of malignancies that are linked by the presence of blasts displaying morphologic and immunophenotypic features of myeloid cell differentiation. With the development of genome-wide gene expression profiling (GEP), array-base comparative genomic hybridization methodologies, and next generation sequencing technologies, the field has gained a greater understanding of the molecular features of this malignancy. Several pathologic lesions have been found to have prognostic implications contributing to a continuous refinement of risk stratification in the context of modern therapy. Recently, the Children's Oncology Group (COG)-National Cancer Institute (NCI) TARGET AML initiative molecularly characterized 993 pediatric AML cases including 197 specimens that underwent comprehensive whole genome sequencing. Of these, 94 carried one of three oncogenic fusions known to be strong drivers of leukemogenesis: RUNX1-RUNX1T1, CBFB-MYH11 and KMT2A rearrangements (KMT2Ar). Among all the alterations detected only ten occurred in more than 5% of subjects, all of which had been previously described. This suggested that low-frequency molecular subsets may exist that require larger cohorts to fully elucidate. To address this limitation, we selected 122 pediatric AML specimens that lacked RUNX1-RUNX1T1, CBFB-MYH11 and KMT2Ar by clinical testing for whole genome (WGS), exome (WES) and RNA (RNAseq) sequencing to enrich for cases that carry low-frequency events. GEP coupled with somatic mutation calls and outcome data were utilized to identify distinct molecular subtypes with prognostic implications. Structural variations, copy number alterations, single nucleotide variations and indels were determined by our established pipelines, as well as an evaluation for regulatory rearrangements driving oncogene overexpression through enhancer hijacking. In addition to known AML somatic mutations and rearrangements in genes such as CEBPA, GATA2, NPM1, WT1, FLT3, NRAS, KRAS, ETV6, Cohesin, NUP98 and KAT6A, we identified rare novel events in known oncogenic drivers. These include a GATA2-ITD as well as the repositioning of a distal MYC enhancer to ectopically activate either the MECOM or BCL11B loci. Interestingly, several AML cases carrying loss of function mutations in polycomb repressive complex 2 (PRC2) genes were found to resemble an early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) GEP by gene set enrichment analysis. ETP-ALL exhibits aberrant expression of stem cell and myeloid markers and has been shown to have a GEP consistent with transformation of a stem cell progenitor. Further, mixed phenotype acute leukemias (MPAL) with T and myeloid lineage characteristics have been previously suggested to be in this spectrum of immature leukemias. We therefore hypothesized that these PRC2-mutated AML cases represented the myeloid end of this continuum. To provide global transcriptional context to these ETP-like AMLs and evaluate a comprehensive cohort encompassing a range of pediatric myeloid malignancies, we integrated results from previously published AML, MPAL, acute megakaryoblastic Leukemia (AMKL), and ETP-ALL datasets that had RNAseq and either WES or WGS available for a total of 436 cases. t-SNE visualization using a 381 gene list derived from the top 100 most variably expressed transcripts within each cohort revealed a clear molecular classifier identifying groups that had consistent mutational compositions and disease outcomes but were agnostic of immunophenotype. This approach allowed the distinction of 63 ETP-like cases comprising a mixture of AML, MPAL, and ETP-ALL leukemias which fell into two subgroups distinguished by FLT3-ITD and PRC2 alterations. Irrespective of treatment approach, FLT3-ITD positive ETP-like leukemias enjoyed a favorable outcome whereas those with PRC2 mutations had a poor prognosis. Our data support a refined classification of pediatric myeloid malignancies based on molecular determinants that can be used for risk stratification in therapeutic trials. Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding. Reinhardt:Jazz: Other: Participation in Advisory Boards, Research Funding; CSL Behring: Research Funding; Novartis: Other: Participation in Advisory Boards; Roche: Research Funding. Locatelli:bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zwaan:Roche: Consultancy; Servier: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy; Sanofi: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Celgene: Consultancy, Research Funding; Jazz pharmaceuticals: Other: Travel support; Janssen: Consultancy; Incyte: Consultancy.

Therapy-related myeloid neoplasms (tMN) occur in children secondary to cytotoxic therapies used to treat pediatric malignancies, are typically resistant to conventional chemotherapy, require hematopoietic cell transplantation as the only curative option, and have a dismal prognosis. The genomic alterations that drive tMN in children have yet to be comprehensively described, and it is unclear if particular genomic lesions hold prognostic value. We have characterized the genomic profile of 62 pediatric tMN cases (tMDS: n=23, tAML: n=39) obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1987 and 2018. These cases arose following treatment for a variety of primary tumors (hematological (74%), bone and soft tissue (23%), and brain (3%)). Acute lymphoblastic leukemia was the most frequent primary tumor (n=39, 63%). Conventional cytogenetics (n=60) showed a complex karyotype (≥3 structural alterations) in 19 (32%) cases, and 7 of these cases contained a deletion involving chromosome 7 (del(7)). Eleven (18%) other cases without complex karyotypes had del(7). Deletions of chromosome 5 were present in 9 (15%) cases, but only in the context of a complex karyotype. We hypothesized that the patients' younger age and the different spectrum of primary tumor types and chemotherapy would give rise to a mutational spectrum distinct from adult tMN. We used whole exome (WES), whole genome (WGS), and RNA sequencing (RNA Seq) to describe the mutational profile of our pediatric tMN cohort. WES was completed for 58 tumor/normal pairs using Nextera Rapid Capture Expanded Exome (Illumina). Fifteen cases were analyzed by WGS (11 also had WES). Normal comparator genomic DNA was obtained from flow-sorted lymphocytes. An average of 21 coding variants/patient (range: 1-131) was observed from the gene-coding region, and these include synonymous, non-synonymous, and splice site variants. Ras/MAPK pathway mutations were present in 44% of the cases (43 mutations in 27 cases). Canonical KRAS (n = 16), NF1 (n = 10), and NRAS (n = 7) mutations were the most frequent coding mutations. Eleven (18%) patients had either heterozygous deletion or a copy neutral loss of heterozygosity event involving chromosome 17p and the TP53 locus; 5 of these cases had concurrent TP53 missense mutations identified at allele frequencies near 100%. Unlike tMN in adults, mutations in PPM1D were not identified. RNA-Seq completed on 56 evaluable cases identified 28 (50%) cases with KMT2A rearrangement (KMT2Ar). MLLT3 was the most common fusion partner (n=13, 46%). In addition to KMT2A rearrangements, RNA-Seq also identified a RUNX1-MECOM fusion. Alterations involving the MECOM locus have been described in some myeloid neoplasms like tMN, and its overexpression is associated with a poor prognosis and some AMLs with KMT2Ar. MECOM expression levels were variable in this cohort (FPKM range: 0.004 - 38.4) with 24 cases (43%) having an FPKM>5 (MECOMHigh). In addition to the RUNX1-MECOM event, these 24 MECOMHigh cases included 18 with KMT2Ar (64% of KMT2Ar group) and 1 with a NUP98 fusion (NUP98-HHEX). The remaining 4 MECOMHigh cases demonstrate allele-specific MECOM expression, suggesting a cis-regulatory element is driving this expression. Two of these 4 cases have WGS and were found to contain a t(2;3)(p21;q26.2) involving MECOM on chromosome 3 and noncoding regions of chromosome 2 adjacent to ZFP36L2, a gene highly expressed in hematopoietic cells. ENCODE data supports that this region of the genome is an active enhancer in hematopoietic cells, suggesting a proximity effect in which this enhancer has been hijacked to drive high levels of MECOM expression. In our cohort, MECOM expression levels are predictive of a worse outcome (overall survival (OS) at 2 years: High=14.6% vs. Low=46.3%; log rank p In conclusion, we report the genomic profile of a large cohort of pediatric tMN cases and show that high levels of MECOM expression, a portion of which is driven by enhancer hijacking, predicts a worse outcome. Disclosures Gruber: Bristol-Myers Squibb: Consultancy.

Infants diagnosed with KMT2A-rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) have a poor prognosis with an event free survival of 23-44%. To identify new treatment approaches we previously performed in vitro and in vivo assays to evaluate the activity of FDA approved compounds in 15 primary KMT2Ar infant leukemia samples. Three classes of agents were found to be active in these assays: proteasome inhibitors, anthracyclines, and histone deacetylase inhibitors (HDACi). KMT2Ar infant leukemia samples were exquisitely sensitive to the proteasome inhibitor bortezomib, requiring 10-100 fold less drug to achieve 50% toxicity when compared to non-KMT2Ar childhood ALL. Bortezomib is FDA approved for multiple myeloma and laboratory studies using this model system have previously demonstrated responses to be mediated through several mechanisms including NFKB inhibition, stabilization of cell cycle regulatory proteins, and perhaps most importantly the induction of an unfolded protein response (UPR) and endoplasmic reticulum (ER)-stress-induced apoptosis. To evaluate global protein dynamics in KMT2Ar ALL cells treated with bortezomib, we performed tandem mass tag (TMT) quantitative mass spectrometry on synchronized SEM cells exposed to either 50nM of bortezomib or DMSO at 0 hours (hr), 6hr, 12hr, 16hr, and 20hr. Applying pairwise comparison for 9232 unique proteins measured over the time course compared to untreated controls, we identified 1593 proteins with a log2 fold change >1.5 in bortezomib treated cells compared to 101 proteins in the DMSO control (FDR While GADD34 has been shown to be the main phosphatase that functions in a negative feedback loop to resolve cell stress, recent data suggests that stabilization of CReP mRNA by ER stress is able to reverse eIF2α phosphorylation at later stages of UPR leading to re-expression of key UPR proteins. Further, p-eIF2α-attenuated protein synthesis, and not ATF4 mRNA translation has been shown to promote cell survival. Our data support a model whereby the UPR and ER-stress in KMT2Ar ALL cells is induced upon exposure to bortezomib leading to increased levels of ATF4 and CHOP. Attenuation of p-eIF2α by CReP further contributes to cell death through the recovery of protein synthesis in a setting of limited protein folding capacity. These results support the use of proteasome inhibitors in KMT2Ar leukemia which is currently being formally evaluated in a Phase II clinical trial for newly diagnosed patients with infant ALL (NCT02553460). Disclosures Gruber: Bristol-Myers Squibb: Consultancy.

The homeobox (HOX) genes are a highly conserved family of transcription factors involved in embryonic patterning as well as adult hematopoiesis. Dysregulation of HOX genes, in particular upregulation of HOXA cluster genes, is a frequent event in Acute Myelogenous Leukemia (AML). Recently, we performed a detailed genomic analysis on pediatric non-Down Syndrome Acute Megakaryoblastic Leukemia (non-DS-AMKL) and identified novel fusions involving a HOX cluster gene in 14.9% of the cases. While most fusions were predicted to lead to an in-frame functional protein, several fusions included a non-coding HOX antisense gene (PLEK-HOXA11-AS, C8orf76-HOXA11-AS, HOXA10-AS-CD164) that were predicted to result in a loss of function of these regulatory transcripts. The functional consequence of these events, however, remain unknown. HOXA11-AS (human) and Hoxa11os (mouse) have been previously shown to have mutually exclusive expression with the Hoxa11 transcript throughout development. We therefore hypothesized that loss of function of non-coding HOX antisense genes as a result of these structural variations would cause upregulation of nearby coding HOXA genes that in turn promote leukemogenesis. To test this hypothesis, using CRISPR-Cas9 technology, we genome edited the human AMKL cell line CMK to carry the PLEK-HOXA11-AS translocation. qRT-PCR of HOXA11-AS and HOXA9-11 transcripts in this cell line recapitulated the pattern seen in patient specimens. Specifically, HOXA11-AS expression was significantly diminished while HOXA10 and HOXA11 transcripts were upregulated 1.8-2.5-fold when compared to parental CMK cells (p=0.0385 and p=0.006 respectively). To further investigate the loss of HOXA11-ASin vivo a CRISPR-Cas9 Hoxa11os knockout mouse model was established. qRT-PCR on bone marrow confirmed the loss of Hoxa11os transcripts in heterozygous (Hoxa11os1+/-) and homozygous (Hoxa11os-/-) mice of both genders (p= Combined these data demonstrate that loss of function alterations in Hoxa11os transcripts lead to upregulation of Hoxa11 and gender specific hematopoietic progenitor cell perturbations. Ongoing efforts include competitive transplant studies as well as RNA and ChIP sequencing to identify gender specific downstream targets of Hoxa11 in the hematopoietic compartment in order to understand the selective expansion of progenitor subsets and male specific self-renewal capacity of this protein. These data will contribute to our understanding on how HOXA11-AS translocations promote oncogenesis. Disclosures Zwaan: Daiichi Sankyo: Consultancy; Sanofi: Consultancy; Roche: Consultancy; Pfizer: Research Funding; BMS: Research Funding; Incyte: Consultancy; Celgene: Consultancy, Research Funding; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Janssen: Consultancy. Gruber:Bristol-Myers Squibb: Consultancy.

Introduction: Resistance and relapse remain major obstacles in the treatment of acute myeloid leukemia (AML). Pre-existence and persistence of drug resistant leukemia stem cells (LSCs) is considered one of the major causes of relapse. A previous study (Ng et al., 2016) reported a prognostic signature of 17 genes (LSC17 score) differentially expressed in LSC+ compared to LSC- cell fractions that predicted outcome in patients with AML thereby classifying patients into high and low risk groups. The goal of this study is to determine the validity of LSC17 score in pediatric AML patients and to enhance its clinical utility by exploring a new score with limited number of stem cell genes. Methods: 150 pediatric patients with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression data and clinical data were included in the study. Since only 14 of the 17 genes were represented on the Affymetrix U133A gene chip we tested the validity of the LSC14 score using the previously defined equation (Ng et al, 2016) with multiple clinical endpoints such as minimum residual disease (MRD), event free survival (EFS) and overall survival (OS). To reduce the model complexity, we applied a penalized regression algorithm called the least absolute shrinkage and selection operator (LASSO) implemented in the glmnet R-package using event free survival (EFS) as an outcome variable. Score of the new equation, which included three genes, was designated as pediatric-LSC3 (pLSC3). pLSC3 was tested in the AML02 cohort for association of high or low pLSC3 (based on the median value) with clinical endpoints mentioned above. pLSC3 score equation was validated using publically available gene-expression data from 117 pediatric relapse enriched AML patient cohort enrolled in Children's Oncology Group (COG) protocol (TARGET database). COX-proportional hazard models and Log rank test were used for survival data analysis. Results: AML02 cohort: Patients with high LSC14 scores (greater than median), had significantly worse MRD (p In a multivariate COX regression model, pLSC3 score groups was the only significant covariate (table 1). It explained 13.1% of variability in EFS and 11.6% of variability in OS, while other prognostic factors such as risk groups, FLT3 status, treatment arm and age collectively explained 15.1 and 12.1 % of variability. Discussion: In summary, our results show validity of a previously defined LSC14 score in a pediatric AML population from the multicenter AML02 clinical trial. To enhance the clinical utility, score equation was further simplified and the final score (pLSC3) was derived from three genes: DNMT3B, which encodes for DNA methyltransferase; CD34, an important cell surface marker for early-undifferentiated LSCs; and GPR56, a G protein coupled receptor of significance in AML. Given that there is need to refine classification of a highly heterogeneous group of patients with standard risk AML, we show that differentiating standard risk patients based on pLSC3 score should be considered in the future. We show the relevance of pLSC3 in two independent cohorts, opening up opportunities to improve treatment outcomes of pediatric patients with AML. Disclosures No relevant conflicts of interest to declare.

Pediatric cancers are distinct from adult cancers in both their genomic alterations and therapeutic responses. Fms-like tyrosine kinase 3 (FLT3) mutations, especially internal tandem duplications (ITD), are among the most common mutations in acute myeloid leukemia (AML). FLT3-ITD mutations occur in approximately 15% of pediatric and 25-30% of adult AML, and are generally associated with poor prognosis. However, a number of studies have suggested that FLT3-ITD-positive(+) AML requires additional cooperative mutations. The objective of this study was to characterize the mutational landscape in a cohort of FLT3-ITD+ pediatric AML patients (median age,12.6 years; range, 2.8-19.2 years) enrolled to the AML02 and AML08 trials using samples obtained at diagnosis (n=34) and paired diagnosis/relapse samples (n=5). Children with promyelocytic leukemia were excluded. Samples were analyzed by RNASeq, a targeted 95 gene next generation sequencing (NGS) panel, and whole exome sequencing (WES). At diagnosis, 58.8% of the samples contained fusion genes; 41.2% were NUP98-NSD1, 11.8% were novel fusions (NSD1-CAPRIN1, NSD1-RALBP1, RUNX1-BCL11B, ZEB2-BCL11B), and 5.9% were previously reported fusions (CBFB-MYH11, DEK-NUP214). The NGS panel identified that WT1 and NPM1 were routinely mutated at a frequency of 32.4% and 20.6% respectively. While the NPM1 mutation was either a 4bp insertion at amino acid (a.a.) 287 or 288, WT1 mutations were heterogenenous with missense mutations, insertions and deletions all being reported. WT1 mutations and NUP98-NSD1 co-associated in 7 patients, 1 patient also harbored a TYK2 mutation; in the remaining 7 patients with NUP98-NSD1 fusions, a mutation in RAD21 or NRAS was observed in 2 patients. For samples with other fusions (n=6), we detected an average of 1 additional mutation per sample, which included mutations (variant allele frequency; VAF) in DNMT3A (0.44), IDH2 (0.49), KIT (0.37), NPM1 (0.51), PLCG2 (0.44), RAD21 (0.55), and SMC1A (0.47). No fusion genes were observed in 13 patients. In this latter subset, mutations in NPM1 (n=6) and WT1 (n=3) were observed. Other alterations that were identified in these samples included mutations in DNMT3A, IDH2, PLCG2, and PRKCB, which co-occurred with NPM1 mutations. Three patients did not harbor a fusion gene or a gene mutation by our analysis. When looking at cumulative incidence of relapse or resistant disease, our study results are concordant with previous reports where a NUP98-NSD1 fusion associated with worse prognosis (hazard ratio [HR] = 3.2, p = 0.02), but FLT3-ITD allelic ratio >0.4 was not prognostic (HR = 1.1, p=0.87). NPM1 mutations were not significantly associated with better prognosis (HR = 0.2, p = 0.11). We next sought to identify relapse specific alterations by analysis of paired diagnosis/relapse samples by RNASeq, NGS panel, and WES. Notably, the FLT3-ITD mutation was maintained at relapse in all samples. From the NGS panel, we observed the emergence of a MED12 mutation (P1751Q, VAF 0.37) and WT1 mutation (p.S152*, VAF 0.19) at relapse; a mutational switch in WT1 from diagnosis to relapse was also observed (5bp insertion at a.a. 157 to 2bp insertion at a.a. 158). By RNASeq analysis, we found a novel relapse specific fusion gene, LUZP6-OSBL1A. From exome sequencing, mutations in transcription factors were observed at relapse such as CREBBP, GLI3, and TBX20. Our analysis of relapse specific genes showed recurrent mutations in HUWE1, OGT, NACAD, and UNC13A. Intriguingly, both OGT and HUWE1 have been implicated in cancer metabolic reprogramming, and regulate MYC transcriptional programs. OGT is an O-Linked β-N-acetylglucosamine (O-GlcNAc) transferase involved post-translational modification of serine and threonine residues. HUWE1 is an E3 ubiquitin ligase that has been established as a tumor suppressor and previously reported to be mutated in AML. In conclusion, we demonstrate that additional genomic alterations are observed in the majority of pediatric FLT3-ITD+ AML samples evaluated, with a high proportion of samples containing fusion genes, WT1 and NPM1 mutations. We also identified novel fusion genes and mutations that have not been previously reported in pediatric FLT3-ITD+ AML, including relapse specific mutations. These results provide further biological insight into the genomic heterogeneity of pediatric FLT3-ITD+ AML, warranting further investigations in larger patient cohorts. Disclosures Inaba: Arog: Research Funding.

Introduction: Post-transcriptional modification of RNA, known as RNA-editing, has been shown to occur in many species including human. A recent study using genomic data from adult solid tumors generated by The Cancer Genome Atlas project (TCGA) investigated the potential effects of RNA editing on cancer cell viability, invasion potential, cancer pathogenesis and drug sensitivity (Han, et al., Cancer Cell 2015). Historically, there have been mixed reports regarding the prevalence of RNA editing in human cells, partly due to substantial difficulties in distinguishing RNA editing events from mapping artifacts in next-generation sequencing (NGS) data. In this study, we developed a suite of computational analysis tools to enable precise mapping of RNA-Seq in order to carry out the first systematic investigation of RNA editing events affecting coding regions in pediatric leukemia. Methods: We developed a knowledge-guided accurate RNA-Seq mapping pipeline named StrongArm to maximize mapping accuracy and efficiency. StrongArm performs multiple mappings with different aligners and databases, and uses a set of competition heuristics to choose an optimal mapping, thereby reducing the mapping error rate and bias introduced by any single aligner, especially for error-prone splice junction sites and paralogs. The analysis was performed on 17 leukemia samples including 10 acute myeloid leukemia subtype M7 (AMLM7) and 7 Ph-like acute lymphoblastic leukemia (Ph-like ALL), which were profiled using RNA-Seq of tumor samples and whole-genome sequencing or whole-exome sequencing of paired tumor and normal DNA samples. The single nucleotide variants (SNVs) detected in RNA-Seq, but absent in DNA samples, were considered putative editing events and were further processed to remove additional false positives that could not be corrected by the mapping pipeline alone. These false positive variants, arising from paralog mapping artifacts, genetic polymorphisms, nano exons, and sequencing errors at homopolymer loci introduced by reverse transcription, account for 96% to 99% of putative DNA-RNA coding variants in the leukemia samples. Results: Using 17 leukemia samples, we identified a total of 103 RNA editing events in coding regions affecting 43 unique loci, 92% of which were canonical A-to-G or C-to-T editing; 62 (61%) and 66 (64%) of the 103 editing events match those in the RNA editing database DARNED and RADAR, respectively. Seventy-eight (76%) of 103 editing events resulted in missense variants, suggesting that they may potentially affect protein function. The four most prevalent RNA editing events were present in 30% our leukemia samples, including COG3 I635V (n=12), BLCAP Q5R (n=10), CDK13 Q103R (n=9) and AZIN1 S367G (n=6). Previous studies have shown that AZIN1 S367G and COG3 I635V impact cell proliferation, and that BLCAP Q5R is correlated with survival rate in renal clear cell carcinoma (Han, et al., Cancer Cell 2015), while the impact of CDK13 Q103R in leukemia is unknown. Interestingly, three of four candidate "master" driver editing sites identified in TCGA solid tumors, AZIN1 S367G, COPA I164V, and COG3 I635V were also present in our data set, while GRIA2 R764G is absent, as GRIA2 is not expressed in leukemia. Conclusions and Discussion: Leveraging an accurate mapping pipeline for RNA-seq data, we found that pediatric leukemia samples have fewer RNA-editing events (3 to 14 per sample) in coding exons, comparable to the informative coding RNA-editing events identified in adult solid tumors from the TCGA. Notably, 3 out of the 4 most common RNA-editing sites in our leukemia samples have been reported to have functional effects on cell survival / proliferation or have been correlated with patient survival rate in adult solid tumors, indicating that RNA-editing in coding regions may have a functional impact on leukemia tumorigenesis. Disclosures Mullighan: Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding.

Acquired activating mutations in kinase/PI3K/RAS signaling pathways occur in about half of pediatric acute leukemia cases with Mixed Lineage Leukemiagene rearrangements (MLL-R; MLL, also known as KMT2A). Mutations resulting in activated signaling cooperate with the MLL-R in mouse leukemia models, however, detailed insight on the effect on the transcriptional and proteomic landscape is lacking. In infant MLL-R acute lymphoblastic leukemia (ALL), a subtype with a very poor prognosis, a majority of the activating mutations are subclonal, but the biological mechanisms by which subclonal mutations affect leukemogenesis remain unclear. Here we show that NRASG12D, FLT3internal tandem duplication(ITD)and FLT3N676K,cooperates with MLL-MLLT3 in myeloid leukemogenesis using a competitive murine retroviral bone marrow transplantation model. The addition of an activating mutation remodels the gene expression patterns of the MLL-R leukemia with distinct profiles for each mutation as determined by RNA-sequencing. Gene set enrichment analysis revealed enrichment of genes involved in chromatin assembly, transcription and stemness in leukemia induced by MLL-MLLT3 and NRASG12D, FLT3ITD or FLT3N676K. Leukemia induced by only MLL-MLLT3 displayed upregulation of genes involved in signal transduction suggesting activation of such pathways by alternative mechanisms. Upon secondary transplantation, mice receiving MLL-MLLT3-only leukemic cells succumbed to disease at a similar latency to those receiving MLL-MLLT3 and an activating mutation, supporting that fully transformed MLL-MLLT3 leukemias have sustained active signaling via high expression of genes involved in signal transduction. Using the same model as above but with significantly reduced numbers of transplanted cells containing an activating mutation, we could further show that a subclonal mutation, exemplified by FLT3N676K, causes significantly reduced disease latency as compared to mice receiving MLL-MLLT3 only (34 vs 50 days). The size of the double positive (MLL-MLLT3+FLT3N676K) and single positive (MLL-MLLT3) clones within each mouse was determined by flow cytometry revealing that 8/24 mice had a double positive subclone (≤50%). The clonal evolution of the double positive cells from 22/24 mice was assessed in secondary recipients showing three distinct patterns 1) increase in size, 19/22 mice 2) maintained, 2/22 mice or 3) decreased in size, 1/22 mice. Targeted gene re-sequencing of the latter leukemia that lost its MLLT3+FLT3N676K subclone, identified a de-novo CblA308T in the SH2-like domain, in the MLL-MLLT3 onlycells that had gained clonal dominance in the secondary recipient. The decreased disease latency in mice with subclonal activating mutations raises the possibility that cells with an activating mutation support the growth of other leukemic cells by direct cell-cell contact or through secreted factors. Transcriptome and proteomic analyses identified a high expression of the macrophage inhibitory factor (MIF), a pro-inflammatory cytokine, in mice with MLL-MLLT3 and NRASG12D, FLT3ITD or FLT3N676K. Addition of rMIF increased the survival of MLL-MLLT3 murine leukemic cells in vitro. Although additional factors likely mediate pro-leukemic effects in vivo, our data suggest that MIF could be one of these factors. In summary, our data demonstrate that activating mutations cooperate with the MLL-R in murine leukemogenesis and cause widespread changes in the transcriptional landscape. In addition, our results suggest that cells containing an activating mutation in addition to an MLL-fusion positively influence the survival and likely also the growth of other leukemic cells, suggesting a pro-leukemic effect mediated by interclonal cooperation between clones carrying distinct mutational set-ups in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Acute Megakaryoblastic Leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) that morphologically resembles abnormal megakaryoblasts. While extremely rare in adults, pediatric cases comprise 4-15% of newly diagnosed AML patients. Clinical outcomes for Down syndrome (DS) patients with AMKL are uniformly excellent, whereas studies on non-DS patients (non-DS-AMKL) are more variable with the majority reporting inferior survival rates compared to other AML subtypes. Furthermore, the recommendation for stem cell transplant (SCT) in first remission for non-DS-AMKL patients is not uniform among pediatric cooperative groups. Previous efforts have identified chimeric oncogenes in non-DS-AMKL cases, including RBM15-MKL1, CBFA2T3-GLIS2, MLL gene rearrangements and NUP98-KDM5A. The etiology of 30-40% of cases, however, remains unknown. To better understand the genomic landscape of non-DS-AMKL and its contribution to clinical outcomes, we performed RNA and exome sequencing on specimens from 115 patients compiled from eight institutions and three cooperative groups including 90 pediatric and 25 adult cases. Of the 104 patients for whom RNA was available, 27.8% (5/18) adult and 72% (62/86) pediatric cases carried a high confidence fusion event by RNAseq. The most frequent fusions in the pediatric cohort when combining RNAseq data, cytogenetics and RT-PCR include CBFA2T3-GLIS2 (17/90), MLL r (13/90), NUP98-KDM5A (9/90), and RBM15-MKL1 (9/90). Previously described low frequency fusions identified in this expanded cohort, include a case of NIPBL-HOXB9 and a novel but functionally analogous NIPBL-HOXA9 fusion. Similarly, a case carrying GATA2-HOXA10 was identified, which is functionally equivalent to the GATA2-HOXA9 fusion that has been reported in a single case. Chimeric transcripts not previously described include several fusions involving genes within the HOX cluster (HOTAIRM1-HOXA3, HOXA_AS3-HOXA9, EWSR1-HOXB8, PLEK-HOXA11-AS, and BMP2K-HOXD10 each in a single case). Collectively, fusions involving a HOX cluster gene (HOX r) occurred in 11% of the pediatric cohort. Single Nucleotide Variation (SNV) analysis of exome and RNAseq data on the cohort revealed the presence of truncating GATA1 mutations in one adult and 10 pediatric specimens lacking fusion genes. Patients carrying GATA1 mutations did not have stigmata of DS or evidence of mutant reads in germline DNA, suggesting they are not mosaics. To determine if these fusion events contribute significantly to gene expression patterns, samples with greater than 60% purity were subjected to unsupervised clustering. Confirming the strength of the fusions in altering gene expression signatures, samples clustered according to fusion subtype and were distinct from those carrying GATA1 mutations. Specifically MLL r, HOX r, NUP98-KDM5A, and CBFA2T3-GLIS2 cases formed distinct clusters. When analyzing differentially upregulated genes within these subgroups, HOX r cases demonstrated upregulation of a HOX gene signature. Combined with MLL r and NUP98-KDM5A, chimeric oncogenes also known to upregulate HOX cluster genes, roughly one-third of pediatric non-DS-AMKL patients carry a HOX gene expression program. These cases were distinct from those carrying the CBFA2T3-GLIS2 inversion. HOX genes play a significant role in normal hematopoietic development and data suggests that deregulated expression has a central role in the etiology of several subtypes of acute leukemia, in part through the acquisition of enhanced self-renewal. We evaluated our identified HOXr for their ability to serially replate in murine colony formation assays as a surrogate marker of this characteristic. Confirming their pathogenicity, chimeric transcripts conferred an enhanced ability to replate. We conclude that chimeric transcripts involving HOX cluster genes comprise a distinct subset of pediatric AMKL. Clinical outcome analyses between genomic subgroups of this heterogeneous malignancy may allow us to more effectively risk stratify these patients and determine those that may benefit from SCT in first remission. JdR and CB contributed equally FL, DR, MH-E, MF, CMZ, and TAG co-corresponding authors on behalf of AIEOP, BFM, DCOG, and SJCRH study groups Disclosures Shih: Novartis: Research Funding.

Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of the BRAF V600E mutation in ~50% of patients with LCH and the non-LCH Erdheim-Chester Disease (ECD) provided the first molecular target in these patients and novel insights into the pathogenesis of these disorders. However, recurrent mutations in the majority of the ~50% of BRAF V600E-wild type patients with non-LCH are unknown. Moreover, recurrent mutations outside of the MAP kinase pathway are undefined throughout histiocytic neoplasms. To address these issues, we performed whole exome sequencing (WES) of frozen biopsies from 24 patients with LCH (n=10) or ECD (n=14) paired with peripheral blood mononuclear cells. 13/24 patients also underwent RNA sequencing (RNA-seq). All mutations in activating kinases were validated by droplet-digital PCR, while targeted-capture next-generation sequencing validated all others. Both adult (n=18; n=2 with LCH) and pediatric cases (n=9; n=8 with LCH) were included. Using combined WES/RNA-seq, activating kinase alterations were identified in 100% of patients. In LCH, 60% and 40% had BRAF V600E and MAP2K1 mutations, respectively. In non-LCH 51%, 14%, 14%, and 7% were BRAFV600E, ARAF, MAP2K1, and NRAS mutant (Fig1A). Overall, a mean of 7 non-synonymous mutations per adult patient was identified (range 1-22) compared with 5 mutations per pediatric patient (range 4-9; p =ns). Mutations affecting diverse cellular processes were found to co-exist with kinase mutations including mutations in epigenetic modifiers and the p38/MAPK pathway. In addition to kinase point mutations, RNA-seq identified recurrent, in-frame kinase fusions-a first for these disorders. All identified fusions were validated using FISH and RT-PCR. This includes novel fusions in BRAF (RNF11-BRAF and CLIP2-BRAF), as well as therapeutically important fusions in ALK (2 separate KIF5B-ALK fusions) and NTRK1 (LMNA-NTRK1;Fig1B). Expression of each fusion in Ba/F3 cells conferred cytokine-independent growth. Importantly, the BRAF fusions were found to be sensitive to MEK inhibition but resistant to vemurafenib while the ALK fusions conferred sensitivity to the ALK inhibitors crizotinib or alectinib. We next interrogated a validation cohort of 37 BRAF V600E-wild type, non-LCH, formalin-fixed, paraffin-embedded tissue samples using targeted mutational profiling for MAP2K1, ARAF, NRAS, KRAS, and PIK3CA. This revealed activating mutations in MAP2K1 (32%; n=12), NRAS (16%; n=6), KRAS (11%; n=4), PIK3CA (8%; n=3), and ARAF (3%; n=1). Three of the investigated non-LCH patients with refractory disease and progressive organ dysfunction were treated with targeted therapies based on the discovery of novel kinase alterations described above. Treatment of 2 refractory MAP2K1- mutant, non-LCH patients with MEK inhibitors (trametinib or cobimetinib) resulted in dramatic clinical improvement (Fig1C). Both patients have been maintained on MEK inhibitor single-agent therapy with a sustained clinical response for >100 days. Further evidence of effective targeted inhibition was found in a refractory ECD patient carrying an ARAF S214A mutation. This patient failed to respond to 3 lines of prior therapies and suffered near blindness due to disease infiltration in the retina and optic nerves. Given a recent report of complete response to sorafenib in a lung cancer patient with an ARAF S214C mutation, we initiated sorafenib. Within 12 weeks, there was improvement in the patientÕs eyesight and decreased infiltrative disease, coinciding with >50% decrease in mutant ARAF DNA in plasma cell-free DNA. Whole exome and transcriptome sequencing identified activating kinase mutations or translocations in all patients with the common downstream effect of activating the MAPK pathway. The preliminary, dramatic, clinical efficacy observed with use of MEK and RAF inhibitors in MAP2K1 - and ARAF-mutated, non-LCH patients further supports the central role of targeting the MAPK pathway in these tumors. The discovery of the discussed mutations and fusions in diverse kinases provides critical new insights into the genetic events central to a spectrum of adult and pediatric histiocytic neoplasms. Figure 1. Figure 1. Disclosures Off Label Use: This abstract describes use of MEK inhibitors (both tremetinib and cobimetinib) as well as sorafenib for MEK1 and ARAF mutant histiocytosis. . Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc.: Employment. Ali:Foundation Medicine Inc.: Employment. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria.

Systemic histiocytic disorders, including Langerhans cell histiocytosis (LCH) and the non-Langerhans cell histiocytic disorder Erdheim-Chester Disease (ECD) are clinically heterogeneous diseases whose underlying etiology has long been obscure. Recent identification of BRAF V600E mutations in ~50% of LCH and ECD patients, as well as mutations in MAP2K1 in ~25% of BRAF-wild type LCH patients has refined our understanding of these disorders as clonal malignancies driven by constitutive MAP kinase signaling. However, the compendium of mutations co-occurring with BRAF V600E and MAP2K1 mutations in ECD/LCH have yet to be defined since unbiased genomic sequencing studies have not been performed in ECD. Moreover, the molecular bases of the phenotypic differences between LCH and ECD have remained elusive. Therefore, we performed whole exome sequencing (WES) of adult and pediatric LCH and ECD patients to further elucidate our understanding of the molecular bases of these disorders. Nineteen histiocytic disorder cases including fresh-frozen tissue biopsies from 10 adult (2 LCH and 8 ECD) and 8 pediatric patients (all LCH), along with paired normal tissue from blood were evaluated by WES (1 adult ECD patient had synchronous biopsies from 2 different anatomic sites- these 2 samples are treated independently in this analysis). Percentage of tumor involvement ranged from 25%-50% based on review of histologic sections by board-certified hematopathologists. High quality sequence variants unique to the tumor DNA including SNVs and indels were identified for subsequent analyses. All candidate missense genetic variants were evaluated by 3 in silico analysis methods (PROVEAN; SIFT; PolyPhen-2) for predicted mutational effects on protein function. For missense variants, only those determined to be damaging to protein function in 2 or more in silico methods were retained for further evaluation. Mutations passing these analytical steps were further evaluated for biological significance using several web-based, freely available pathway analysis databases. We first examined mutations in the MAP kinase pathways identified by the WES of LCH and ECD patients (Figure). Of LCH cases, 40% harbored BRAF V600E mutations while 30% had mutations in MAP2K1. Three patients carried in-frame deletion mutations in the negative regulatory domain of MAP2K1. Interestingly, mutations in the JNK and p38 MAP kinase pathways were identified in 20% of LCH patients, one of which was BRAF/MAP2K1 wild type (Figure). Of the ECD patients, 44% were BRAF V600E mutant, and we identified one BRAF V600E-wild type ECD patient as having a MAP2K1 mutation (MAP2K1 K57N) and another as having an NRAS (NRAS Q61R) mutation. As with LCH patients, WES analysis again noted mutations in p38 and JNK MAP kinase pathway members in BRAF/NRAS/MAP2K1-wild type ECD patients further highlighting involvement of all 3 MAP kinase pathways in ECD/LCH pathogenesis. We next evaluated mutated genes not known to be directly involved in MAP kinase signaling in order to understand how non-MAP kinase signaling pathways might contribute to LCH/ECD pathogenesis. Recurrent mutations in members of the Notch, WNT/β-catenin, NF-κB, and FGF signaling pathways were identified (Figure). Interestingly, loss-of-function mutations in Notch pathway mediators identified in this study occurred only (i) in MAML3 and MAMLD1 and (ii) in LCH (60% of patients) but not ECD. These findings, combined with recent identification of the monocytic-origin of LCH and the discovery that loss of Notch signaling results in monocytic clonal disorders, highlights a potential role for Notch signaling in the pathogenesis of LCH. Likewise, identification of a DNMT3A mutation in a BRAF V600E-mutant ECD patient and the association of Dnmt3a loss with clonal dominance of Dnmt3a-deficient hematopoietic cells suggests a potential role for disordered epigenetic regulation in ECD. This WES study of somatic mutations in LCH and ECD confirms previously reported frequencies of BRAF V600E and MAP2K1 mutations in LCH, identifies MAP2K1 mutations in ECD for the first time, and reveals additional candidate mutations in MAPK signaling that are mutually exclusive of BRAF/MAP2K1 mutations. Furthermore, this first WES study of ECD reveals somatic mutations in multiple genes that regulate diverse cellular processes co-occurring with recurrent mutations in MAPK signaling pathways. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Infants with MLL rearranged (MLLr) acute lymphoblastic leukemia (ALL) have a poor prognosis, with an event free survival of only 23-44%. Whole genome sequencing (WGS) of this subtype has revealed a paucity of cooperating mutations, with an average of 2.2 somatic single nucleotide variations and/or insertions/deletions per case. Despite recent progress in defining the epigenetic alterations that result from the expression of the MLL fusion protein, these insights have only recently begun to be extrapolated into the development of new therapeutic approaches whose benefits have yet to be defined. Thus, there remains an urgent need for the development of alternative approaches to improve outcomes in these patients. To identify compounds that are active in MLLr disease, we established in vitro and in vivo assays to evaluate drug sensitivity of primary infant ALL patient samples. 15 infant MLLr leukemia samples that have previously undergone WGS were xenografted into NOD/SCID/IL2Rγnull (NSG) mice. All samples engrafted and expanded in NSG mice, leading to overt leukemia with a latency of 49 to 276 days. Purification of leukemic blasts from a single moribund mouse yielded on average 108 cells, providing sufficient material to screen large numbers of compounds. In vitro conditions were defined that support growth in 40% of the patient specimens, allowing for a more accurate determination of drug sensitivity. Growth in vitro was associated with early onset of disease in NSG xenografts and younger age at presentation, allowing us to evaluate patient samples that represent aggressive high risk disease. Using this system, we tested bortezomib in addition to 28 other drugs, including standard ALL therapeutic agents as well as targeted kinase inhibitors and inhibitors of epigenetic marks. Three classes of agents were active in this system: anthracyclines, histone deacetylase inhibitors (HDACi), and the proteasome inhibitor bortezomib. In contrast to anthracyclines and HDACi, where IC50 values were on par with those reported in the literature for primary childhood ALL samples, MLLr infant samples required 10-100 fold less bortezomib to induce toxicity. Bortezomib has been shown to mediate responses through several mechanisms, including NFKB inhibition, stabilization of cell cycle regulatory proteins, and induction of apoptosis. Recently, proteasome inhibition has been demonstrated to lead to accumulated MLL fusion protein levels, triggering apoptosis and cell cycle arrest in MLLr cell lines. To determine if NFKB inhibition also plays a role, we evaluated cellular concentrations of the activated NFKB transcription factor, but failed to see decreased levels when MLLr cells were treated with bortezomib. Bortezomib has also been shown to deregulate ubiquitin stores and deplete histone H2B ubiquitination (H2Bub), an epigenetic mark that is linked to histone methylation and expression. Recently, several groups have demonstrated that H2Bub is required for DOT1L activity and HOX gene expression. We therefore evaluated H2Bub levels in bortezomib-treated patient samples and confirmed depletion of this epigenetic mark. Furthermore, patient samples treated with bortezomib downregulated both the MLL gene expression signature and signatures of downstream targets, such as cMYC, demonstrating that the MLL transcriptional program is inhibited in the presence of bortezomib. ChIP-seq is underway to map H2Bub and H3K79 methylation changes genome wide in response to treatment with bortezomib. The HDACi vorinostat and bortezomib have both been evaluated in Phase I and II pediatric leukemia clinical trials. Based on the safety and efficacy from these earlier studies, we treated 6 relapsed/refractory MLLr leukemia patients with a chemotherapy regimen that included mitoxantrone, vorinostat, and bortezomib. 4 patients had a complete response (CR), 1 patient had a partial response (PR) and 1 patient had stable disease for an overall response rate of 5/6 (83%). Clinical trials are in development to assess this combination further for both relapsed MLLr disease as well as newly diagnosed infant ALL. Our data suggests that these three classes of drugs, identified in our laboratory assays, are clinically active thus validating our system. We are now using this platform to proceed with a high throughput drug screen to identify additional compounds for future clinical development. Disclosures Off Label Use: Vorinostat and Bortezomib for the treatment of pediatric leukemia.

Acute megakaryoblastic leukemia (AMKL) accounts for ~10% of childhood AML. AMKL patients without Down syndrome have a poor outcome with a 3 year survival of less than 40%. To gain insight into the biology of this disease, we previously performed transcriptome sequencing on diagnostic blasts from a discovery cohort of 14 pediatric cases and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult samples. This analysis identified novel fusion transcripts restricted to pediatric AMKL including CBFA2T3-GLIS2,GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9. To confirm their role in oncogenesis and gain insight into the mechanism whereby these fusions promote disease, we introduced each of them into murine hematopoietic cells and assessed their effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a control retrovirus failed to form colonies after the second replating. By contrast, expression of each of the fusion genes resulted in a marked increase in self-renewal capacity, with colony formation persisting through 10 replatings. Immunophenotypic analysis revealed evidence of megakaryocytic differentiation in CBFA2T3-GLIS2 and MN1-FLI1 cohorts, whereas NIPBL-HOXB9 and GATA2-HOXA9 cells carried markers consistent with myeloid progenitors. Transplantation of fusion gene modified bone marrow cells into syngeneic recipients induced overt leukemia in all cohorts with the exception of CBFA2T3-GLIS2, suggesting an essential requirement for cooperative mutation(s) in cases expressing this chimeric gene. To assess self-renewal activity of the leukemia generated in our murine models, we conducted secondary transplants for all cohorts. In all cases, the leukemia was transplantable with a shorter latency than in the primary transplant setting. To characterize the tumors at the molecular level, 5 samples from each of the 3 fusions underwent array comparative genomic hybridization, transcriptome, and whole exome sequencing. Samples demonstrated a small number of cooperating mutations with 1.5 copy number alterations (range 0-6) and 6.4 single nucleotide variations (range 2-13) per case. Overall, cases carried an average of 7.9 mutations (range 2-14). Despite the low number of lesions, recurrently mutated genes were identified. These include activating mutations in Flt3, Kras, and cMet, as well as loss of function mutations in the tumor suppressors Phactr4, Wt1, and Tet2. A comparison between fusion subtypes did not reveal any statistically significant differences, although there was a trend towards a greater number of mutations in the GATA2-HOXA9 cohort. Transcriptome sequencing of cohorts, along with normal hematopoietic progenitor subsets, confirmed unique gene expression patterns between each of the fusions. Consistent with immunophenotyping, MN1-FLI1 demonstrated enrichment of the MEP signature while NIPBL-HOXB9 and GATA2-HOXA9 were enriched for CMP and monocyte precursor signatures respectively. ChIP-seq analysis of each of the fusions is underway to definitively identify the genomic targets whose expression is directly altered by their binding. A common characteristic between all fusions is the presence of protein interaction domains contributed by the N term partner, and DNA binding domains contributed by the C term partner. To determine if these fusions have a novel gain of function distinct from their independent counterparts, we introduced each partner gene into murine bone marrow cells for transplantation experiments. As previously described, introduction of MN1 into hematopoietic cells led to a highly penetrant leukemia. In contrast, HOXA9, HOXB9, and FLI1 all had >75% disease free survival with few myeloid leukemias resulting from their over expression, while GATA2 failed to induce any disease at all. NIPBL’s size precluded transplant assays. Therefore, to evaluate its contribution we introduced a point mutation previously shown to disrupt binding of NIPBL to the cohesion component MAU2. This alteration abrogated the ability of the fusion to induce leukemia in our transplant model, demonstrating the importance of this interaction in the pathogenesis of disease. In conclusion, our data confirms a pathogenic role for GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9 in AMKL. Further studies delineating the cooperating mutations required for CBFA2T3-GLIS2 are indicated. Disclosures No relevant conflicts of interest to declare.

Pathologic germ line mutations that predispose patients to cancer are estimated to occur in 4-30% of all pediatric oncology cases. In addition to leukemia specific familial predisposition syndromes, children with rare constitutional syndromes, heterogeneous dysmorphic syndromes, and multiple-cancer hereditary predisposition syndromes are all at an increased risk for hematologic malignancies. However, to date no genome-wide analysis has been done to define the range of germ line mutations that occur in pediatric patients with hematological malignancies. To determine the frequency of pediatric cancer patients that have germ line variants of pathological significance in genes that predisposed to cancer, we analyzed the germ line and tumor DNA from 1120 pediatric cancer patients that were enrolled in the St. Jude – Washington University Pediatric Cancer Genome Project (PCGP). Samples were analyzed by whole-genome sequencing (n = 595), whole-exome sequencing (n = 456), or both (n = 69). Single nucleotide variants (SNVs), insertions/deletions (indels), structural variations (SV) and copy number alterations (CNAs) were detected using our analytical pipeline and all single nucleotide polymorphisms (SNPs) previously identified in non-cancer populations were filtered out. Our analysis then focused on the 23 cancer predisposition genes recently recommended for germ line analysis by the American College of Genetics and Genomics, along with an additional 8 genes that have been previously shown to predispose to pediatric cancer at a high penetrance. All variants in these 31 genes were classified as pathologic, likely pathologic, uncertain significance, likely benign, and benign based on literature review and in-silico predictions on the effect of novel mutations. An expanded analysis including a total of 565 genes known to play a role in oncogenesis was also evaluated. Pathologic or likely pathologic germ line variants in one of the 31 genes were detected in 8% (90/1120) of patients, including: 16% (46/287) of patients with solid tumors, 8.6% (21/245) with brain tumors, and 3.9% (23/588) with leukemia. Expanding this analysis to 565 cancer gene resulted in only a slight increase, with a pathologic or likely pathologic variant being detected in 8.6% (97/1120) of patients. The most frequently effected genes included TP53 (n=48), APC (n=7) and BRCA2(n=6). Importantly, in >50% of these patients, analysis of their tumor DNA revealed the absence of a wild type allele for the cancer predisposition gene that was altered in the germ line. The 588 pediatric patients with leukemia included 116 acute myeloid leukemias (AMLs: FAB M7 n=20; Core Binding Factor leukemias n=86; MLL-R n=10) and 472 acute lymphoblastic leukemias (ALLs: E2A-PBX1 n=53; ERG-R n=39; TEL-AML1 n=53; Hyperdiploid n=69; Hypodiploid n=47; BCR-ABL1 n=40; T-ALL n=32; MLL-R n=40; BCR-ABL-like n=31; and Other n=68). Across this cohort, 3.9% (23/588) of leukemia patients harbored a pathologic germ line mutations in one of the 31 cancer pre-disposing genes. This number increased to 4.6% (27/588; 28 mutations) when the expanded gene list was evaluated. TP53 (n=10) was the most frequently altered germ line gene in pediatric leukemia patients and was found predominantly in low-hypodiploid ALL, as previously reported. Germ line pathologic variants were also identified in KRAS, RUNX1, APC, BRCA2, and RET (2 cases each), and NRAS, SH2B3, BRCA1, MUTYH, PTCH1, SDHA,VHL, and NF2 (1 case each). Although germ line mutations in RUNX1 and SH2B3are typically associated with myeloid neoplasms, we identified these lesions in 3 cases of B lineage ALL suggesting an association with a wider spectrum of leukemia. In conclusion, a small but significant proportion of pediatric patients with leukemia carry a germ line variant of pathologic significance in a cancer predisposition gene. These results suggest that these germ line lesions likely play a direct role in the pathogenesis of the patient’s presenting leukemia. Moreover, our results suggest that these patients would benefit from future clinical surveillance for the development of a second cancer. Lastly, these data demonstrate the power of comprehensive next generation DNA/RNA sequencing for the identification of pediatric patients who carry a germ line pathologic variant in a cancer predisposition gene. Disclosures No relevant conflicts of interest to declare.

Abstract 69 Infant (< 1 year of age) acute lymphoblastic leukemia (ALL) is a rare disease characterized by rearrangements of the Mixed Lineage Leukemia (MLL) gene at 11q23 and a poor prognosis. In an effort to determine the total complement of somatic mutations occurring in this high risk leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 22 infants with MLL rearranged ALL using the Illumina platform. In addition, we sequenced 2 paired relapse samples. Somatic alterations, including single nucleotide variations (SNV), and structural variations (SV) including insertions, deletions, inversion, and inter- and intra-chromosomal rearrangements were detected using complementary analysis pipelines including Bambino, CREST and CONSERTING. Validation of identified somatic mutations was performed using PCR amplification of the leukemia and germ line DNA followed by Sanger or 454-based sequencing, or by array-based capture followed by Illumina-based sequencing. Analysis of the structure of MLL rearrangements at the base pair level revealed that over half had complex rearrangements that involved either three or more chromosomes, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In five of the complex cases, chromosomal rearrangements were predicted to generate not only a MLL-partner gene fusion, but also novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B; MLLT10-CTNNAP3B; MLLT10-ATP5L / ATP5L-YPEL4; and CRTAM-GNL3. An analysis of the sequence surrounding the breakpoints of MLL and its partner genes suggest that the predominant mechanism of rearrangement involved non-homologous end joining. An analysis of the total number of non-silent mutations revealed infant ALL to have the lowest frequency of non-silent somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLL rearrangements, a mean of only 2 somatic SVs and 2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case, with a range of non-silent mutation of between 0 and 11 per case (0–7 SV and 0–5 SNV). Despite the paucity of mutations several pathways were recurrently targeted. Mutations leading to activation of signaling through the PI3K/RAS pathway was observed in 45% of the cases with mutation of individual components including KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and the GTPase activating protein ARHGAP32 (p200Rho/GAP), which mediates cross-talk between RAS and Rho signaling. Other pathways altered include B cell differentiation, with 23% of cases containing mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2. WGS of two infant ALL relapse samples and comparison with the data from their matched diagnostic samples revealed a marked increase in the number of mutations at relapse with additional SVs, SNVs, and CNAs identified. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone. Because of the exceedingly low frequency of mutations detected in infant ALL, we decided to define the frequency of non-silent SNVs in MLL rearranged leukemia occurring in older children (7–19 years of age). Exome sequencing was performed on 13 MLL leukemias (8 ALLs and 5 AMLs). This analysis revealed that non-infant pediatric MLL rearranged leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 8/case in older patients versus 2/case in infants, p In summary our analysis demonstrated an exceedingly small number of mutations required to generate infant MLL rearranged leukemia. The number of detected somatic mutations may represent the lower limit of mutations required to transform a normal human cell into cancer. Disclosures: Fioretos: Cantargia AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Qlucore AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract 181 Activation-Induced Cytidine Deaminase (AID) is required for somatic hypermutation and immunoglobulin (Ig) class switch recombination in germinal center (GC) B cells. Occasionally, AID can target non-Ig genes and thereby promote GC B cell lymphomagenesis. We recently demonstrated that the oncogenic BCR-ABL1 kinase induces aberrant expression of AID in pre-B acute lymphoblastic leukemia (ALL). Compared to other ALL subtypes, BCR-ABL1 ALL is considered high risk and is characterized by a high degree of genetic instability. Because aberrant mutational activity of AID is associated with malignant transformation in B cell lymphoma, we sought to determine whether aberrant AID expression contributes to clonal evolution and genetic instability in Ph+ ALL. To investigate the function of AID expression in Ph+ ALL, we established a genetic loss-of-function model for Ph+ ALL: Bone marrow cells from AID−/− mice and AID+/+ controls were transformed by retroviral transduction with BCR-ABL1 under B lymphoid culture conditions and subsequently injected into lethally irradiated congenic recipients. Mice transplanted with AID−/−BCR-ABL1 ALL had prolonged median survival as compared to mice transplanted with leukemia cells generated from AID+/+ bone marrow (AID−/− 34 days (n=18) vs AID+/+ 13 days (n=21); p In support of enzymatic activity of AID in BCR-ABL1-transformed ALL cells, we observed that aberrant somatic hypermutation of non-immunoglobulin genes in these leukemias was largely dependent on AID: mutations in the known hypermutation target genes Pax5 and Rhoh were increased in AID+/+ but not AID−/−BCR-ABL1 ALL cells. Mutations in the first intron of Rhoh as observed here are relevant because they interfere with Rhoh transcription. Indeed, we found that Rhoh mRNA levels are significantly higher in AID−/− compared to AID+/+BCR-ABL1 ALL cells. Rhoh is a hematopoietic specific GTPase that negatively regulates Rac-mediated signaling downstream of the oncogenic BCR-ABL1 kinase. AID-dependent mutation and transcriptional inactivation of Rhoh in BCR-ABL1 ALL therefore likely augments oncogenic BCR-ABL1 signaling. Consistent with a causative role of AID in genetic instability, AID−/− leukemia had a lower frequency of amplifications (17+2 vs 45+7; p=0.002) and deletions (11+2 vs 40+7; p=0.003) as compared to AID+/+ leukemias. AID−/− and AID+/+ ALL cells showed a markedly distinct gene expression pattern with 2,365 differentially expressed genes (p=0.003; FDR 0.05). A detailed analysis of these differences in gene expression revealed that AID−/−BCR-ABL1 ALL cells failed to downregulate a number of tumor suppressor genes including p53, Rhoh, Cdkn1a (p21), and Blnk (SLP65). AID-dependent downregulation of p53 in BCR-ABL1 ALL cells is of particular importance, because previous work demonstrated that transcriptional repression of p53 in normal GC B cells is required to make these cells permissive to high levels of AID expression. AID-induced DNA damage would otherwise activate p53 and rapidly induce apoptosis. Compared to AID-deficient BCR-ABL1 ALL, AID+/+BCR-ABL1 ALL cells are more resistant to Imatinib-treatment. However, acquisition of BCR-ABL1 kinase domain mutations does not appear to be the main cause of drug-resistance in this experiment, since only one relevant mutation was amplified from AID+/+ ALL cells (no mutations in AID−/− ALL cells). We conclude that AID accelerates clonal evolution in BCR-ABL1 ALL by enhancing genetic instability, aberrant somatic hypermutation, and by negative regulation of tumor suppressor genes. Disclosures: No relevant conflicts of interest to declare.

Pre-B cells within the bone marrow are destined to die unless they are rescued through survival signals from the pre-B cell receptor. Studying the configuration of the immunoglobulin heavy chain locus (IGHM) in sorted human bone marrow pre-B cells by single-cell PCR, we detected a functional IGHM allele consistent with the expression of a functional pre-B cell receptor in the vast majority of normal human pre-B cells. However, only in 10 of 57 cases of BCR-ABL1-transformed pre-B cell-derived acute lymphoblastic leukemia (ALL), we detected a functional IGHM allele. While normal pre-B cells respond vigorously to pre-B cell receptor engagement by Ca2+ release, the pre-B cell receptor was unresponsive even in the few cases of BCR-ABL1-driven ALL, in which we amplified a productively rearranged IGHM allele. For this reason, we studied the function of the pre-B cell receptor during early B cell development and progressive transformation in a BCR-ABL1-transgenic mouse model: Interestingly, BCR-ABL1-transgenic mice that have not yet undergone leukemic transformation show almost normal pre-B cell receptor selection. In these pre-leukemic pre-B cells, however, expression of the BCR-ABL1-transgene is very low as compared to full-blown ALL, suggesting that high levels of BCR-ABL1 expression are not compatible with normal expression of the pre-B cell receptor. Consistent with our observations in human ALL, full-blown ALL clones in BCR-ABL1-transgenic mice show defective pre-B cell receptor selection and the pre-B cell receptors expressed on few leukemic cells are not functional. Treatment of leukemic mice with the BCR-ABL1 kinase inhibitor AMN107, however, reinstated normal pre-B cell receptor selection and pre-B cell receptor function within seven days. These data suggest that the transforming signal through BCR-ABL1 and normal survival signals through the pre-B cell receptor are mutually exclusive. To test whether functional pre-B cell receptor signaling prevents transformation by BCR-ABL1, we transformed murine pre-B cells carrying a deletion of the SLP65 gene, which is required for functional pre-B cell receptor signaling. Unlike SLP65-wildtype pre-B cells, SLP65−/− pre-B cells can be transformed by BCR-ABL1 at a high efficiency. Reconstitution of SLP65 using a retroviral vector, however, induced rapid cell death of BCR-ABL1-transformed pre-B cells. We next investigated the potential impact of Slp65-reconstitution on leukemic growth of BCR-ABL1-transformed pre-B cells from SLP65−/− mice in vivo. To this end, SLP65−/− BCR-ABL1-transformed pre-B cells were labeled with firefly-luciferase and then transduced with retroviral vectors encoding SLP65/GFP or GFP alone. NOD/SCID mice were sublethally irradiated and injected with either SLP65/GFP+ or GFP+ ALL cells. Engraftment as monitored by bioluminescence imaging was delayed by more than three weeks in mice injected with SLP65/GFP+ ALL cells as compared to mice injected with GFP+ ALL cells. 36 days after injection, the first mice that were inoculated with GFP-transduced leukemia cells, became terminally ill and also the other mice in this group showed weight loss at that time. In contrast, the mice injected with SLP65-GFP-transduced ALL cells showed no signs of disease and no significant weight loss. At this time, all mice were sacrificed: Whereas mice injected with GFP-transduced ALL cells showed splenomegalia and leukemic infiltration into multiple organs, there was only mild splenic enlargement, when SLP65-reconstituted ALL cells were injected. Reconstitution of SLP65 also reduced the frequency of BCR-ABL1-transformed leukemia cells about 15-fold in the bone marrow, 5-fold in the spleen and >100-fold in the peripheral blood. We conclude that deficiency of the pre-B cell receptor-related signaling molecule SLP65 not only represents a frequent feature in human ALL cells but also represents a critical requirement for BCR-ABL1-driven leukemic growth in vivo. We conclude that pre-B cell receptor signaling renders B cell progenitor cells non-permissive to BCR-ABL1-mediated transformation. Only crippled pre-B cells with a non-functional pre-B cell receptor are susceptible to BCR-ABL1-mediated transformation.

Current intensive chemotherapy regimens have dramatically increased survival in acute lymphoblastic leukemia (ALL) patients compared to the 1950s when single agent chemotherapy was used. Despite this success certain subsets of patients have a high rate of relapse such as those with the Philadelphia chromosome (Ph+). Because the Bcr-Abl oncogene is a novel protein product and uniquely expressed in the leukemia clone, it has the potential to generate anti-leukemic immune responses. Our lab has been studying immunotheraputic approaches for Ph+ ALL using a murine model. Previous data have demonstrated that transduction of leukemia cells with the immunomodulators CD40Ligand, CD80, and GM-CSF generate T and NK cell immune responses. When irradiated and given as a vaccine these gene-modified cells are able to protect a portion of mice from an otherwise lethal dose of leukemia. We looked at the ability to systemic IL-12 treatments to potentiate this immune response and found that IL-12 alone was able to eliminate pre-existing disease in mice. IL-12 treatments, however, did not establish immunologic memory and did not protect mice from subsequent re-challenge with a lethal dose of leukemia. IL-12 protection was primarily mediated by CD4 and CD8 T cells as demonstrated by a decrease in survival in nude mice. When CD4 or CD8 T cells were depleted individually, however, protection was maintained indicating that one cell type can compensate for the other in its absence. Depletion of NK cells from Nude mice further decreased survival indicating a role for these cells in the protection. Thus protection was mediated in part by CD4 T lymphocytes, CD8 T lymphocytes, and Natural Killer cells. The ability of IL-12 to activate three different cell types may explain the efficacy seen in this model, where other cytokines alone have failed. In combination, IL-12 and our leukemia cell vaccine are effective in eliminating pre-established aggressive Philadelphia chromosome positive leukemia and establishing long lasting immunity from subsequent lethal doses of wild type leukemia. As expected, the immunologic memory generated by vaccination with gene modified leukemia cells was mediated by CD4 T cells as indicated by depletion studies. These studies demonstrate the feasibility of immunotheraputic approaches in the treatment of Ph+ ALL.