Leukemia Samples Research Articles

Cytotoxicity of Gymnopilus purpureosquamulosus extracts on hematologic malignant cells through activation of the SAPK/JNK signaling pathway.

Treatment of hematologic malignancies is a formidable challenge for hematologists and there is an urgent need to identify safe and efficacious agents either via synthesis in the laboratory or isolation from natural products. Here, we report the cytotoxicity of extracts from mushroom Gymnopilus purpureosquamulosus Høil (G. pps) and describe its molecular mechanisms. Using leukemia, lymphoma and multiple myeloma cell lines, 28–35 ppm G. pps extract inhibited cell proliferation by ~46–79%, which correlates with activation of apoptosis as indicated by increase in annexin V-positive cells (~5–8-fold), production of reactive oxygen species (~2–3-fold), cells in sub G0/G1 phase (~3–13-fold), caspase 3 enzymatic activity (~1.6–2.9-fold), DNA fragmentation, PARP1 cleavage and down-regulation of prosurvival proteins. Mitochondrial membrane potential decreased and leakage of pro-apoptotic factors to cytoplasm was observed, consistent with the activation of intrinsic apoptosis. Western blot analysis showed activation of the ASK1-MEK-SAPK/JNK and ASK1-P38 MAPK pathways possibly due to changes in the cellular redox status as suggested by decreased protein levels of peroxiredoxin, thioredoxin and thioredoxin reductase. Moreover, antioxidant N-acetylcysteine alleviated the cytotoxicity of G. pps. Pharmacological inhibition of SAPK/JNK and P38 alleviated the G. pps-mediated cytotoxicity. The extract activated apoptosis in leukemia and lymphoma patient cell samples but not in mononuclear cells from healthy donors further supporting the therapeutic values of G. pps for hematologic malignancies.

Clinical utility of whole genome sequencing in hematological neoplasms.

7028 Background: Hematological neoplasms are often characterized by acute onset and rapid disease progression. Cytogenetics, FISH, SNP arrays, targeted DNA and RNA sequencing are performed to inform diagnosis, risk stratification and guide treatment decisions. Whole genome sequencing (WGS) offers the opportunity to comprehensively characterize all putative biomarkers in a single assay. However, a limitation in current WGS implementation is the requirement for a germline sample, as sources of control tissue are frequently contaminated with leukemic cells resulting in false negative calls. Methods: To evaluate the clinical utility and feasibility of WGS in the diagnostic work up of leukemias, we analyzed 57 B-cell acute lymphoblastic leukemia (B-ALL) from the UKALL14 trial (NCT01085617) with no informative biomarkers at diagnosis. WGS analysis was performed on the leukemic sample and a matching control sample (with minimal residual disease level of <1%). Using this dataset, we trained the development of an unmatched (uWGS) analytical workflow (Isabl) for a tumor only WGS study. This workflow was validated across 20 hematologic neoplasms (12 B-ALL, 6 AML and 2 T-ALL). Results: Among the 57 cases, 5 failed QC owing to low tumor content (<25%). Of the remaining 52, putative biomarkers of clinical relevance were identified by WGS in 69% (36/52). These included delineation of aberrant karyotypes where conventional chromosome banding failed (4/52), the detection of newly described fusion genes (such as IGH-DUX4 and EP300-ZNF384 in 21/52) and recurrent gene mutations (i.e. PAX5 P80R, ZEB2 H1038R in 11/52). uWGS workflow in our training dataset captured 86% of biomarkers identified in the matched analysis (3/3 ploidy, 21/22 fusion and 7/11 coding). Concordance between the matched and uWGS workflow for arm-level and focal copy number alterations (CNAs), structural variants (SVs) and annotated hotspot mutations were 94%, 84%, 83% and 100% respectively. Independent validation of the uWGS workflow across 20 myeloid and lymphoid neoplasms, recapitulated all clinically reported biomarkers (14/15 CNAs, 16/16 SVs) as well as captured two novel findings not previously detected in two B-ALL patients, to include a focal deletion in BTG1 and the fusion gene P2RY8-CRLF2, as well as a NOTCH1 translocation in T-ALL. Conclusions: Our findings demonstrate that comprehensive WGS allows for the detection of the same biomarkers as a range of clinical assays using a single test, as well as the opportunity to discover novel clinical and research findings to support future correlative research and biomarker development. Additionally, we developed and validated an uWGS workflow that allows WGS analysis of hematopoietic neoplasms at diagnosis, enabling detection and reporting of clinically relevant biomarkers.

The Role of Truncated SOS1 in Acute Lymphoblastic Leukemia

Abstract Progenitor B cell acute lymphoblastic leukemia (B-ALL) remains the most common form of childhood cancer and has a poor survival in adult patients. Therefore, efforts to identify further driver mutations in B-ALL are critical. Previously, we identified a truncated form of SOS1 that cooperated with constitutively active STAT5b to induce leukemia. Expression of truncated Sos1 transcript correlated inversely with overall survival. Using a new model of B-ALL characterized by heterozygous expression of Pax5 and Ebf1, we identified increased expression of a truncated form of Sos1 transcript in RNA-Seq analyses. Using western blot analyses, we confirmed the over-expression of smaller SOS1 protein. To identify the truncated Sos1 starting site, we performed 5′ RACE and found a small region of a Riken gene fused to Sos1 at the junction of exon 8 and 9. Further, there is an almost 3 kb truncation of the 3′ untranslated region of Sos1. Next, we used a 10X Genomics approach to identify gene arrangements that showed clear evidence of a linkage between exon 9 of Sos1 and 3′ region of Map4k3. WGS on three independent leukemia samples identified an inversion event between Sos1 and a region downstream of Cdkl4, the location of the RIKEN we identified earlier. These results provide evidence that the truncated Sos1 arises from a micro-inversion event. SOS1 is known to play a critical role in the RAS-ERK signaling pathway and is important in PreBCR signaling. We examined both pERK and pp38. Surprisingly, we found reduced levels of pERK correlated with increased pSTAT5b. Thus, novel chromosomal rearrangements generate a truncated form of SOS1 that plays an important role in transformation of progenitor B cells in B-ALL.

SIRT5 IS A DRUGGABLE METABOLIC VULNERABILITY IN ACUTE MYELOID LEUKEMIA.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.

Drug ranking using machine learning systematically predicts the efficacy of anti-cancer drugs

Artificial intelligence and machine learning (ML) promise to transform cancer therapies by accurately predicting the most appropriate therapies to treat individual patients. Here, we present an approach, named Drug Ranking Using ML (DRUML), which uses omics data to produce ordered lists of >400 drugs based on their anti-proliferative efficacy in cancer cells. To reduce noise and increase predictive robustness, instead of individual features, DRUML uses internally normalized distance metrics of drug response as features for ML model generation. DRUML is trained using in-house proteomics and phosphoproteomics data derived from 48 cell lines, and it is verified with data comprised of 53 cellular models from 12 independent laboratories. We show that DRUML predicts drug responses in independent verification datasets with low error (mean squared error < 0.1 and mean Spearman’s rank 0.7). In addition, we demonstrate that DRUML predictions of cytarabine sensitivity in clinical leukemia samples are prognostic of patient survival (Log rank p < 0.005). Our results indicate that DRUML accurately ranks anti-cancer drugs by their efficacy across a wide range of pathologies.

Genome-wide interference of ZNF423 with B-lineage transcriptional circuitries in acute lymphoblastic leukemia

Aberrant expression of the transcriptional modulator and early B-cell factor 1 (EBF1) antagonist ZNF423 has been implicated in B-cell leukemogenesis, but its impact on transcriptional circuitries in lymphopoiesis has not been elucidated in a comprehensive manner. Herein, in silico analyses of multiple expression data sets on 1354 acute leukemia samples revealed a widespread presence of ZNF423 in various subtypes of acute lymphoblastic leukemia (ALL). Average expression of ZNF423 was highest in ETV6-RUNX1, B-other, and TCF3-PBX1 ALL followed by BCR-ABL, hyperdiploid ALL, and KMT2A-rearranged ALL. In a KMT2A-AFF1 pro-B ALL model, a CRISPR-Cas9–mediated genetic ablation of ZNF423 decreased cell viability and significantly prolonged survival of mice upon xenotransplantation. For the first time, we characterized the genome-wide binding pattern of ZNF423, its impact on the chromatin landscape, and differential gene activities in a B-lineage context. In general, chromatin-bound ZNF423 was associated with a depletion of activating histone marks. At the transcriptional level, EBF1-dependent transactivation was disrupted by ZNF423, whereas repressive and pioneering activities of EBF1 were not discernibly impeded. Unexpectedly, we identified an enrichment of ZNF423 at canonical EBF1-binding sites also in the absence of EBF1, which was indicative of intrinsic EBF1-independent ZNF423 activities. A genome-wide motif search at EBF1 target gene loci revealed that EBF1 and ZNF423 co-regulated genes often contain SMAD1/SMAD4-binding motifs as exemplified by the TGFB1 promoter, which was repressed by ZNF423 outcompeting EBF1 by depending on its ability to bind EBF1 consensus sites and to interact with EBF1 or SMADs. Overall, these findings underscore the wide scope of ZNF423 activities that interfere with B-cell lymphopoiesis and contribute to leukemogenesis.

Loss of H3K27 methylation identifies poor outcomes in adult-onset acute leukemia

BackgroundAcute leukemia is an epigenetically heterogeneous disease. The intensity of treatment is currently guided by cytogenetic and molecular genetic risk classifications; however these incompletely predict outcomes, requiring additional information for more accurate outcome predictions. We aimed to identify potential prognostic implications of epigenetic modification of histone proteins, with a focus on H3K4 and H3K27 methylation marks in relation to mutations in chromatin, splicing and transcriptional regulators in adult-onset acute lymphoblastic and myeloid leukemia.ResultsHistone 3 lysine 4 di- and trimethylation (H3K4me2, H3K4me3) and lysine 27 trimethylation (H3K27me3) mark expression was evaluated in 241 acute myeloid leukemia (AML), 114 B-cell acute lymphoblastic leukemia (B-ALL) and 14T-cell ALL (T-ALL) patient samples at time of diagnosis using reverse phase protein array. Expression levels of the marks were significantly lower in AML than in B and T-ALL in both bone marrow and peripheral blood, as well as compared to normal CD34+ cells. In AML, greater loss of H3K27me3 was associated with increased proliferative potential and shorter overall survival in the whole patient population, as well as in subsets with DNA methylation mutations. To study the prognostic impact of H3K27me3 in the context of cytogenetic aberrations and mutations, multivariate analysis was performed and identified lower H3K27me3 level as an independent unfavorable prognostic factor in all, as well as in TP53 mutated patients. AML with decreased H3K27me3 demonstrated an upregulated anti-apoptotic phenotype. In ALL, the relative quantity of histone methylation expression correlated with response to tyrosine kinase inhibitor in patients who carried the Philadelphia cytogenetic aberration and prior smoking behavior.ConclusionThis study shows that proteomic profiling of epigenetic modifications has clinical implications in acute leukemia and supports the idea that epigenetic patterns contribute to a more accurate picture of the leukemic state that complements cytogenetic and molecular genetic subgrouping. A combination of these variables may offer more accurate outcome prediction and we suggest that histone methylation mark measurement at time of diagnosis might be a suitable method to improve patient outcome prediction and subsequent treatment intensity stratification in selected subgroups.

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a “sample-in and result-out” approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Sequence Variations of Epstein-Barr Virus-Encoded Small Noncoding RNA and Latent Membrane Protein 1 in Hematologic Tumors in Northern China

Objective: To investigate the relationship between hematologic tumors and Epstein-Barr virus (EBV)-encoded small noncoding RNA (EBER) variations as well as latent membrane protein 1 (LMP1) variations. Methods: Patients with leukemia and myelodysplastic syndrome (MDS) were selected as subjects. Genotypes 1/2 and genotypes F/f were analyzed using the nested PCR technology, while EBER and LMP1 subtypes were analyzed by the nested PCR and DNA sequencing. Results: Type 1 was more dominant than type 2, found in 59 out of 82 (72%) leukemia and in 31 out of 35 (88.6%) MDS, while type F was more prevalent than type f in leukemia (83/85, 97.6%) and MDS (29/31, 93.5%) samples. The distribution of EBV genotypes 1/2 was not significantly different among leukemia, MDS, and healthy donor groups, neither was that of EBV genotypes F/f. EB-6m prototype was the dominant subtype of EBER in leukemia and MDS (73.2% [30/41] and 83.3% [10/12], respectively). The frequency of EB-6m was lower than that of healthy people (96.7%, 89/92), and the difference was significant (p < 0.05). China 1 subtype was the dominant subtype of LMP1 in leukemia and MDS (70% [28/40] and 90% [9/10], respectively), and there was no significant difference in the distribution of LMP1 subtypes among the 3 groups (p > 0.05). Conclusion: The distribution of EBV 1/2, F/f, EBER, and LMP1 subtypes in leukemia and MDS was similar to that in the background population in Northern China, which means that these subtypes may be rather region-restricted but not associated with leukemia and MDS pathogenesis.

Elevated nuclear phospho-eIF4E body levels are associated with tumor progression and poor prognosis for acute myeloid leukemia

Uncontrolled proliferation is a hallmark of cancer cells, yet the molecular mechanisms that contribute to this proliferation are unclear. Therapeutic treatment of cancer is suboptimal in many cases, with no accurate index by which to evaluate the success of treatment or patient prognosis. In this study, we explored the protein levels of nuclear phosphoeIF4E in acute myeloid leukemia (AML) cell lines and primary leukemia samples by Western blot and immunofluorescence and as well analyzed transcriptomes by RNA-seq. We found nuclear phospho-eIF4E, an exporter of oncogenic mRNAs, to be abundant in AML. Further, nuclear phospho-eIF4E abundance was significantly associated with tumor burden as well as the response of AML patients to chemotherapy. The results demonstrate “massive clustering and export of oncogenic mRNAs to the translation machinery” by highly abundant RNA-nuclear phospho-eIF4E bodies. This is an efficient mechanism that may drive the proliferation of cancer cells. Herein, nuclear phospho-eIF4E bodies were identified as potential markers of AML, which may be useful for prognosis and as targets for cancer therapy.

Malignant Progenitors From Patients With Acute Myelogenous Leukemia Are Sensitive to a Diphtheria Toxin–Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractWe have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 × 10−12 mol/L (range, 5 to >4,000 × 10−12 mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing ≥35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 × 10−9 mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.

Cyclin A1 Expression in Leukemia and Normal Hematopoietic Cells

Human cyclin A1 is a newly cloned, tissue-specific cyclin that is prominently expressed in normal testis. In this study, we showed that cyclin A1 was highly expressed in a subset of leukemia samples from patients. The highest frequency of cyclin A1 overexpression was observed in acute myelocytic leukemias, especially those that were at the promyelocyte (M3) and myeloblast (M2) stages of development. Cyclin A1 expression was also detected in normal CD34(+) progenitor cells. The expression of cyclin A1 increased when these cells were stimulated to undergo myeloid differentiation in vitro. Taken together, our observations suggest that cyclin A1 may have a role in hematopoiesis. High levels of cyclin A1 expression are especially associated with certain leukemias blocked at the myeloblast and promyelocyte stages of differentiation.

Finding a suitable library size to call variants in RNA-Seq

BackgroundRNA sequencing allows the study of both gene expression changes and transcribed mutations, providing a highly effective way to gain insight into cancer biology. When planning the sequencing of a large cohort of samples, library size is a fundamental factor affecting both the overall cost and the quality of the results. Here we specifically address how overall library size influences the detection of somatic mutations in RNA-seq data in two acute myeloid leukaemia datasets.Results We simulated shallower sequencing depths by downsampling 45 acute myeloid leukaemia samples (100 bp PE) that are part of the Leucegene project, which were originally sequenced at high depth. We compared the sensitivity of six methods of recovering validated mutations on the same samples. The methods compared are a combination of three popular callers (MuTect, VarScan, and VarDict) and two filtering strategies. We observed an incremental loss in sensitivity when simulating libraries of 80M, 50M, 40M, 30M and 20M fragments, with the largest loss detected with less than 30M fragments (below 90%, average loss of 7%). The sensitivity in recovering insertions and deletions varied markedly between callers, with VarDict showing the highest sensitivity (60%). Single nucleotide variant sensitivity is relatively consistent across methods, apart from MuTect, whose default filters need adjustment when using RNA-Seq. We also analysed 136 RNA-Seq samples from the TCGA-LAML cohort (50 bp PE) and assessed the change in sensitivity between the initial libraries (average 59M fragments) and after downsampling to 40M fragments. When considering single nucleotide variants in recurrently mutated myeloid genes we found a comparable performance, with a 6% average loss in sensitivity using 40M fragments.ConclusionsBetween 30M and 40M 100 bp PE reads are needed to recover 90–95% of the initial variants on recurrently mutated myeloid genes. To extend this result to another cancer type, an exploration of the characteristics of its mutations and gene expression patterns is suggested.

The effects of sample handling on proteomics assessed by reverse phase protein arrays (RPPA): Functional proteomic profiling in leukemia

Reverse phase protein arrays (RPPA) can assess protein expression and activation states in large numbers of samples (n > 1000) and evidence suggests feasibility in the setting of multi-institution clinical trials. Despite evidence in solid tumors, little is known about protein stability in leukemia. Proteins collected from leukemia cells in blood and bone marrow biopsies must be sufficiently stable for analysis. Using 58 leukemia samples, we initially assessed protein/phospho-protein integrity for the following preanalytical variables: 1) shipping vs local processing, 2) temperature (4 °C vs ambient temperature), 3) collection tube type (heparin vs Cell Save (CS) preservation tubes), 4) treatment effect (pre- vs post-chemotherapy) and 5) transit time. Next, we assessed 1515 samples from the Children's Oncology Group Phase 3 AML clinical trial (AAML1031, NCT01371981) for the effects of transit time and tube type. Protein expression from shipped blood samples was stable if processed in ≤72 h. While protein expression in pre-chemotherapy samples was stable in both heparin and CS tubes, post-chemotherapy samples were stable in only CS tubes. RPPA protein extremes is a successful quality control measure to identify and exclude poor quality samples. These data demonstrate that a majority of shipped proteins can be accurately assessed using RPPA. SignificanceRPPA can assess protein abundance and activation states in large numbers of samples using small amounts of material, making this method ideal for use in multi-institution clinical trials. However, there is little known about the effect of preanalytical handling variables on protein stability and the integrity of protein concentrations after sample collection and shipping. In this study, we used RPPA to assess preanalytical variables that could potentially affect protein concentrations. We found that the preanalytical variables of shipping, transit time, and temperature had minimal effects on RPPA protein concentration distributions in peripheral blood and bone marrow, demonstrating that these preanalytical variables could be successfully managed in a multi-site clinical trial setting.

Association of aberrant ASNS imprinting with asparaginase sensitivity and chromosomal abnormality in childhood BCP-ALL

AbstractKaryotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remain unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, but ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. The ASNS CpG island is largely unmethylated in normal hematopoietic cells, but it is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knockin mice. In 3 childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL patients with favorable karyotypes but is mostly unmethylated in BCP-ALL patients with poor prognostic karyotypes. Higher ASNS methylation is associated with higher L-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene as a result of aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.

Novel DNMT3A Germline Variant in a Patient with Multiple Paragangliomas and Papillary Thyroid Carcinoma.

Simple SummaryThe use of next generation technologies has helped to unravel the genetics of rare inherited diseases, facilitating the discovery of new susceptibility genes. Nonetheless, the sequencing of all protein-coding genes of an individual may lead to doubtful assignments of causality for non-pathological variants, so it is mandatory to perform comprehensive studies and screening of additional patients for new mutations. Here we describe a novel DNMT3A germline variant identified by whole-exome sequencing in a patient with multiple paragangliomas and papillary thyroid carcinoma. The increased methylation of DNMT3A target genes observed in the proband’s sample points towards a gain-of-function effect of the variant, contrasting with the inactivation caused by loss-of-function alterations commonly seen in other neoplasia and in patients with Tatton-Brown-Rahman syndrome. This finding stresses the diverse molecular outcomes and suggests a heterogeneous phenotypic spectrum related to DNMT3A germline variants.Over the past few years, next generation technologies have been applied to unravel the genetics of rare inherited diseases, facilitating the discovery of new susceptibility genes. We recently found germline DNMT3A gain-of-function variants in two patients with head and neck paragangliomas causing a characteristic hypermethylated DNA profile. Here, whole-exome sequencing identifies a novel germline DNMT3A variant (p.Gly332Arg) in a patient with bilateral carotid paragangliomas, papillary thyroid carcinoma and idiopathic intellectual disability. The variant, located in the Pro-Trp-Trp-Pro (PWWP) domain of the protein involved in chromatin targeting, affects a residue mutated in papillary thyroid tumors and located between the two residues found mutated in microcephalic dwarfism patients. Structural modelling of the variant in the DNMT3A PWWP domain predicts that the interaction with H3K36me3 will be altered. An increased methylation of DNMT3A target genes, compatible with a gain-of-function effect of the alteration, was observed in saliva DNA from the proband and in one independent acute myeloid leukemia sample carrying the same p.Gly332Arg variant. Although further studies are needed to support a causal role of DNMT3A variants in paraganglioma, the description of a new DNMT3A alteration in a patient with multiple clinical features suggests a heterogeneous phenotypic spectrum related to DNMT3A germline variants.

Novel Germline TRAF3IP3 Mutation in a Dyad with Familial Acute B Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common single malignancy in children, representing 25% of all new cancer diagnoses. Advances in genomic sequencing has demonstrated that inherited genetic risk factors play a larger role in leukemia development than previously appreciated. We identified a father-daughter dyad with childhood B cell acute lymphoblastic leukemia (B-ALL) and obtained their diagnostic bone marrow samples from the Children's Oncology Group (COG) bank in addition to germline samples via remission marrow (daughter) or buccal swab (father). Whole Exome Sequencing (WES) was performed and compared to a panel of normal to identify large genomic changes and single nucleotide variants. In parallel, RNA-sequencing (RNA-seq) was performed on the diagnostic marrows. We discovered a novel germline chromosomal structural variant in chromosome 1q32.2 within the TRAF3IP3 gene. TRAF3IP3 regulates B cell lymphopoiesis and this mutation likely resulted in a predisposition to leukemia by causing expansion of immature B-cell precursors which are highly vulnerable to secondary somatic mutations. This mutation has not been previously described in ALL, however based on the function of TRAF3IP3 in B cell lymphopoiesis, we conclude this likely represents a mutation predisposing to the development of leukemia. WES revealed this dyad had no shared SNVs that are associated with leukemia in the literature and that they had only a few shared SNVs within the leukemia samples, none of which were identified as clinically significant; which suggests the spectrum of their somatic mutations were distinctly different. Given the lack of concordance in their somatic mutational spectrum, we wondered if the two leukemia samples would exhibit a shared transcriptome, implying convergent leukemogenic pathways were altered within the two specimens since they were both clinically reported as hyperdiploid. In comparing the leukemia gene expression profiles identified by RNA-seq to 216 cases of sporadic B-ALL from the TARGET database (The Therapeutically Applicable Research to Generate Effective Treatments program), we discovered that these two leukemia samples did not cluster together but did cluster with other cases of childhood B-ALL. We suspect that this germline TRAF3IP3 mutation increased this dyad's susceptibility to leukemia development but that the somatic mutational spectrum drove the leukemia development and dictated its phenotype. This research may have identified a novel gene involved in leukemogenesis which may also be involved in de novo cases of ALL. Additional studies are needed to further characterize this TRAF3IP3 structural variant, the co-occurring somatic mutations within these leukemia samples and their combined role in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

3D Genomics of Acute Meyloid Leukemia Reveals the Imbalance between DNA Methylation Canyon Interactions and Leukemic Specific Enhancer Network Interactions

Changes in 3D chromatin organization like enhancer hijacking are believed to the driver for disease development like leukemia. Here we performed high-resolution HiC assays on primary acute myeloid leukemia (AML) samples and cell lines to dissect the abnormal 3D chromatin organization in AML. Our data set covers 5 AML samples and 3 AML cell lines. This dataset includes the common genetic abnormalities in AML: MLL-rearrangement, NPM1 mutation, RUNX1 mutation, and IDH1/TET2 mutations. We have recently generated high-resolution map for normal human hematopoietic stem cells (HSC) (Zhang et al. Mole Cell. 2020). In comparison with the HSC 3D chromatin organization, we found TADs and loops are very stable in both primary leukemia samples and cell lines. Less than 5% of all TADs in HSC fuse in AML, mimicking the enhancer hijacking scenario. These fusion events do not cause the gene expression changes of genes in the fused TAD. Interestingly, in TET2 or IDH1 mutated AML blast, two-fold more TAD fusion events occurred in primary AML blast in comparison with RUNX1 and MLL-r leukemia, with a loss in the CTCF sites on the TAD fusion break point. We previously found in HSC, the Polycomb marked DNA methylation Canyons (DMC) form multi-Mb size long-range interactions. DMC interactions in general decrease in primary AMLs. AMLs with IDH1 or TET2 mutations shows the biggest reduction in DMC interactions. Hypermethylation in the DMCs is observed in the AML samples with IDH1/2 or TET2 mutations, suggesting DNA methylation level in DMCs controls DMC 3D interactions directly. In leukemia cell lines, the DMC interactions almost disappear, with further hypermethylation in DMCs. Compared with normal HSC, we found in AML, the AML-specific H3K27ac marked regions form leukemia specific loops and transcription stripes in both cell lines and primary samples. Particularly in MLL-r primary leukemias, we found broad H3K27ac covered, hyperacetylated domains (10kb to 200kb). 22 such hyperacetylated domains were identified and associated with leukemogenic genes such as SATB1, ZEB2 and HOXA. All these domains formed distinct 3D micro TAD in the MLL-r primary leukemia in comparison with the HSPC, and CTCFs are not located at the border of these domains. Taken together, suggest active leukemia specific transcription created new 3D genomic interactions which is independent of cohesion-CTCF mediated loop extrusion. Interestingly, in HOXA cluster, we found a geneless DMC 1.3MB upstream of HOXA switched from Polycomb binding site to active enhancer site in the leukemia cells. By applying CRISPR/Cas9 editing, we found this canyon is essential for survival of HOXA high expressing leukemia cell lines like OCI-AML3 and MV4:11. In summary, we found the 3D chromatin organization in human leukemia significantly alters in two opposite way 1. The significant loss of Polycomb marked DMC interactions caused by the DNA hypermethylation and 2. The leukemic specific hyperacetylated domains form its own distinct micro TAD and stripes in the 3D chromatin organization. Disclosures No relevant conflicts of interest to declare.

In Vitro Drug Response Profiling in BCP- and T-ALL Primary Samples Adds a Robust Functional Layer Enabling Optimized Guidance of Individualized Therapy in Relapsed and Refractory Pediatric Acute Leukemia Patients

In Vitro Drug Response Profiling in BCP- and T-ALL Primary Samples Adds a Robust Functional Layer Enabling Optimized Guidance of Individualized Therapy in Relapsed and Refractory Pediatric Acute Leukemia Patients

A Novel Anti-Cancer Vaccine Approach for the Treatment of High-Risk Leukemia in Children

Introduction: There is strong experimental and clinical data to indicate the critical involvement of immune evasion in relapsed leukemia in children. A well-defined characteristic of refractory leukemia is the accumulation of genetic aberrations and mutations that may act as drivers or passengers in the process of tumor recurrence. Many of these mutations get translated into proteins that contain tumor-specific immune-stimulatory epitopes (neoantigens) that can elicit host antitumor immune responses. Although, in general, the mutation rate is lower in pediatric tumors, recent studies have shown that almost 90% of pediatric leukemias carry potentially actionable neoepitopes. In this study, we describe the results from a comprehensive experimental approach of neoantigen prediction coupled with antigen processing and HLA-binding prediction algorithms with in vitro validation assays for the generation of neoantigen vaccines against high-risk leukemias in children. Methods: DNA and RNA from leukemia cells and matched fibroblasts were obtained. Raw reads were aligned to human reference genome and somatic variants (SNVs) were called using Strelka v1.0.1441. RNA-seq data from leukemic cells were used to predict neoantigen expression levels resulting from SNVs using STAR (2.4.1)12 and Cufflinks v2.2.1. Normalized expression data were then cross-referenced with the list of SNVs to identify leukemia-specific mutant proteins. HLA typing for each sample was carried out from RNA-seq data using seq2HLA v2.2. Using the patient's HLA phenotype, we then used NetMHCons v1.1 to predict short peptides derived from leukemia-specific mutant proteins that will bind to autologous HLA Class I molecules. These 8/9-mers were filtered to predict a high likelihood of proteasomal or immune-proteasomal processing and transporter associated with antigen processing (TAP) using NetChop v3.1 and the immune epitope database (IEDB), respectively. The peptides identified were rank-ordered based on the composite immunogenicity score derived from MHC class I binding affinities, proteasomal processing and TCR binding predictions and synthesized accordingly. Peripheral blood derived dendritic cells (DCs) and CD8+ T-cells were isolated and expanded in culture with relevant cytokines. The DCs were pulsed with peptides and then co-cultured with CD8+ T-cells. After five days, the primed CD8+ T-Cells were separated, washed and exposed to the patient's leukemic cells at varying ratios and the leukemia specific CD8+ T-cell activation was quantified by IFN gamma secretion using ELISpot assays. Results: In the leukemia specimen studied, approximately 5% of all on-target germline mutations were found only in leukemic cells. Tumor mutational burden was, on average, 0.34 mut/Mb. Analysis of the highest ranking synthetic peptides (approximately 10 per leukemia sample) showed leukemia-specific activation of patient's T-cells as measured by the mean number of spots observed in ELISpot assays. For example, in patient one (15 year old male, high-risk ALL, one year off therapy), 14 individual short sequences were identified and corresponding peptides were synthesized. Among these, three peptides were not soluble and three peptides showed significant activity above controls. Maximum leukemia specific T-cell activation was noted with peptide #7 QQSALVLL (mean 135 ELISpots compared to 72 in controls, p&amp;lt;0.05, triplicate) indicating a strong nonantigenic potential in this region. Furthermore, this activity was significantly diminished when an extra amino acid was added to this peptide (LQQSALVLL, mean 79 spots) showing the specificity of the approach. A number of other peptides and combinations in non-overlapping regions gave intermediate activities. Discussion: Completed data, including the vaccine peptide sequences and corresponding activities showed the feasibility of identifying pediatric leukemia neoantigen sequences in personalized mutational landscapes of these patients. In addition, we have provided an in vitro experimental approach to validate the potential of such vaccines in future clinical studies and this methodology can also be used to identify agents for effective combinations such as immune checkpoint inhibitors. A clinical trial using these strategies is in development for the treatment of high-risk leukemia in children. Disclosures No relevant conflicts of interest to declare.