ALBERT

Description: Natural killer (NK) cells are components of the innate immunity and play an important role in cancer surveillance through their cytolytic and immunomodulatory capabilities. Infusion of NK cells is a promising tool for cell therapy of hematologic malignancies and solid tumors. However, the potent cytotoxicity of NK cells might be hampered by tumor immune escape mechanisms and intrinsic resistance. We and others previously demonstrated intrinsic resistance of leukemia cells to NK cell lysis can be overcome by the transduction of artificial antigen receptor into NK cells. The genetic engineering of primary NK cells with chimeric antigen receptor improved cytotoxic activity and cytokine production, and this enhanced function was target-specific. Thus, a novel method to enhance NK cell activity against a wide range of tumors is also required. Several cytokines are associated with enhanced cytotoxicity, in vivo survival, and proliferation of NK cells. In particular, interleukin (IL)-21, which shares the common cytokine-receptor gamma chain with IL-2, was reported to enhance the cytotoxicity of human NK cells. In the present study, we investigated whether the enforced expression of human IL-21 in primary human NK cells enhanced their cytotoxicity against leukemic cells and allowed prolonged survival. We collected peripheral blood samples from healthy adult donors, and mononuclear cells were isolated by density gradient centrifugation. Primary NK cells were expanded by stimulation with K562-mbIL15-41BBL cell line following standard procedures. After 7 days of expansion, residual T cells were removed with magnetic beads and NK cells were transduced with a retroviral vector containing human IL-21 cDNA and GFP. Fourteen days after transduction, more than 95% of cells were CD56+CD3- NK cells. Median GFP expression in the CD56+CD3- cells was 84.2% (74.5%-97.1%, n=6). We confirmed that NK cells transduced with human IL-21 cDNA (NK-IL21) had intracellular expression of IL-21 as assessed by flow cytometry, while NK cells transduced with a vector containing GFP only (NK-mock) did not. 4-hour cytotoxicity assays revealed significantly enhanced cytotoxicity exerted by NK-IL21 (Fig. 1). Cytotoxic activity of NK-IL21 against K562 cells and Jurkat cells was significantly higher than that of NK-mock. We found that the intracellular expression levels of both perforin and granzyme B were higher in NK-IL21 cells than in NK-mock cells, in accordance with their higher cytotoxicity against target cells. However, NK-IL21 did not show increased expression of the apoptosis-inducing molecule TRAIL, NK cell activating receptor NKG2D, or natural cytotoxicity receptors p30, p44, or p46. The success of NK cell infusions might rely on the in vivo persistence of NK cells. We therefore tested whether the enforced expression of IL-21 in NK cells enhanced their proliferation and survival, and found that IL-21 expression in NK cells did not prevent apoptosis induced by IL-2 withdrawal and therefore did not favorably alter cell proliferation without IL-2. In contrast to the favorable results obtained by short-time cytotoxicity assays, NK-IL21 did not exert effective tumor control in long-term coculture experiments. The residual leukemic cell burden in NK-IL21 cocultures was not decreased and did not differ from that in NK-mock coculture experiments where cocultures were extended to 7 days without IL-2. However, by adding IL-2 (100 U/ml) to the culture, we demonstrated a dramatic suppression of residual leukemia burden exerted by NK-IL21. As shown in Figure 2, the number of residual K562 cells in the NK-IL21 cocultures was much lower than in the NK-mock cocultures (1.9% ± 0.4% vs 61.5% ± 3.8% of control culture without NK cells at a 1:1 E:T ratio, p

Description: Introduction Clinical outcome of relapsed pediatric B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remains poor, although survival rate for children with BCP-ALL has greatly increased over time and is now reached 90%. To clarify the molecular pathogenesis of relapsed ALL may provide novel prognostic markers and therapeutic targets. Some genome-wide analyses for specific patients group with poor prognosis, such as early-relapsed patients and Ph- or BCR-ABL-like patients, were reported. They described important insights to understand genetic background of poor prognosis. However, the majority of relapsed cases did not have any poor prognostic marker, and the molecular mechanisms of relapse in these cases still remained unclear. Therefore we performed whole exome sequencing (WES) to describe clonal evolution in 21 relapsed pediatric BCP-ALL patients. Our cohort included various cases whose time to relapse from diagnosis were between 6 months to over 10 years. We also analyzed the clonality of leukemia cells using immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements. Patients and Methods Genomic DNA was isolated from 21 cases whose median time to relapse was 33 months. Somatic mutations including SNVs (single nucleotide variants), insertions / deletions and CNVs (copy number variants) were detected by WES using Agilent SureSelect and illumine HiSeq systems. To evaluate accurate VAF (valiant allele frequency), targeted deep sequencing was performed in candidate somatic mutations. The clonality analysis of leukemia cells was performed by standard PCR methods using Ig and TCR rearrangements. Results WES was performed in samples obtained at diagnosis, remission and relapse from 21 pediatric BCP-ALL patients. Tumor specific mutations had been identified by WES. Three of 21 were hypermutated with over 150 somatic mutations at relapse. Mutation of DNA mismatch repair gene, MSH3, was detected in 2 of them. Except for these hypermutated cases, the median number of somatic mutations detected at relapsed phase was 22 (range 8 to 53), which was higher than that at diagnosis (median 16, range 6 to 31). Sixteen recurrently mutated genes were identified in 21 cases by WES. Some known leukemia associated genes were detected, including KRAS and WHSC1 observed only at diagnosis and IKZF1 and CREBBP observed at relapse. Then we compared VAFs of these mutations between at diagnosis and relapse to solve the clonal architectures over time. Three patterns of clonal evolution were estimated from VAFs using targeted deep sequencing; (i) In 7 cases, all mutations described at diagnosis were shared at relapse, suggesting that relapse clone derived from predominant clone at diagnosis with additional mutations in these cases. (ii) In other 13 cases, most of mutations in predominant clone at diagnosis were not detected at relapse except for some shared mutations at diagnosis and relapse, indicating that relapsed clone occurred from founder clone existing as subclone at diagnosis. (iii) In one very late-relapsed case, there were no shared mutations at diagnosis and relapse. According to clonality analysis of Ig and TCR, none of rearrangements identified at diagnosis were conserved at relapse in this case. On the other hand, most rearrangement at diagnosis were conserved at relapse in other 20 cases except one patient who relapsed in 10 years after diagnosis. Relapse from predominant clone at diagnosis were observed in only one out of 8 late-relapsed cases (〉 36 months), whereas a half of the early-relapsed showed this clonal evolution pattern. The number of shared mutations between diagnosis and relapse was very limited in very late-relapsed cases over 10 years. Discussion Our study suggests that the clonal evolution pattern differs according to the time to relapse. In a half of early-relapsed cases, relapsed clone derived from major clone at diagnosis with additional mutations, and clonal selection of resistant clones occurred during treatment. Meanwhile, in late-relapsed cases, relapse was frequently associated with clonal evolution from minor subclone with some conserved mutations and same Ig/TCR rearrangements. The founder clone should be remained dormant for a long period until additional mutations lead to relapse. Towards a better understanding of clonal evolution in ALL, our study will shed light on the early prediction of relapse risk and new treatment strategies for relapsed ALL. Disclosures No relevant conflicts of interest to declare.

Description: Objective: The Myc proteins are transcription factors with essential roles in cell growth and proliferation through their ability to regulate gene expression. MYC binding protein 2(MYCBP2) is probable E3 ubiquitin-protein ligase and its function in leukemia is undetermined. IKZF1 encodes a kruppel-like zinc finger protein Ikaros that is essential for normal hematopoiesis and acts as a tumor suppressor in acute lymphoblastic leukemia(ALL). IKZF1 deletion is associated with the development of ALL and poor clinical outcome. This study aimed to explore the expression of c-MYC and MYCBP2 and their correlation with clinical features in adult ALL, as well as the mechanism by which Ikaros directly regulates c-MYC/MYCBP2 expression in ALL. Methods: Quantitative PCR (qPCR) was performed to explore the expression of c-MYC and MYCBP2 in 151 newly diagnosed adult patients with ALL. The correlations of c-MYC/MYCBP2 expression with clinical parameters and survival status were analyzed. In addition, luciferase assay, quantitative Chromatin Immunoprecipitation (qChIP) and Ikaros shRNA knockdown were performed to further explore the mechanism underlying regulation of c-MYC/MYCBP2 expression. Results: Expression of c-MYC is significantly higher and MYCBP2 is significantly lower in both B-ALL and T-ALL patients compared with that in normal controls. C-MYC expression is also negatively co-related with the MYCBP2 in ALL cohorts. The patients with c-MYC high and MYCBP2 low expression (c-MYChigh +MYCBP2low) showed higher median white blood cell counts (WBC) (101.5×109/L vs 29.4×109/L, P =0.007), incidence of splenomegaly and liver infiltration (75.0% vs 33.3%, P =0.004;75.0% vs19.4%, P =0.000), percentage of CD34(+) and CD33(+) cells (90.0% vs 61.3%, P =0.025; 80.0% vs 25.8%, P =0.000) and a lower percentage of complete remission (CR) rate (60.0% vs 92.0%,P =0.027) compared with that of patients with c-MYC low and MYCBP2 high expression (c-MYClow +MYCBP2high). Notably, our Ikaros ChIP-seq data showed strong Ikaros binding peaks in the promoter region of both c-MYC and MYCBP2. The qChIP assay showed that Ikaros significantly binds to c-MYC and MYCBP2 promoter regions in both Nalm6 B-ALL and Molt4 T-ALL cells. Moreover, expression of Ikaros suppressed c-MYC but increased MYCBP2 expression in both Nalm6 and CEM T-ALL cells. Conversely, Ikaros knockdown induced the increase of c-MYC but decrease of MYCBP2 in Nalm6 and CEM cells. Ikaros activator,Ck2 inhibitor TBB suppress c-MYC and increase MYCBP2 expression in a dose-dependent manner in Nalm6 and CEM cells. Ikaros knockdown with shRNA could block the TBB-induced suppression of c-MYC and increase of MYCBP2 expression. These data indicated that both c-MYC and MYCBP2 are direct Ikaros targets in ALL and Ikaros regulates their expression. Importantly, we also observed Ikaros binding to c-MYC and MYCBP2 promoters in primary B-All and T-ALL. The expression of c-MYC significantly increased and MYCBP2 decreased in patients with Ikaros deletion compared to that of Ikaros wild type. These data indicated Ikaros regulatory effect on c-MYC and MYCBP2 in ALL patients and Ikaros deletion is one of the reasons for expression change of c-MYC and MYCBP2 in the patients. Conclusion: We observed the expression of c-MYC significantly increased and MYCBP2 decreased in adult ALL patients. C-MYC high and MYCBP2 low expression is correlated with high-risk leukemia. Ikaros dysfunction is one of the reasons underlying c-MYC high and MYCBP2 low expression in the patients. Our data revealed the oncogenic effect of Ikaros/MYCBP2/c-MYC on oncogenesis in adult ALL, also suggested CK2 inhibitor exert its anti-leukemia effect through Ikaros-mediated regulation on c-MYC and MYCBP2 expression in leukemia. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Genetic abnormalities are important to predict prognosis and sometimes can be therapeutic target in pediatric acute lymphoblastic leukemia (ALL). Although the cell lines with recurrent chromosomal abnormalities or leukemic fusions derived from ALL patients are useful tool for various in vitro experiments, it has not been fully investigated whether there is the difference of genetic alterations between clinical samples and cell lines.Here, we performed MLPA analysis of 86 ALL cell lines to determine copy number abnormalities (CNA) and compare with those of the patient's clinical samples. Methods: We performed MLPA analysis of 86 cell lines of ALL (14 with BCR-ABL, 11 with MLL rearrangement, 18 with TCF3-PBX1, 4 with TCF3-HLF, 4 with ETV6-RUNX1 and 35 B-other ALL cell lines) to determine CNA of IKZF1, PAX5, CDKN2A, CDKN2B, ETV6, RB1, BTG1 and EBF1. Then, CNAs were compared to those of patients' samples such as UK cohort (Schwab C, et al. Haematologica, 2013) and Japanese cohort (Asai D, et al. Cancer Med, 2013) according to each specific genetic abnormality, such as BCR-ABL, ETV6-RUNX1, TCF3-PBX1 and MLL-related fusions. In addition, we performed multiplex PCR and RNA-seq to determine fusion transcripts related to Ph-like ALL for the six Ph-negative cell lines with IKZF1 deletions. To determine the expression level of IKZF1 isoform in these cell lines, we performed real time PCR analysis of IKZF1 isoform 1 (IK1) and isoform 6 (IK6). Results: In the BCR-ABL positive cell lines, the frequencies of CDKN2A/2B and BTG1 deletion significantly higher than those in UK cohort (CDKN2A/2B: 100 vs 48%, P

Description: T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL). Although gene fusions generated through chromosomal translocations, deletions, and inversions are the most frequent genetic abnormalities detected in other types of leukemia, recurrent gene fusions except for SIL-TAL1 have been poorly defined in T-ALL. To discover driver mutations or fusion genes, which involved in the pathogenesis of childhood T-ALL and to identify novel prognostic markers of childhood T-ALL, we performed whole-exome sequencing (WES) and transcriptome sequencing (WTS) in 31 and 46 cases, respectively. We also performed SNP array karyotyping in 46 cases. To detect somatic mutations or fusion transcripts, we used our pipeline "Genomon-exome" and "Genomon-fusion" algorithm. Subsequently, somatic mutations were validated using deep amplicon sequencing. Candidate fusion transcripts were validated by reverse - transcription polymerase-chain-reaction and Sanger sequencing.Recurrent mutations observed in more than 3 cases were detected in NOTCH1 (n = 18, 58%), FBXW7 (n = 7, 23%), PHF6 (n = 5, 16%), GATA3 (n = 4, 13%), MYB (n = 3, 10%), and NRAS (n = 3, 10%), respectively. We identified previously known fusion genes, such as MLL-ENL (n = 2), CALM-AF10 (n = 2), NUP214-ABL1 (n = 1) and FGFROP1-FGFR1 (n = 1). MLL-ENL is one of the frequent translocation in infant multilineage leukemia, but also reported in non-infant B cell precursor ALL and T-ALL. FGFR1OP is ubiquitously expressed, and the predicted chimeric FGFR1OP-FGFR1 protein contains the catalytic domain of FGFR1. It is thought to promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1. CALM-AF10 leukemia is reported to increase HOXA cluster gene transcription, we could also confirm elevated HOXA genes expression by FPKM value. Four SIL-TAL1 fusions were detected in our cohort. Recently, a novel mutation in TAL1 enhancer region which introduce de novo MYB biding site has been reported. Since this abnormality lead high expression of TAL1, we also analyzed expression data obtained from WTS. Among 46 specimens, 19 samples showed high expression of TAL1 (FPKM value ≥5). In those cases, 4 cases had SIL-TAL1 fusions (8%), and 3 cases (6%) had insertions in enhancer region of TAL1. Subsequent analysis using Gene Set Enrich Analysis (GSEA) between TAL1 high and low expression samples revealed that "LEE_EARLY_T_LYMPHOCYTE_UP" was enriched in TAL1 high expression samples (Enrichment score = 0.73, FDR = 0.073). This gene set includes genes up-regulated at early stages of progenitor T lymphocyte maturation compared to the late stages, and MYB was included in this gene set. Intriguingly, MYB mutation samples were not represented TAL1 high expression. TAL1 related rearrangement or enhancer insertion was not detected in the rest of 12 cases with TAL1 high expression, suggesting that other mechanisms of TAL1 high expression might be exist. In conclusion, although NOTCH1 and FBXW7 mutations were relatively frequently detected in our series, we also found recurrent MYB mutations. SIL-TAL1 was known as most frequent rearrangement, TAL1 enhancer insertions were also frequent in TAL1 overexpressed samples. TAL1 enhancer insertion and MYB mutation was exclusive, suggesting that TAL1 and MYB have a key role in childhood T-ALL. Consistent with other reports, frequent translocations were not observed in T-ALL, suggesting the genetic differences between T-ALL and other hematological malignancies. Further studies will be necessary to unravel oncogenic mechanisms that implicated in new therapeutic strategy for childhood T-ALL. Disclosures No relevant conflicts of interest to declare.