Acute Lymphoblastic Leukemia Patient Samples Research Articles

Evaluation of lipopolymer as therapeutic siRNA delivery agents in non-malignant and malignant primary cells.

e15189 Background: Targeting leukemia-driving oncogenes via short interfering RNA (siRNA) offers an alternative approach to leukemia treatment that can overcome the limitations of current therapies. Low molecular weight polyethyleneimine grafted with lipids (lipopolymers) have emerged as promising carriers for siRNA delivery to leukemia cells. Lipopolymers harboring select lipids were recently shown to downregulate target oncogene leading to significant anti-leukemic activity in both in vitro and in vivo cell line-based leukemia models. The goal of this study was to evaluate these lipopolymers in (i) PBMCs obtained from healthy donors to determine the potential impact on non-malignant cells, and (ii) primary leukemia patient cells to assess therapeutic efficacy. Methods: Target gene levels were determined by RT-qPCR and the effect of treatment on progenitor cells was assessed by measuring changes in colony forming ability. Results: At day 1 post-treatment, no changes in FLT3 mRNA levels were observed in all 6 PBMC samples with the 2 candidate lipopolymers (LeuFectB and LeuFectC) tested. Among 11 PBMC samples evaluated at day 2, only one sample showed a significant reduction in oncogene transcript levels on treatment with LeuFectB. The impact of lipopolymer/siRNA complex treatment on progenitor cells in the PBMC population was then assessed. Of the 7 PBMC samples tested, significant reduction in colony numbers was observed in one sample with LeuFectB and two samples with LeuFectC. On an average, the lipopolymer/siRNA complexes were well tolerated by normal healthy cells as changes observed in oncogene expression and colony forming ability were not consistent across the whole pool. Primary acute lymphoblastic leukemia (ALL) patient cells treated with complexes targeting STAT5A showed marked reduction in STAT5A transcript levels in 4 out of 7 samples with both the lipopolymers tested. The subsequent impact of STAT5A downregulation on the clonogenicity of leukemia stem cells was observed in the significant decrease in colony numbers seen in 3 samples (out of 8) with LeuFects. Additionally, combined downregulation of STAT5A and BCR-ABL genes in BCR-ABL+ ALL patient samples led to a notable decrease in cell proliferation and viability. Moreover,2 out of 3 acute myeloid leukemia (AML) patient samples harboring the FLT3-ITD mutation showed significant retardation in their colony forming ability on treatment with FLT3-targeting siRNA complexes. Collectively, the ALL and AML primary samples demonstrated promising preliminary results, suggesting a therapeutic effect on the stem cell population which is critical for mitigating relapse or prolonging remission. Conclusions: These findings provide further proof-of-concept for clinical translation of lipopolymer mediated siRNA therapy of leukemia, showing relative safety and selectivity of these lipopolymer/siRNA complexes.

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL

AbstractThe t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre–B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Comprehensive Characterisation of the Copy Number Landscape in Acute Lymphoblastic Leukemia Using Digitalmlpa

Introduction: Acute lymphoblastic leukemia (ALL) is a heterogeneous disease affecting various signaling pathways including cell differentiation and cell cycle control. ALL subtypes are defined by their genetic abnormalities, encompassing gross chromosomal aberrations, ploidy changes, translocations or rearrangements, regional or focal (intragenic) copy number alterations (CNAs), gene fusions and point mutations. Novel biomarkers for risk stratification are still being discovered, further mapping the diverse genetic ALL landscape. Testing for these newly identified genetic biomarkers and characterising the copy number (CN) landscape in ALL samples can provide useful new insights for risk stratification and patient-tailored treatment regimens. Methods: For multiplex detection of CNAs in ALL samples, a new test version of SALSA® digitalMLPA™ Probemix D007 Acute Lymphoblastic Leukemia was developed. Hereafter, this new test version will be referred to as D007 Acute Lymphoblastic Leukemia. This assay contains probes for CNA detection of 70 key genes and regions frequently altered in B-cell ALL and T-cell ALL ( Figure 1). In addition, this probemix contains digital karyotyping probes covering each chromosomal arm for the detection of gross chromosomal aberrations (except the p-arm of acrocentric chromosomes). A selection of these digital karyotyping probes also serve as reference probes. Finally, a set of single nucleotide polymorphism (SNP) probe pairs, targeting both alleles of each of the SNPs, was included for sample identification and guiding the correct CN baseline determination, facilitating optimal data normalisation in samples with complex karyotypes. To test the performance of this new test version of the probemix, DNA samples from 29 ALL cell lines were analysed. digitalMLPA-derived PCR amplicons were sequenced on a NextSeq 1000 Sequencing System (Illumina). FastQ files were analysed using Coffalyser digitalMLPA™ (MRC Holland) and a custom-built algorithm was used for copy number determination using SNP probe pairs. Results: digitalMLPA data of all 29 cell lines was analysed and compared to Cancer Cell Line Encyclopedia (CCLE) copy number data (Depmap, Copy Number Public 23Q2 download). CNAs were detected with D007 Acute Lymphoblastic Leukemia, including clinically relevant biomarkers such as the IKZF1 plus profile, PAX5alt, TCF3 fusions, focal 22q11.22 deletion (incl. VPREB1), intrachromosomal amplification of chromosome 21 (iAMP21), hypo- and hyperdiploidy. Other CNAs of emerging significance in ALL risk stratification included 6q15-q16 deletions, DNMT3B, CDKN1B and KRAS alterations. SNP probe pairs were effectively used to facilitate correct CN baseline determination, and results were confirmed using CCLE data (Depmap, CCLE_ABSOLUTE_combined 2019 download). Correct CN baseline detection is particularly important for samples with complex karyotypes, such as the P12-ICHIKAWA cell line. In this cell line, our data revealed that most chromosomal regions targeted by the SNP probe pairs had a copy number ≥3 ( Figure 2), consistent with the hypotetraploid karyotype previously described for this cell line. Conclusions: Using this new test version of SALSA® digitalMLPA™ D007 Acute Lymphoblastic Leukemia, numerous CNAs can be detected in a single reaction. Results obtained with this probemix showed a high correlation with CN data from the CCLE database in a panel of 29 ALL cell lines. digitalMLPA provides valuable insights into the CN of key target sequences relative to reference sequences in the genome. Moreover, our novel approach using pairs of probes that target both SNP alleles provides an additional layer of information, guiding CN and ploidy status determination in highly complex karyotypes. The SNP probe pair based approach for detection of CN levels in complex samples shows promising results and will be further validated in an independent cohort of 140 ALL patient samples. These results will be presented at the ASH meeting.

Implications of mitochondrial DNA variants in pediatric B-cell acute lymphoblastic leukemia

BackgroundResearch on the role of variations in the mitochondrial genome in pathogenesis of acute lymphoblastic leukemia (ALL) has been unfolding at a rapid rate. Our laboratory has previously described higher number of copies of the mitochondrial genomes per cell in pediatric ALL patients as compared to the healthy controls. In the current study, we evaluated the pattern of mitochondrial genome variations in 20 de-novo pediatric B-ALL cases and seven controls. Quantitative real-time Polymerase Chain Reaction was used for estimation of mitochondrial genomes’ copy number in bone marrow samples of each ALL patient and peripheral blood samples of controls. The complete mitochondrial genomes of all samples were sequenced using the Illumina platform.ResultsSequencing data analysis using multiple mitochondrial genome databases revealed 325 variants in all 27 samples, out of which 221 variants were previously known while 104 were unassigned, new variants. The 325 variants consisted of 7 loss-of-function variants, 131 synonymous variants, 75 missense variants, and 112 non-coding variants. New, missense variants (n = 21) were identified in genes encoding the electron transport chain complexes with most of them encoding ND4, ND5 of complex I. Missense and loss-of-function variants were found to be deleterious by many predictor databases of pathogenicity. MuTect2 identified true somatic variants present only in tumors between patient-sibling pairs and showed overlap with missense and loss-of-function variants. Online MtDNA-server showed heteroplasmic and homoplasmic variants in mitochondrial genome.ConclusionsThe data suggest that some of these variations might have a deleterious impact on the expression of mitochondrial encoded genes with a possible functional relevance in leukemia.

Multi-Cohort Transcriptomic Subtyping of B-Cell Acute Lymphoblastic Leukemia.

RNA sequencing provides a snapshot of the functional consequences of genomic lesions that drive acute lymphoblastic leukemia (ALL). The aims of this study were to elucidate diagnostic associations (via machine learning) between mRNA-seq profiles, independently verify ALL lesions and develop easy-to-interpret transcriptome-wide biomarkers for ALL subtyping in the clinical setting. A training dataset of 1279 ALL patients from six North American cohorts was used for developing machine learning models. Results were validated in 767 patients from Australia with a quality control dataset across 31 tissues from 1160 non-ALL donors. A novel batch correction method was introduced and applied to adjust for cohort differences. Out of 18,503 genes with usable expression, 11,830 (64%) were confounded by cohort effects and excluded. Six ALL subtypes (ETV6::RUNX1, KMT2A, DUX4, PAX5 P80R, TCF3::PBX1, ZNF384) that covered 32% of patients were robustly detected by mRNA-seq (positive predictive value ≥ 87%). Five other frequent subtypes (CRLF2, hypodiploid, hyperdiploid, PAX5 alterations and Ph-positive) were distinguishable in 40% of patients at lower accuracy (52% ≤ positive predictive value ≤ 73%). Based on these findings, we introduce the Allspice R package to predict ALL subtypes and driver genes from unadjusted mRNA-seq read counts as encountered in real-world settings. Two examples of Allspice applied to previously unseen ALL patient samples with atypical lesions are included.

High-Throughput Drug Library Screening in Primary KMT2A-Rearranged Infant ALL Cells Favors the Identification of Drug Candidates That Activate P53 Signaling.

KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) represents an aggressive type of childhood leukemia characterized by a poor clinical outcome with a survival chance of <50%. Implementing novel therapeutic approaches for these patients is a slow-paced and costly process. Here, we utilized a drug-repurposing strategy to identify potent drugs that could expeditiously be translated into clinical applications. We performed high-throughput screens of various drug libraries, comprising 4191 different (mostly FDA-approved) compounds in primary KMT2A-rearranged infant ALL patient samples (n = 2). The most effective drugs were then tested on non-leukemic whole bone marrow samples (n = 2) to select drugs with a favorable therapeutic index for bone marrow toxicity. The identified agents frequently belonged to several recurrent drug classes, including BCL-2, histone deacetylase, topoisomerase, microtubule, and MDM2/p53 inhibitors, as well as cardiac glycosides and corticosteroids. The in vitro efficacy of these drug classes was successfully validated in additional primary KMT2A-rearranged infant ALL samples (n = 7) and KMT2A-rearranged ALL cell line models (n = 5). Based on literature studies, most of the identified drugs remarkably appeared to lead to activation of p53 signaling. In line with this notion, subsequent experiments showed that forced expression of wild-type p53 in KMT2A-rearranged ALL cells rapidly led to apoptosis induction. We conclude that KMT2A-rearranged infant ALL cells are vulnerable to p53 activation, and that drug-induced p53 activation may represent an essential condition for successful treatment results. Moreover, the present study provides an attractive collection of approved drugs that are highly effective against KMT2A-rearranged infant ALL cells while showing far less toxicity towards non-leukemic bone marrow, urging further (pre)clinical testing.

Selective Inhibition of Class II HDAC Isoforms 4 and 5 Provides a Promising Therapeutic Intervention for MLL-Rearranged Acute Lymphoblastic Leukemia in Infants

▪MLL-rearranged acute lymphoblastic leukemia (ALL) is characterized by deregulation of the epigenome and shows susceptibility towards epigenetic perturbators such as histone deacetylase (HDAC) inhibitors. Hence, HDACs represent attractive drug targets and a variety of small molecule HDAC inhibitors have been developed and evaluated for the treatment of hematological malignancies. However, most broad-spectrum inhibitors, which simultaneously target the majority of human HDAC isoforms, often induce toxicity, especially in combination with other therapeutic agents. Therefore, selective inhibition of only one or two HDAC isoforms may represent a better alternative, provided that disease-specific dependency on specific HDACs has been identified.We examined the effects of shRNA-mediated knock-down of the class II HDACs (i.e. HDAC4, HDAC5, HDAC6, HDAC7 and HDAC9) in the MLL-rearranged ALL cell lines SEM and ALL-PO. Except for HDAC9, loss of expression (both on the mRNA and protein level) of all HDACs led to strong reductions in viable cells (0.70 to 0.19-fold; p=0.02-0.0016) in both models due to apoptosis, cell cycle arrest, or a combination thereof.Next, we evaluated the in vitro efficacy of a variety of class II HDAC-specific inhibitors on a panel of MLL-rearranged ALL (n=5) using 4-day viability MTT assays. This revealed that the selective HDAC4/5 inhibitor LMK-235 was able to recapitulate the loss-of-function phenotype of HDAC4 and HDAC5. Dose response curves showed complete growth inhibition in MLL-rearranged ALL cell lines (n=5), as well as in primary MLL-rearranged infant ALL patient samples (n=4), with IC 50 values of ~100 nM and 40-100 nM, respectively. Importantly, at these concentrations, LMK-235 hardly affected whole bone marrow samples derived from healthy individuals (n=2), for which IC 50 values were ~1 µM.To further explore the potential of class II HDAC inhibitor-based therapeutic strategies, we performed a combinatorial drug screen to identify compounds that synergize with LMK-235. For this, a compound library (comprising >200 unique agents) was screened in the absence and presence of varying concentrations of LMK235 in the MLL-rearranged cell line models SEM and ALL-PO. This, and subsequent validation experiments in additional cell line models, revealed that Venetoclax (BCL2 inhibitor), Trametinib (MEK/ERK inhibitor), Ponatinib (multi-tyrosine kinase inhibitor) and Omipalisib (a PI3K/mTOR inhibitor) strongly synergized with LMK-235. Average ZIP synergy scores ranged from 10-30, with peak ZIP scores up to 40. Importantly, synergistic effects were consistent over all concentration combinations tested. The addition of 50-100 nM LMK-235 strongly reduced IC 50 values for Omipalisib, Ponatinib and Venetoclax (0.27-fold p=0.003, 0.11-fold p=0.0005, 0.75-fold p=0.0004, respectively) in both models.In preparation to assess the in vivo efficacy of LMK-235 in patient-derived xenograft (PDX) mouse models of MLL-rearranged infant ALL, pharmacokinetic/pharmacodynamic (PK/PD) analysis was performed in immunodeficient NSG mice (n=5). For this, mice were treated with 20 mg/kg of LMK-235, daily administered via intraperitoneal injections for a total of 29 days. While none of the mice showed signs of toxicity or weight loss, LMK-235 plasma levels were stably maintained at concentrations that are highly effective against MLL-rearranged ALL cells in vitro.Taken together, these data demonstrate that various class II HDAC isoforms are targetable vulnerabilities in MLL-rearranged ALL and that pharmaceutical inhibition of HDAC4/5 by LMK-235 represents an attractive therapeutic option. Moreover, high levels of synergy observed between this HDAC inhibitor and various agents belonging to drug classes already reported to be effective against MLL-rearranged ALL, warrants pre-clinical evaluation in vivo. Currently, the assessment of the in vivo efficacy of LMK-235 monotherapy in MLL-rearranged infant ALL PDX models is in progress, after which promising synergistic HDAC inhibitor-based drug combinations will be evaluated. To determine the additional therapeutic value, the efficacy of LMK-235 and promising synergistic combinations will be evaluated in the background of conventional combination chemotherapy, where PDX models will receive a mouse-adapted version of induction therapy currently applied for treatment of MLL-rearranged infant ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Chromatin Accessibility Landscapes of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients spanning both B- and T-cell lineages. ALL also occurs less commonly in adults but with cure rates of only around 30%. Past work has characterized ALL into molecular subtypes spanning a range of aberrant chromosomal rearrangements and oncogene chimeric fusions driving malignancy. While transcriptional profiling of these subtypes has been extensively examined, the accompanying chromatin accessibility landscape and corresponding gene regulatory repertoire is not well characterized for many subtypes. To better profile the ALL epigenomic and gene regulatory repertoire we examined chromatin accessibility of 12 distinct molecular subtypes (BCR-ABL1, ETV6-RUNX1, Hyperdiploid, Hypodiploid, KMT2A rearranged, Ph-Like, PAX5, DUX4/ERG, TCF3-PBX1, T-ALL, Early T Precursor and B-other) across 189 primary patient samples of pediatric ALL (n = 106) and adult ALL (n = 83) origin using ATAC-seq. To our knowledge, this represents the largest collection of chromatin accessibility data in primary ALL samples spanning multiple molecular subtypes to date. Collectively, we identified over 600,000 accessible chromatin sites in the ALL genome with over 50,000 regions of differentially accessible chromatin encompassing both common and subtype-specific modalities. Further, transcription factor (TF) footprint profiling of ATAC-seq yielded tens of thousands of candidate TF binding events and identified key TF drivers within distinct molecular subtypes. We additionally performed H3K27ac ChIP-seq in a subset of 12 primary ALL patient samples, with integration of these histone data for select patient samples allowing inferences about candidate super-enhancer drivers of ALL molecular subtypes. Overall, these analyses and data offer a window into the gene regulatory and epigenetic landscape of ALL, and further highlight the complexity and heterogeneity of accessible chromatin landscapes among distinct molecular subtypes of ALL. DisclosuresStock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee. Evans: Princess Máxima Center for Pediatric Oncology, Scientific Advisory Board, Chair: Membership on an entity's Board of Directors or advisory committees; 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.

Cell-Free DNA As a Biomarker for Acute Lymphoblastic Leukemia Relapse in the Central Nervous System

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with a relapse rate of 15%. The presence of lymphoblasts in the central nervous system (CNS) is a negative prognostic indicator and a frequent site of disease relapse. CNS disease is defined as more than five white blood cells in the cerebrospinal fluid (CSF) with positive lymphoblast cytomorphology. Often leukemic blasts are detected by flow cytometry of the CSF prior to reaching the five white blood cell threshold, requiring repeat lumbar punctures at 2-4 week intervals until the arbitrarily defined clinical criteria for diagnosis are met. Additionally, more than 50% of autopsied brains from ALL patients show evidence of CNS disease, despite non-detectable CNS involvement via cytology of CSF. These data suggest that patients may be harboring CNS disease before it expands enough to be clinically diagnosed. We have developed a new assay that may allow for earlier detection of CNS disease and relapse by quantifying cell-free, circulating tumor DNA (ctDNA) in the CSF. ctDNA is a proven biomarker of relapse and metastasis in solid tumors in pre-clinical testing; however, its utility at predicting CNS disease in ALL has not been examined. We examined two possible methods for using ctDNA as a biomarker: leukemia cell clonality and DNA methylation profiling. We collected bone marrow aspirate, blood, and CSF samples from 11 newly diagnosed patients prior to the start of chemotherapy treatment to use as a training data set. ctDNA was isolated from blood and CSF samples at diagnosis and throughout treatment. Genomic DNA was isolated from bone marrow and peripheral blood mononuclear cell (PBMC) samples at diagnosis. For leukemia cell clonality assays, Invivoscribe Lymphotrack PCR assays combined with MinION (Oxford Nanopore Technologies) sequencing were used to identify the VDJ sequence of the immunoglobulin (B-ALL) or T-cell receptor (T-ALL) rearrangements in the clones comprising each leukemia. Throughout the course of treatment, ctDNA samples were run on the MinION sequencer, examining the abundance of major clones present and ctDNA quantification was done on patient plasma and CSF samples over time. While this assay relies on patient specific VDJ sequencing, we are also in the process of developing a more universal assay that utilizes recurrent methylation changes in T-ALL or B-ALL, compared to normal PBMCs. We performed methylation sequencing on 3 control PBMC samples and 7 ALL patient samples to identify differentially methylated regions (DMRs) present specifically in ALL samples. This sequencing identified 9,222 DMRs present in the ALL samples. These sites were then compared with publicly available datasets, yielding 55 overlapping regions and 19 specific overlapping methylation sites commonly present in ALL samples and not in PBMCs. We are now in the process of validating these differentially methylated sites by droplet digital polymerase chain reaction (ddPCR) to come up with a panel of biomarkers to track ALL disease over time. The end goal of our study is to develop an assay to rapidly detect relapse and CNS disease in ALL that is more sensitive and less invasive than the current clinical assays. Earlier detection of relapse and CNS disease will provide patients with more treatment options, which may ultimately improve patient outcome. Results will ultimately be correlated with patient response to therapy, the presence of CNS disease, and overall outcomes as determined by standard clinical diagnostic procedures. Disclosures No relevant conflicts of interest to declare.

MRD Xenotransplantation Prospectively Identifies Treatment-Selected Acute Lymphoblastic Leukemia Subpopulations with Relapse-Initiating Potential

Despite complete remissions being achieved in most newly diagnosed acute lymphoblastic leukemia (ALL) patients, relapse remains a significant clinical challenge. Interrogation of matched ALL samples has provided insights into the cellular characteristics and evolutionary trajectory of relapse-fated clones, but requires blasts obtained at time of relapse to provide a reference point for the retrospective analysis. Despite the strong prognostic value of minimal residual disease (MRD), MRD-positive samples have been under-utilized for investigating disease progression as the low leukemia burden presents significant challenges to the evaluation of the residual blasts. To overcome this limitation, we performed patient-derived xenografting (PDX) with MRD samples to enable deeper characterization of residual blasts, including functional assessment of their leukemia propagating ability. In marked contrast to a previous study in adult ALL, we achieved engraftment of MRD blasts from 5 of 7 pediatric ALL patient samples, obtained at the end of induction (EOI) therapy, by intravenous injection into non-conditioned NSG mice. MRD blasts expanded with patient-specific kinetics, reaching overt leukemia in recipients of three of the samples and remaining at MRD levels in recipients of the remaining two. The absence of normal human hematopoiesis in the MRD-engrafted mice (termed EOI-PDX), combined with the expansion of residual blast numbers, enabled clear phenotypic definition of therapy-selected, leukemia-propagating subpopulations, including those present at &amp;lt;1%. Subsequent reanalysis of diagnosis (DX) and EOI clinical samples for the identified subpopulations exposed a treatment response-mediated disruption of the established population hierarchy that explained the phenotypic shifts observed during disease progression. The relevance of the subpopulations identified as potential leukemia drivers was confirmed by their dominance in subsequent clinical relapses. Furthermore, phenotype-based sorting of viable blast subpopulations enabled the identification and comparison of metabolic characteristics of treatment sensitive and resistant blasts. This immunophenotypic study recapitulates the enrichment of minor clones during ALL progression described by retrospective sequencing studies, but it does so prospectively. By expanding on the quantitative and qualitative information that can be gathered from MRD samples, EOI-PDX identify the blast populations that are most likely to drive relapse, prior to clinical disease progression. By providing a platform to achieve early identification and ongoing monitoring of therapy-resistant subpopulations with strong leukemia-propagating capacity, this approach could refine treatment-response assessment and relapse-risk stratification. Further, by expanding the numbers of viable treatment-selected MRD blasts available for interrogation, EOI-PDX could enable early identification of vulnerabilities in chemotherapy-resistant blasts. Disclosures No relevant conflicts of interest to declare.

Preclinical Investigation of the p53-MDM2 Antagonist Idasanutlin (RG7388) Demonstrates Significant Activity in High Risk Adult Acute Lymphoblastic Leukemia

Outcomes for adult patients with acute lymphoblastic leukemia (ALL) are dismal and have not kept pace with their pediatric counterparts, with five-year survival rates of less than 45%. TP53 mutations are infrequent in ALL, but activity of the oncoprotein MDM2 may otherwise phenotypically disrupt and circumvent normal p53 function, positing the p53-MDM2 signaling axis as a potential therapeutic target for the engagement of intrinsic cell death programs. Given the clinical safety and responses to p53-MDM2 antagonist idasanutlin (RG7388) in other hematological cancers and solid tumors, we aimed to evaluate the therapeutic potential of idasanutlin in ALL. Single-agent activity of idasanutlin was investigated in 17 high-risk ALL patient and patient-derived xenograft samples (aged 4 to 51 years), both B-ALL (n=15) and T-ALL (n=2), including KMT2A-rearranged, TCF3-rearranged, and Philadelphia-positive ALL. An ex vivo coculture of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSC) was employed to support growth of the ALL blasts during short-term culture, complemented by a fluorescent image-based microscopy platform which identifies and discriminates the two cellular compartments using random forest machine learning algorithms based on cellular nuclear staining. Idasanutlin demonstrated sub-micromolar, dose-dependent anti-leukemic activity against 15 of 17 samples tested, with half maximal effective concentrations (EC50) in the range of 10 to 220 nM (mean EC50 = 45.1nM); the two exceptions were later determined to harbor homozygous inactivating TP53 mutations; p.Y220C and p.S241P within the p53 DNA binding domain. The idasanutlin EC50 concentrations determined are clinically achievable, well below the peak plasma concentrations reported in patients for other disease indications. Furthermore, idasanutlin concentrations below 10µM had no impact upon MSC survival. Conforming to p53-MDM2 auto-regulatory feedback mechanisms, we demonstrated that idasanutlin efficiently stabilized and activated p53 at the protein level within 6 hours when treated with their respective idasanutlin EC50, to a level greater than 4-fold increased relative to their respective vehicle-only controls (p=0.001, n=7). Further, p53-regulated transcriptional target gene products, MDM2 and p21, were increased by 5-fold and 2-fold respectively, validating engagement of the p53 pathway by idasanutlin (p=0.036 and 0.125, respectively). By contrast, idasanutlin did not elicit increased expression of either p53-regulated transcriptional target gene product in the identified TP53-mutant patient samples (n=2). On-target specificity of idasanutlin was further confirmed in a NALM6 isogenic cell line model, whereby the TP53 wildtype line was sensitive to idasanutlin (EC50 = 74nM) and effectively activated p53 signaling whereas the TP53 homozygous null line was highly resistant (EC50 = ~10µM). To determine whether the decreased cell numbers and engagement of p53 signaling observed were accompanied by cell death, the capacity of idasanutlin to induce apoptosis in the ALL samples was next investigated. Patient-derived ALL samples (n=6) were treated with vehicle or idasanutlin at their respective EC50s for 24 and 48 hours, and then analyzed by flow cytometry. There was an increase in annexin-V positive cells within 24 hours compared to the vehicle-only treated cells (mean±SD 14.3±6.6% vs 27.0±21.2% respectively (p=0.125). By 48 hours significant apoptosis was attained, with a mean±SD of 59.0±23.8% annexin-V positive cells compared to the mean of vehicle-treated cells at 29.1±11.6% (p=0.004). Cleaved poly(ADP-ribose) polymerase (PARP) levels were also increased greater than 3-fold compared to vehicle-only control cells as assessed by immunoblotting (p=0.045, n=3), corroborating these findings. These data emphasize the potential of pharmacologically targeting the p53-MDM2 axis in ALL, demonstrating potent, on-target, cytotoxic activity in a range of high-risk ALL cytogenetic subgroups. Taken together, these findings support further preclinical investigations into idasanutlin and other p53-MDM2 antagonists and potential combinations to improve the treatment of adult ALL. Disclosures Irving: F. Hoffmann-La Roche: Research Funding.

Abstract 5399: The NSD2 p.E1099K mutation is enriched at relapse and confers drug resistance in a cell context dependent manner in pediatric acute lymphoblastic leukemia

Abstract Introduction: Outcomes for children with relapsed acute lymphoblastic leukemia (ALL) remain poor. Mutations in epigenetic modifiers are commonly found at relapse, including the activating p.E1099K (EK) mutation within the conserved SET domain of NSD2, which has been reported in 10% of patients at relapse. NSD2 has 3 isoforms, two of which (Types II and REIIBP) contain the SET domain. Our aim was to determine the role of NSD2 in driving relapse and further elucidate the prevalence of NSD2 EK in patient samples. Methods: Whole genome/exome sequencing was performed on diagnosis/relapse ALL pairs and data were aggregated for somatic SNVs/indels. NSD2 EK positive cases at relapse were subject to ultra-deep targeted re-sequencing to determine the presence of small subclones at diagnosis. To investigate the role of NSD2 and its isoforms in mediating relapse, short hairpin RNAs (shRNAs) were used to knockdown (KD) NSD2 in three NSD2 EK mutant cell lines with non-targeting (NT) shRNA used as controls. Cells were treated with chemotherapy commonly used in pediatric ALL and assessed for cytotoxicity by CellTiter- Glo®. To determine NSD2's impact on gene expression and the epigenetic landscape, RNAseq, ChIPseq, ATACseq, and Hi-C were performed using standard techniques. Results: Examination of paired ALL patient samples revealed enrichment of NSD2 EK clones at relapse. B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM) revealed that KD led to decreased proliferation in all lines and decreased clonal growth in RCH-ACV relative to NT. Furthermore, increased sensitivity to various chemotherapeutic agents was observed in the KD lines compared to NT controls, but the pattern of drug sensitivity varied among lines implying cell context specific drug responses. Simultaneous knockdown of Type II and REIIBP had a greater impact than Type II alone, indicating both SET containing EK isoforms contribute to phenotypic changes. When comparing gene expression changes in NT vs KD, minimal overlap in differential genes was noted among the three cell lines (4.4% upregulated, 1.7% downregulated) indicating significant diversity in transcriptional reprogramming. ATACseq of the NT and KD for all three lines revealed intergenic regions as the most affected by NSD2 with RCH-ACV and RS4;11 NT lines having more open chromatin compared to their KD whereas SEM NT displayed marked peak loss compared to its KD. Less than 2% of differential intergenic regions were shared among the lines. Hi-C analysis demonstrated disparity in chromosome architecture among NSD2 EK lines as well. Shifts in intergenic regions was further supported by ChIPseq, which revealed increased H3K36me2 across intergenic regions in RS4;11 NT compared to KD. Lastly, we noted a strong correlation of increased gene expression with increased H3K36me2, H3K9ac, and H3K27ac at promoters and gene bodies. Conclusions: NSD2 EK plays a unique role in mediating clonal fitness through pleiotropic mechanisms that are dependent on the underlying cellular context. Citation Format: Joanna Pierro, Jason Saliba, Sonali Narang, Gunjan Sethia, Shella Saint Fleur-Lominy, Ashfiyah Chowdhury, Takaya Moriyama, Kjeld Schmiegelow, Jun J. Yang, Mignon L. Loh, Patrick A. Brown, Jinghui Zhang, Xiaotu Ma, Aristotelis Tsirigos, Nikki A. Evensen, William L. Carroll. The NSD2 p.E1099K mutation is enriched at relapse and confers drug resistance in a cell context dependent manner in pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5399.

A Knowledge-Reserved Distillation with Complementary Transfer for Automated FC-based Classification Across Hematological Malignancies.

Acute leukemia often comes with life-threatening prognosis outcome and remains a critical clinical issue today. The implementation of measurable residual disease (MRD) using flow cytometry (FC) is highly effective but the interpretation is time-consuming and suffers from physician idiosyncrasy. Recent machine learning algorithms have been proposed to automatically classify acute leukemia samples with and without MRD to address this clinical need. However, most prior works either validate only on a small data cohort or focus on one specific type of leukemia which lacks generalization. In this work, we propose a transfer learning approach in performing automatic MRD classification that takes advantage of a large scale acute myeloid leukemia (AML) database to facilitate better learning on a small cohort of acute lymphoblastic leukemia (ALL). Specifically, we develop a knowledge-reserved distilled AML pre-trained network with ALL complementary learning to enhance the ALL MRD classification. Our framework achieves 84.5% averaged AUC which shows its transferability across acute leukemia, and our further analysis reveals that younger and elder ALL patient samples benefit more from using the pre-trained AML model.

Rapid Identification of Key Copy Number Alterations in B- and T-Cell Acute Lymphoblastic Leukemia by Digital Multiplex Ligation-Dependent Probe Amplification.

Recurrent clonal genetic alterations are the hallmark of Acute Lymphoblastic Leukemia (ALL) and govern the risk stratification, response to treatment and clinical outcome. In this retrospective study conducted on ALL patient samples, the purpose was to estimate the copy number alterations (CNAs) in ALL by digitalMLPA (dMLPA), validation of the dMLPA data by conventional MLPA and RT-PCR, and correlation of CNAs with Minimal Residual Disease (MRD) status. The ALL patient samples (n = 151; B-ALL, n = 124 cases and T-ALL, n = 27 cases) were assessed for CNAs by dMLPA for detection of sub-microscopic CNAs and ploidy status. This assay allowed detection of ploidy changes and CNAs by multiplexing of karyotyping probes and probes covering 54 key gene targets implicated in ALL. Using the dMLPA assay, CNAs were detected in ~89% (n = 131) of the cases with 66% of the cases harboring ≥3 CNAs. Deletions in CDKN2A/B, IKZF1, and PAX5 genes were detectable in a quarter of these cases. Heterozygous and homozygous gene deletions, and duplications were observed in genes involved in cell cycle control, tumor suppression, lineage differentiation, lymphoid signaling, and transcriptional regulators with implications in treatment response and survival outcome. Distinct CNAs profiles were evident in B-ALL and T-ALL cases. Additionally, the dMLPA assay could reliably identify ploidy status and copy number-based gene fusions (SIL-TAL1, NUP214-ABL, EBF1-PDGFRB). Cases of B-ALL with no detectable recurrent genetic abnormalities could potentially be risk stratified based on the CNA profile. In addition to the commonly used gene deletions for risk assessment (IKZF1, EBF1, CDKN2A/B), we identified a broader spectrum of gene alterations (gains of- RUNX1, LEF1, NR3C2, PAR1, PHF6; deletions of- NF1, SUZ12, MTAP) that significantly correlated with the status of MRD clearance. The CNAs detected by dMLPA were validated by conventional MLPA and showed high concordance (r = 0.99). Our results demonstrated dMLPA to be a robust and reliable alternative for rapid detection of key CNAs in newly diagnosed ALL patients. Integration of ploidy status and CNAs detected by dMLPA with cytogenetic and clinical risk factors holds great potential in further refinement of patient risk stratification and response to treatment in ALL.

TCF7L2 activated HOXA-AS2 decreased the glucocorticoid sensitivity in acute lymphoblastic leukemia through regulating HOXA3/EGFR/Ras/Raf/MEK/ERK pathway

Acute lymphoblastic leukemia (ALL) is characterized by abnormal lymphoblasts accumulation in the bone marrow and blood. Despite great efforts have been made in exploring novel therapeutic method, the prognosis of children with ALL is still unsatisfied. Glucocorticoid (GC) resistance is a great obstacle for the clinical treatment of ALL. Therefore, it is essential to investigate the molecular mechanism underlying the GC resistance. According to previous reports, long noncoding RNAs (lncRNAs) are involved in drug resistance of various human cancers. LncRNA HOXA cluster antisense RNA2 (HOXA-AS2) has been reported in several human malignancies due to its oncogenic property. However, the molecular mechanism of HOXA-AS2 involved in the GC resistance of ALL still needs to be further clarified. At first, we found that lncRNA HOXA-AS2 was highly expressed both in prednisone insensitive ALL cell lines and patient samples. Gain or loss-of-function assays revealed that HOXA-AS2 enhanced GC resistance via promoting cell proliferation and inhibiting cell apoptosis. Furthermore, we validated that HOXA-AS2 upregulated HOXA3, thereby activating EGFR/Ras/Raf/MEK/ERK signaling pathway. Our findings showed that HOXA-AS2 may be a potential therapeutic target for ALL patients with poor GC resistance.

Transcriptome sequencing in pediatric acute lymphoblastic leukemia identifies fusion genes associated with distinct DNA methylation profiles

BackgroundStructural chromosomal rearrangements that lead to expressed fusion genes are a hallmark of acute lymphoblastic leukemia (ALL). In this study, we performed transcriptome sequencing of 134 primary ALL patient samples to comprehensively detect fusion transcripts.MethodsWe combined fusion gene detection with genome-wide DNA methylation analysis, gene expression profiling, and targeted sequencing to determine molecular signatures of emerging ALL subtypes.ResultsWe identified 64 unique fusion events distributed among 80 individual patients, of which over 50% have not previously been reported in ALL. Although the majority of the fusion genes were found only in a single patient, we identified several recurrent fusion gene families defined by promiscuous fusion gene partners, such as ETV6, RUNX1, PAX5, and ZNF384, or recurrent fusion genes, such as DUX4-IGH. Our data show that patients harboring these fusion genes displayed characteristic genome-wide DNA methylation and gene expression signatures in addition to distinct patterns in single nucleotide variants and recurrent copy number alterations.ConclusionOur study delineates the fusion gene landscape in pediatric ALL, including both known and novel fusion genes, and highlights fusion gene families with shared molecular etiologies, which may provide additional information for prognosis and therapeutic options in the future.

Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models

Recently, NOD-SCID IL2Rγ-/- (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties.To further improve our humanized niche scaffold model, we genetically engineered human MSCs to secrete human interleukin-3 (IL-3) and thrombopoietin (TPO). Invitro, these IL-3- and TPO-producing MSCs were superior in expanding human cord blood (CB) CD34+ hematopoietic stem/progenitor cells. MLL-AF9-transduced CB CD34+ cells could be transformed efficiently along myeloid or lymphoid lineages on IL-3- and TPO-producing MSCs. Invivo, these genetically engineered MSCs maintained their ability to differentiate into bone, adipocytes, and other stromal components. Upon transplantation of MLL-AF9-transduced CB CD34+ cells, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) developed in engineered scaffolds, in which a significantly higher percentage of myeloid clones was observed in the mouse compartments compared with previous models. Engraftment of primary AML, B-cell ALL, and biphenotypic acute leukemia (BAL) patient samples was also evaluated, and all patient samples could engraft efficiently; the myeloid compartment of the BAL samples was better preserved in the human cytokine scaffold model. In conclusion, we show that we can genetically engineer the ectopic human BM microenvironment in a humanized scaffold xenograft model. This approach will be useful for functional study of the importance of niche factors in normal and malignant human hematopoiesis.

A Novel Multiplex NGS-Based Digital MLPA Assay for Copy Number Detection of 700 Target Sequences in Childhood Acute Lymphoblastic Leukemia

▪Background: Recurrent and clonal genetic alterations are characteristic of different subtypes of pediatric acute lymphoblastic leukemia (ALL) and several of them are strong independent predictors of patient outcome. Multiplex Ligation-dependent Probe Amplification (MLPA) has become one of the standard methods for detection of common copy number alterations (CNAs), including IKZF1 deletions, which are associated with a poor outcome. However, standard MLPA assays only accommodate a maximum of 60 MLPA probes. In order to increase the number of target sequences in one assay, a next generation sequencing (NGS)-based MLPA variant has been developed. This digitalMLPA assay is based on the well-known MLPA procedure but can include up to 1000 probes in a single reaction, uses minute amounts of DNA (≥20ng) and can be analysed on all Illumina NGS platforms.Methods: A digitalMLPA assay was designed and optimised to detect CNAs of 55 key target genes and regions in childhood ALL, including deletions of B-cell differentiation and cell cycle control genes (PAX5, IKZF1/2/3, EBF1, RB1, CDKN2A/B, ETV6, and BTG1), iAMP21 and rearrangements of the PAR1 region, T-cell ALL associated aberrations (STIL-TAL1, LEF1, CASP8AP2, MYB, EZH2, MLLT3, NUP214-ABL1, PTEN, LMO1/2, NF1, SUZ12, PTPN2, PHF6), ERG and TP53 deletions. Several genetic alterations of potential prognostic and/or therapeutic relevance in ALL have also been included (NOTCH1, CD200/BTLA, VPREB1, TBL1XR1, PDGFRB-EBF1, IGHM, NR3C1/2, CREBBP, CTCF, ADD3, EPHA1, FHIT, SPRED1 and TOX). All target genes are covered by at least 3 digitalMLPA probes (~450 probes in total). Moreover, a set of 200 probes was designed for genome-wide detection of gross ploidy changes (high hyper- or hypodiploidy) and to determine the extent of CNAs, while also acting as reference probes for data normalization. Performance of all probes has been extensively tested on genomic DNA from healthy individuals and positive cell lines. Only probes with a standard deviation <0.12 were included. All probes were also tested under various experimental conditions, such as different salt, probe and polymerase concentrations. A series of 76 pediatric ALL patient samples (including both B-ALL and T-ALL samples), previously characterized for specific genetic aberrations by array CGH (aCGH) and/or SNP array, has been analysed single-blinded using the digitalMLPA assay. Results were compared to those obtained from standard MLPA assays (P335 ALL-IKZF1 and P327 iAMP21-ERG), which contain probes with different ligation sites for the respective genes. A dilution series of three B-ALL patient samples was analysed to determine the detection limit for subclonal aberrations.Results: All aberrations previously identified by other methods in 67 pediatric ALL samples, were also identified using the digitalMLPA assay. These included whole chromosome gains and losses, whole gene deletions or gains, iAMP21, fusion genes (STIL-TAL1, NUP214-ABL1 and PDGFRB-EBF1) and intragenic gene deletions (IKZF1, ERG, CDKN2A/B, ETV6, PAX5 RUNX1, RB1, LEF1, NR3C1, RAG2, VPREB1, MLLT3, BTG1 and PTEN). Of interest was a case of high hyperdiploidy, which was correctly identified by digitalMLPA, while by aCGH analysis this case was misinterpreted as having multiple deletions. Among several other intragenic deletions, a heterozygous deletion of ERG exons 5-9 was observed in one patient. In addition, several cases of homozygous single-exon deletions were observed (e.g. IKZF1 exon 8, LEF1exon 3) and confirmed by aCGH. These findings should be further investigated with respect to their clinical impact. Results from the dilution series indicated that subclonal aberrations can be reliably detected by this digitalMLPA assay if present in at least 20- 30% of tumour cells.Conclusions: This study demonstrates that digitalMLPA is a reliable technique that can be used to genetically characterize clinical samples of ALL patients. Experiments can be performed on small amounts of DNA in a high-throughput and cost-effective fashion. Because of the targeted approach, data analysis will be much easier as compared to array or other sequencing platforms. These results merit further consideration of (digital)MLPA as a valuable alternative for genetic work-up of newly diagnosed ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Disruption of Annexin II /p11 Interaction Suppresses Leukemia Cell Binding, Homing and Engraftment, and Sensitizes the Leukemia Cells to Chemotherapy.

The bone marrow microenvironment plays an important role in acute lymphoblastic leukemia (ALL) cell proliferation, maintenance, and resistance to chemotherapy. Annexin II (ANX2) is abundantly expressed on bone marrow cells and complexes with p11 to form ANX2/p11-hetero-tetramer (ANX2T). We present evidence that p11 is upregulated in refractory ALL cell lines and patient samples. A small molecule inhibitor that disrupts ANX2/p11 interaction (ANX2T inhibitor), an anti-ANX2 antibody, and knockdown of p11, abrogated ALL cell adhesion to osteoblasts, indicating that ANX2/p11 interaction facilitates binding and retention of ALL cells in the bone marrow. Furthermore, ANX2T inhibitor increased the sensitivity of primary ALL cells co-cultured with osteoblasts to dexamethasone and vincristine induced cell death. Finally, in an orthotopic leukemia xenograft mouse model, the number of ALL cells homing to the bone marrow was reduced by 40–50% in mice injected with anti-ANX2 antibody, anti-p11 antibody or ANX2T inhibitor compared to respective controls. In a long-term engraftment assay, the percentage of ALL cells in mouse blood, bone marrow and spleen was reduced in mice treated with agents that disrupt ANX2/p11 interaction. These data show that disruption of ANX2/p11 interaction results in reduced ALL cell adhesion to osteoblasts, increased ALL cell sensitization to chemotherapy, and suppression of ALL cell homing and engraftment.

Targeted Inhibition of the MLL Transcriptional Complex By Proteosome Inhibitors Elicits a High Response Rate in Relapsed/Refractory MLL Rearranged Leukemia

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. DisclosuresOff Label Use: Vorinostat and Bortezomib for the treatment of pediatric leukemia.