B-lymphoid Cell Lines Research Articles

Cyclin C promotes development and progression of B-cell acute lymphoblastic leukemia by counteracting p53-mediated stress responses.

Despite major therapeutic advances in the treatment of acute lymphoblastic leukemia (ALL), resistances and long-term toxicities still pose significant challenges. Cyclins and their associated cyclin-dependent kinases are one focus of cancer research when looking for targeted therapies. We discovered cyclin C as a key factor for B-ALL development and maintenance. While cyclin C is non-essential for normal hematopoiesis, CcncΔ/Δ BCR::ABL1+ B-ALL cells fail to elicit leukemia in mice. RNA sequencing experiments revealed a p53 pathway deregulation in CcncΔ/Δ BCR::ABL1+ cells resulting in the incapability of the leukemic cells to adequately respond to stress. A genome-wide CRISPR/Cas9 loss-of-function screen supplemented with additional knock-outs unveiled a dependency of human B-lymphoid cell lines on CCNC. High cyclin C levels in B-cell precursor (BCP) ALL patients were associated with poor event-free survival and increased risk of early disease recurrence after remission. Our findings highlight cyclin C as potential therapeutic target for B-ALL, particularly to enhance cancer cell sensitivity to stress and chemotherapy.

Discovery of 2,5-diaminopyrimidine derivatives as the first series of selective monomeric degraders of B-lymphoid tyrosine kinase

B-lymphoid tyrosine kinase (BLK) is an important knot of B cell receptor signaling, and regulates the function and development of B cells subset. Dysfunction of BLK is correlated with autoimmune diseases and cancer. There is an urgent need to develop selective BLK modulators to facilitate the studies of BLK in biological processes. Herein, we report the discovery of a series of 2,5-diaminopyrimidine-based compounds capable of selectively degrading BLK. The optimized compounds 9–11 possess weak biochemical inhibitory activities against BLK, yet they effectively degrade BLK and show high selectivity for BLK over other structurally and functionally related SRC family and TEC family kinases. Furthermore, compounds 9 and 11 demonstrate potent inhibitory activities in several B-lymphoid cell lines. As the first series of effective and selective monomeric BLK degraders, compounds 9–11 serve as valuable tools for further investigation of the functions of BLK.

Fludarabine nucleoside induces major changes in the p53 interactome in human B-lymphoid cancer cell lines

Triple combination FCR (fludarabine, cyclophosphamide and rituximab) is often used as front-line treatment for chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma. Results from our laboratory indicate that 2-FaraAMP (fludarabine) has multiple mechanisms of cytotoxicity that include accumulation of isoforms and phosphorylated derivatives of p53, and induction of the unfolded protein response (UPR). Using protein pull-downs with Dynabeads coated with p53 antibody, we have found that 2-FaraA (fludarabine nucleoside) induces major changes in the p53 interactome in human Raji lymphoma and IM9 multiple myeloma cells. These changes are likely driven by DNA strand breaks induced by 2-FaraA that activate protein kinases such as ATM, ATR and Chk1.

Polymorphic Region-Specific Antibody for Evaluation of Affinity-Associated Profile of Chimeric Antigen Receptor

Antibody applications in cancer immunotherapy involve diverse strategies, some of which redirect T cell-mediated immunity via engineered antibodies. Affinity is a trait that is crucial for these strategies, as optimal affinity reduces unwanted side effects while retaining therapeutic function. Antibody-antigen pairs possessing a broad affinity range are required to define optimal affinity and to investigate the affinity-associated functional profiles of T cell-engaging strategies such as bispecific antibodies and chimeric antigen receptor-engineered T cells. Here, we demonstrate the unique binding characteristic of the developed antibody clone MVR, which exhibits robust binding to B-lymphoid cell lines. Intriguingly, MVR specifically recognizes the highly polymorphic human leukocyte antigen (HLA)-DR complex and exhibits varying affinities that are dependent upon the HLA-DRB1 allele type. Remarkably, MVR binds to the conformational epitope that consists of two hypervariable regions. As an application of MVR, we demonstrate an MVR-engineered chimeric antigen receptor (CAR) that elicits affinity-dependent function in response to a panel of target cell lines that express different HLA-DRB1 alleles. This tool evaluates the effect of affinity on cytotoxic killing, polyfunctionality, and activation-induced cell death of CAR-engineered T cells. Collectively, MVR exhibits huge potential for the evaluation of the affinity-associated profile of T cells that are redirected by engineered antibodies.

Gene Editing in B-Lymphoma Cell Lines Using CRISPR/Cas9 Technology.

Genome editing in eukaryotes has greatly improved through the application of targeted editing tools. The development of theCRISPR/Cas9 technology has facilitated genome editing in mammalian cells. However, efficient delivery of CRISPR components into cells growing in suspension remains a challenge. Here, we present a strategy for sequential delivery of the two essential components, Cas9 and sgRNA, into B-lymphoid cell lines. Stable Cas9 expression is obtained by retroviral transduction, before sgRNA is transiently delivered into the Cas9+ cells. This method improves the on-target efficiency of genome editing and, through the transient presence of sgRNA, reduces the potential off-target sites. The current method can be easily applied to other cell types that are difficult to edit with CRISPR/Cas9.

Abstract 4812: Vorinostat and fenretinide synergize in preclinical models of T-cell lymphoid malignancies via reactive oxygen species

Abstract Introduction: T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. Pan-histone deacetylase (HDAC) inhibitors vorinostat and belinostat, and class I specific HDAC inhibitor romidepsin have achieved FDA registration as 2nd line therapies for peripheral and/or cutaneous T-cell lymphomas. The cytotoxic retinoid fenretinide achieved durable complete responses against T-cell lymphomas in early-phase clinical trials and T-cell lymphoma patients who failed prior HDAC inhibitor treatment responded to fenretinide (Clinical Cancer Res 23:4550-4555, 2017). Fenretinide is currently being evaluated in a Phase IIa clinical trial for relapsed/refractory PTCL patients (NCT02495415). We have previously shown romidepsin and fenretinide synergize in preclinical models of T-cell lymphoid malignancies (Molecular Cancer Therapeutics 16:649-661, 2017). There exist some key differences in the activity of various classes of HDAC's, which have significantly different chemical structures and metabolic profiles. Therefore we determined if the pan-HDAC inhibitor vorinostat synergizes with fenretinide. Methods and Results: Using the DIMSCAN cytotoxicity assay, we demonstrated cytotoxic synergy between vorinostat and fenretinide in nine TCLM cell lines at clinically-achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, vorinostat + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous (COG-LL-317m and TX-LY-183x PDX) TCLM xenograft models than single agent vorinostat or fenretinide + ketoconazole. Fenretinide + vorinostat caused a reactive oxygen species (ROS, measured by DCFDA dye)-dependent increase in apoptosis (via TUNEL assay), and histone acetylation (measured by immunoblotting). The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + vorinostat was abrogated by antioxidant vitamin C. Vorinostat + fenretinide activated p38 and JNK via ROS, and shRNA knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity and apoptosis. Vorinostat + fenretinide also showed synergistic cytotoxicity for six B-lymphoid malignancy cell lines. Conclusion: Like romidepsin, vorinostat combined with fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in non-malignant cells, via a novel molecular mechanism. As vorinostat is an oral agent and not a PGP substrate it may have advantages in such combination therapy. These data support conducting a clinical trial of vorinostat combined with fenretinide in relapsed and refractory TCLMs. Citation Format: Monish Ram Makena, Thinh H. Nguyen, Balakrishna Koneru, Ashly Hindle, Wan-Hsi Chen, Dattesh U. Verlekar, Min H. Kang, C. Patrick Reynolds. Vorinostat and fenretinide synergize in preclinical models of T-cell lymphoid malignancies via reactive oxygen species [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4812.

Abstract 4458: Identification of splicing regulators using retroviral vectors carrying intron-containing expression cassettes

Abstract Exon inclusion is controlled by both positive (e.g., SR proteins) and negative (e.g., hnRNPs) regulators; yet high-throughput methods to comprehensively identify all relevant RNA-binding proteins are currently lacking. Thus, our long-term goal is to develop a genome-wide CRISPR sgRNA library-based screen, where cells with desired phenotypes are identified by flow cytometry and analyzed as pools using next generation sequencing. To achieve this goal, we took advantage of previous work in our laboratory demonstrating expression of the Δex2 CD19 isoform in B-cell leukemias and lymphomas resistant to CD19-directed immunotherapy (Sotillo et al., 2015; Yu et al., 2016). Skipping of exon 2 results in the truncated protein which fails to localize to the plasma membrane, yielding cells that are CD19-negative by flow cytometry (Bagashev et al., 2015). To study splicing regulation of exon 2, we designed a series of retroviral vectors containing the entire CD19 open reading frame retaining intronic sequences flanking exon 2 (i.e., intron 1 and intron 2). It is known that the nuclear splicing of provirus containing introns completely removes intron from the packaged viral RNA genome (Li et al., 1998). However, when viruses were stably transducedto B-lymphoid cell lines rendered null for endogenous CD19, the retroviral long terminal repeat (LTR) promoter-driven CD19 transcription resulted in skipping of exon2. Further addition of cognate 5'UTR sequence downstream of the retroviral LTR did not promote inclusion of this alternative exon. Similarly, the retroviral based expression of CD19 gene containing intronic sequences flanking constitutive exon10 did not allow proper splicing of exon10 and instead resulted into transcript retaining both introns (9 and 10). Interestingly, the flanking intronic sequences of exon10 allowed precise splicing of upstream exon2 when the expression cassette contained both exon2 and exon10 flanked by their respective introns. Thus, downstream placement of constitutive exon10 and its surrounding introns affected the splicing of upstream alternative exon2. Subsequent introduction of sgRNA libraries into cells transduced with such intron-containing expression cassettes will allow us to identify both positive and negative regulators of CD19 exon2 inclusion and potentially - novel general regulators of splicing. Bagashev, A., Sotillo, E., Wu, G., and Thomas-Tikhonenko, A. (2015). The importance of CD19 exon 2 for surface localization: Closing the Ig-like loop. Blood 126, 3433. Sotillo, E., Barrett, D.M., Black, K.L., Bagashev, A., Oldridge, D., Lanauze, C., Gazzara, M.R., et al. (2015). Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov 5, 1282-1295. Li, K., Garoff, H. (1998). Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors. Proc Natl Acad Sci USA. Citation Format: Mukta Asnani. Identification of splicing regulators using retroviral vectors carrying intron-containing expression cassettes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4458.

CDCA7 is a critical mediator of lymphomagenesis that selectively regulates anchorage-independent growth.

Tumor formation involves the acquisition of numerous capacities along the progression from a normal cell into a malignant cell, including limitless proliferation (immortalization) and anchorage-independent growth, a capacity that correlates extremely well with tumorigenesis. Great efforts have been made to uncover genes involved in tumor formation, but most genes identified participate in processes related to cell proliferation. Accordingly, therapies targeting these genes also affect the proliferation of normal cells. To identify potential targets for therapeutic intervention more specific to tumor cells, we looked for genes implicated in the acquisition of anchorage-independent growth and in vivo tumorigenesis capacity. A transcriptomic analysis identified CDCA7 as a candidate gene. Indeed, CDCA7 protein was upregulated in Burkitt’s lymphoma cell lines and human tumor biopsy specimens relative to control cell lines and tissues, respectively. CDCA7 levels were also markedly elevated in numerous T and B-lymphoid tumor cell lines. While CDCA7 was not required for anchorage-dependent growth of normal fibroblasts or non-malignant lymphocytes, it was essential but not sufficient for anchorage-independent growth of lymphoid tumor cells and for lymphomagenesis. These data suggest that therapies aimed at inhibiting CDCA7 expression or function might significantly decrease the growth of lymphoid tumors.

Abstract 2038: Reactive oxygen species-mediated synergism of fenretinide and romidepsin in preclinical models of T-cell lymphoid malignancies

Abstract Introduction: T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. Romidepsin is a histone deacetylase (HDAC) inhibitor that achieved FDA registration as 2nd line therapy for peripheral and cutaneous T-cell lymphomas. The cytotoxic retinoid fenretinide achieved durable complete responses against T-cell lymphomas in early-phase clinical trials and T-cell lymphoma patients who failed prior romidepsin treatment responded to fenretinide. Fenretinide is currently being evaluated in a Phase IIa clinical trial for relapsed/refractory PTCL patients (NCT02495415). We investigated the potential for using these two agents in combination in TCLMs. Methods and Results: Using the DIMSCAN assay, we demonstrated cytotoxic synergy between romidepsin and fenretinide in fifteen TCLM cell lines at clinically-achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, romidepsin + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous (COG-LL-317m and TX-LY-183x PDX) and disseminated (COG-LL-317m Luc expressing the luciferase gene) TCLM xenograft models than single agent romidepsin or fenretinide + ketoconazole. Fenretinide + romidepsin caused a reactive oxygen species (ROS, measured by DCFDA dye)-dependent increase in pro-apoptotic proteins (Bim, tBid, Bax and Bad), apoptosis (via TUNEL assay), and inhibition of HDAC enzymatic activity, which achieved a synergistic increase in histone acetylation. The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + romidepsin was abrogated by antioxidants (vitamins C or E). Romidepsin + fenretinide activated p38 and JNK via ROS, and shRNA knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity and apoptosis. Romidepsin + fenretinide also showed synergistic cytotoxicity for B-lymphoid malignancy cell lines, but did not increase ROS, acetylation of histones, activation of p38 + JNK, or cytotoxicity in fibroblasts and blood mononuclear cells. Conclusion: Romidepsin + fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in non-malignant cells, via a novel molecular mechanism. These data support conducting clinical trials of romidepsin + fenretinide in relapsed and refractory TCLMs. Citation Format: Monish Ram Makena, Balakrishna Koneru, Min H. Kang, C. Patrick Reynolds. Reactive oxygen species-mediated synergism of fenretinide and romidepsin in preclinical models of T-cell lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2038. doi:10.1158/1538-7445.AM2017-2038

Reactive Oxygen Species-Mediated Synergism of Fenretinide and Romidepsin in Preclinical Models of T-cell Lymphoid Malignancies.

T-cell lymphoid malignancies (TCLM) are in need of novel and more effective therapies. The histone deacetylase (HDAC) inhibitor romidepsin and the synthetic cytotoxic retinoid fenretinide both have achieved durable clinical responses in T-cell lymphomas as single agents. We investigated the potential for using these two agents in combination in TCLMs. We demonstrated cytotoxic synergy between romidepsin and fenretinide in 15 TCLM cell lines at clinically achievable concentrations that lacked cytotoxicity for nonmalignant cells (fibroblasts and blood mononuclear cells). In vivo, romidepsin + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous and disseminated TCLM xenograft models than single-agent romidepsin or fenretinide + ketoconazole. Fenretinide + romidepsin caused a reactive oxygen species (ROS)-dependent increase in proapoptotic proteins (Bim, tBid, Bax, and Bak), apoptosis, and inhibition of HDAC enzymatic activity, which achieved a synergistic increase in histone acetylation. The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + romidepsin were abrogated by antioxidants (vitamins C or E). Romidepsin + fenretinide activated p38 and JNK via ROS, and knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity. Romidepsin + fenretinide also showed synergistic cytotoxicity for B-lymphoid malignancy cell lines, but did not increase ROS, acetylation of histones, activation of p38 + JNK, or cytotoxicity in nonmalignant cells. Romidepsin + fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in nonmalignant cells, via a novel molecular mechanism. These data support conducting clinical trials of romidepsin + fenretinide in relapsed and refractory TCLMs. Mol Cancer Ther; 16(4); 649-61. ©2017 AACR.

TET1 Promotes Leukemic Growth in T-ALL Via Maintenance of 5-Hydroxymethylation Marks and Can be Antagonized By the PARP Inhibitor Olaparib

T-cell acute lymphoid leukemia (T-ALL) is an aggressive hematological cancer predominantly occurring in children and adolescents. T-ALL is characterized by aberrant expression of genes involved in T-cell differentiation and self-renewal accompanied by aberrant DNA cytosine methylation patterns. The mechanisms underlying these aberrations are not well understood. The Ten-eleven translocation 1 (TET1) enzyme is an iron-dependent dioxygenase that catalyzes the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation and regulation of gene expression. Using quantitative real time (qRT)-PCR we now demonstrate that T-ALL is characterized by high and aberrant expression of TET1: TET1 was significantly and >20-fold higher expressed in human T-ALL cell lines compared to other B-lymphoid or myeloid leukemia cell lines (p<0.001, n=3). This was confirmed in primary patient samples, in which TET1 was more than 10-fold higher expressed in 95% of T-ALL cases (n=10) compared to other leukemia subtypes (B-ALL, n=29; AML, n=39; CLL, n=23, p<0.001). Furthermore, expression levels of TET1 in T-ALL patient samples were over 50-fold higher than in normal PB and BM derived healthy CD3+ T cells (n=6, p<0.001) in line with published cDNA microarray data from a large patient cohort of 174 T-ALL cases (Haferlach et al., JCO 2010). Knockdown (KD) of TET1 mRNA using two independent shRNAs in T-ALL cell lines adversely affected their cell growth (>70% reduction compared to scrambled, p<0.001, n=4) clonogenicity (>70% reduction compared to scrambled, p<0.001, n=4) and blocked cells in the G2/M phase of the cell cycle in vitro (n=3). Furthermore, TET1 KD significantly reduced the leukemic engraftment potential of T-ALL cell line Jurkat in xenografts (shRNA, n=16; scrambled, n=8). Global analysis of 5hmC levels using hMeDIP-seq performed on TET1 depleted Jurkat cells revealed lower global 5hmC levels as compared to cells transduced with scrambled control (n=2). Promoters (-5kb TSS) of 5,181 genes showed reduction of 5hmC marks (log2 fold diff.; p-value < 1e-10). Global 5mC analysis (MeDIP-seq) revealed 12,007 genes that gained 5mC marks at their promoters as a result of TET1 KD (log2 fold diff. > 1.5; p-value < 1-10). Of note, genes demonstrating a loss of 5hmC marks and corresponding a gain in 5mC marks on their promoters belonged to gene families involved innucleosome organization (HIST family), DNA damage response (DDR)(RAD, PARP), T-cell development (NOTCH3, RUNX1, LEF1) and homeobox genes (q-value < 1e-5). These changes were paralleled by changes in expression after TET1 depletion as measured by RNA-Seq in Jurkat cells: GO TERM analysis (log2 fold diff. > 1.5; p<0.05) revealed deregulation of genes belonging to nucleosome assembly, DNA damage, nucleosome organization and transcriptional deregulation in cancer. In line with these observations these pathways showed differential expression in T-ALL patients divided into the categories high vs. low TET1 expression based on previously published microarray data (Haferlach et al., JCO 2010) (median as cut-off; n=174)(log2 fold diff. >1.5; p<0.05).The aberrantly high TET1 expression in T-ALL cell lines shares a potential causal relationship with the hypomethylated status of the TET1 promoter (determined by LC/MS), and the enrichment of euchromatic marks such as H3K4me3 and acetylated H3 as determined by ChIP-qPCR. Since it was shown that PARP activity positively influences TET1 expression by regulating its promoter epigenetically, we tested the impact of the PARP inhibitor olaparib on TET1 activity in our model: of note, aberrant expression of TET1 could be reverted by the PARP inhibitor olaparib, which induced reduction of euchromatic marks on the TET1 promoter, in parallel with decreased TET1 expression and protein levels as well as 5hmC levels in Jurkat cells without affecting expression of other TET family members. Olaparib treatment caused a >75% reduction in cell growth and clonogenicity of T-ALL cell lines in vitro (n=3) and delayed leukemic engraftment in NSG mice (42%±8 in scrambled vs 9%±7 for the shRNAs, n=4 per arm; p<0.001). In conclusion, these data demonstrate for the first time that TET1 plays an important oncogenic role in T-ALL. We also identify PARPs as upstream regulators of TET1 in T-ALL, opening the way to antagonize TET1 pharmacologically in this poor prognosis leukemia. DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

Dual-targeting triplebody 33-3-19 mediates selective lysis of biphenotypic CD19+ CD33+ leukemia cells.

Simultaneous targeting of multiple tumor-associated antigens (TAAs) in cancer immunotherapy is presumed to enhance tumor cell selectivity and to reduce immune escape.The combination of B lymphoid marker CD19 and myeloid marker CD33 is exclusively present on biphenotypic B/myeloid leukemia cells. Triplebody 33-3-19 binds specifically to both of these TAAs and activates T cells as immune effectors. Thereby it induces specific lysis of established myeloid (MOLM13, THP-1) and B-lymphoid cell lines (BV173, SEM, Raji, ARH77) as well as of primary patient cells. EC50 values range from 3 pM to 2.4 nM. In accordance with our hypothesis, 33-3-19 is able to induce preferential lysis of double- rather than single-positive leukemia cells in a target cell mixture: CD19/CD33 double-positive BV173 cells were eliminated to a significantly greater extent than CD19 single-positive SEM cells (36.6% vs. 20.9% in 3 hours, p = 0.0048) in the presence of both cell lines. In contrast, equivalent elimination efficiencies were observed for both cell lines, when control triplebody 19-3-19 or a mixture of the bispecific single chain variable fragments 19-3 and 33-3 were used. This result highlights the potential of dual-targeting agents for efficient and selective immune-intervention in leukemia patients.

Abstract IA21: The MYC-microRNA pathway in B-cell neoplasms

Abstract MYC is a noncanonical transcription factor that binds to thousands of genomic loci and affects over 15% of the human transcriptome, with surprisingly little overlap between MYC-bound and MYC-regulated genes. This discordance raises the question whether MYC chooses its targets based on their individual biological effects (“a la carte”) or by virtue of belonging to a certain group of genes (on a “prix fixe” basis). Over the last few years, we have developed a model wherein MYC initially deregulates a select number of microRNAs. These microRNAs then target a broad spectrum of genes based solely on the presence in their 3' UTRs (untranslated regions) of distinct “seed” sequences. Existing evidence suggests that there are significant microRNA components to all key MYC-driven phenotypes, including cell-cycle progression, apoptosis, metabolism, angiogenesis, metastasis, stemness, and last but not least – hematopoiesis (1). For example in a human B-lymphoid cell line, Myc-repressed genes are significantly enriched for predicted binding sites of Myc-regulated miRNAs, primarily those comprising the Myc-activated miR-17-92 cluster. Notably, gene set enrichment analysis demonstrates that miR-17-92 is a major regulator of B-cell receptor (BCR) pathway components. Many of them are immunoreceptor tyrosine inhibitory motif (ITIM)-containing proteins, and ITIM proteins CD22 and FCGR2B were found to be direct targets of miR-17-92. Consistent with the propensity of ITIM proteins to recruit phosphatases, either MYC or miR-17-92 expression is necessary to sustain phosphorylation of spleen tyrosine kinase (SYK) and the B-cell linker protein (BLNK) upon ligation of the BCR. Further downstream, stimulation of the BCR response by miR-17-92 resulted in the enhanced calcium flux, activation of the CD19-PI3K axis, and elevated levels of Myc itself (2). Notably, inhibition of the miR-17-92 cluster in diffuse large B-cell lymphoma (DLBCL) cell lines diminished the BCR response as measured by SYK and BLNK phosphorylation. Conversely, human DLBCLs of the BCR subtype express higher Myc and mir17hg transcript levels than other subtypes. Hence, the Myc-miR-17-92-BCR axis, frequently affected by genomic rearrangements, constitutes a novel lymphomagenic feed-forward loop (3).

T cell-recruiting triplebody 19-3-19 mediates serial lysis of malignant B-lymphoid cells by a single T cell.

Triplebody 19-3-19, an antibody-derived protein, carries three single chain fragment variable domains in tandem in a single polypeptide chain. 19-3-19 binds CD19-bearing lymphoid cells via its two distal domains and primary T cells via its CD3-targeting central domain in an antigen-specific manner. Here, malignant B-lymphoid cell lines and primary cells from patients with B cell malignancies were used as targets in cytotoxicity tests with pre-stimulated allogeneic T cells as effectors. 19-3-19 mediated up to 95 % specific lysis of CD19-positive tumor cells and, at picomolar EC₅₀ doses, had similar cytolytic potency as the clinically successful agent Blinatumomab. 19-3-19 activated resting T cells from healthy unrelated donors and mediated specific lysis of both autologous and allogeneic CD19-positive cells. 19-3-19 led to the elimination of 70 % of CD19-positive target cells even with resting T cells as effectors at an effector-to-target cell ratio of 1 : 10. The molecule is therefore capable of mediating serial lysis of target cells by a single T cell. These results highlight that central domains capable of engaging different immune effectors can be incorporated into the triplebody format to provide more individualized therapy tailored to a patient's specific immune status.

Comparison of Reverse Transcription Quantitative Real-Time PCR, Flow Cytometry, and Immunohistochemistry for Detection of Monoclonality in Lymphomas

In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced.

The Myc-miR-17-92 axis amplifies B-cell receptor signaling via inhibition of ITIM proteins: a novel lymphomagenic feed-forward loop

AbstractThe c-Myc oncoprotein regulates >15% of the human transcriptome and a limited number of microRNAs (miRNAs). Here, we establish that in a human B-lymphoid cell line, Myc-repressed, but not Myc-stimulated, genes are significantly enriched for predicted binding sites of Myc-regulated miRNAs, primarily those comprising the Myc-activated miR-17∼92 cluster. Notably, gene set enrichment analysis demonstrates that miR-17∼92 is a major regulator of B-cell receptor (BCR) pathway components. Many of them are immunoreceptor tyrosine inhibitory motif (ITIM)-containing proteins, and ITIM proteins CD22 and FCGR2B were found to be direct targets of miR-17∼92. Consistent with the propensity of ITIM proteins to recruit phosphatases, either MYC or miR-17∼92 expression was necessary to sustain phosphorylation of spleen tyrosine kinase (SYK) and the B-cell linker protein (BLNK) upon ligation of the BCR. Further downstream, stimulation of the BCR response by miR-17-92 resulted in the enhanced calcium flux and elevated levels of Myc itself. Notably, inhibition of the miR-17∼92 cluster in diffuse large B-cell lymphoma (DLBCL) cell lines diminished the BCR response as measured by SYK and BLNK phosphorylation. Conversely, human DLBCLs of the BCR subtype express higher Myc and mir17hg transcript levels than other subtypes. Hence, the Myc-miR-17-92-BCR axis, frequently affected by genomic rearrangements, constitutes a novel lymphomagenic feed-forward loop.

Low Amount Expression of Ku 70 Protein Related to Radiation-induced Chromosome Instability and Radiosensitivity in Normal Lymphocytes, and Leukemia and Lymphoma Cell Lines

Previous our experiment showed that B-lymphoid cell lines established from α ray- and γ-ray irradiated lymphocytes had higher chromosome instabilities through many passages than that from non-irradiated lymphocytes. Present protein study revealed that these B-cell lines had low amount of Ku 70 protein expression before and after second irradiation, longer localization of Ku 70 protein in nucleus, longer period of cell cycle delay at G1 or G2 phases and high activity of DNA-PK. The first αray- or γ-ray irradiation had an effect of persisting impaired DNA repair system in cells, which resulted in a decreased repair capacity after the second irradiation. Leukemia and lymphoma cell lines also showed similar characteristics after irradiation to those of the B-cell lines having high chromosome instability. These similarities are suggesting that characteristics on abnormal Ku 70 protein might persist long term and might be related to initiating leukemogenesis and lymphomagenesis as well as that such an ability of DNA repair protein might apply to prediction of radiosensitivity of leukemia or lymphoma cells.

Germline Allele-Specific Expression of DAPK1 in Chronic Lymphocytic Leukemia

We previously reported a rare germline variant (c.1-6531) that resulted in allele–specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5′ upstream regulatory region, within distinct exons or in the 3′-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.

Fludarabine and Cladribine Induce Changes in Surface Proteins on Human B-Lymphoid Cell Lines Involved with Apoptosis, Cell Survival, and Antitumor Immunity

Fludarabine and cladribine are purine analogues used to treat hematological malignancies. Alone or in combination with therapeutic antibodies, they are effective in treating patients with chronic lymphocytic leukemia and non-Hodgkin's lymphoma. However, the mechanisms of action of these drugs are not well understood. Plasma membrane proteins perform a variety of essential functions that can be affected by malignancy and perturbed by chemotherapy. Analysis of surface proteins may contribute to an understanding of the mechanisms of action of purine analogues and identify biomarkers for targeted therapy. The surface of human cells is rich in N-linked glycoproteins, enabling use of a hydrazide-coupling technique to enrich for glycoproteins, with iTRAQ labeling for quantitative comparison. A number of plasma membrane proteins on human leukemia and lymphoma cells were affected by treatment with a purine analogue, including decreases in CD22 (an adhesion and signaling molecule) and increases in CD205 (a "damaged cell marker") and CD80 and CD50 (T-cell interaction molecules). Purine analogues may affect B-cell receptor (BCR) signaling and costimulatory molecules, leading to multiple signals for apoptosis and cell clearance. Fludarabine and cladribine induce differential effects, with some cell survival proteins (ECE-1 and CD100) more abundant after fludarabine treatment. Cell surface proteins induced by fludarabine and cladribine may be targets for therapeutic antibodies.

Fludarabine nucleoside induces accumulations of p53, p63 and p73 in the nuclei of human B-lymphoid cell lines, with cytosolic and mitochondrial increases in p53

Human Raji cells treated with fludarabine nucleoside (2-FaraA, 3 μM) undergo apoptosis with accumulation of p53 in the nuclei as multiple phosphorylated isoforms and C-terminal truncated derivatives. Changes induced by 2-FaraA in the levels of p53, p63 and p73 in the nuclear, cytosolic and mitochondrial fractions have been determined in four human B-lymphoid cell lines that are TP53-functional (Raji and IM9) and TP53-mutated (MEC1 and U266). The B-lymphoid cell lines were treated with 2-FaraA (3 μM, 24 h, 48 h) and viability determined. Protein extracts of subcellular fractions from 2-FaraA-treated cells were analysed by 1D and 2D electrophoresis; multiple phosphorylated isoforms and truncated derivatives were identified by Western blots for p53, p63 and p73. p53 and p63 were present in all three fractions, while p73 was only detected in nuclei. After treatment with 2-FaraA, nuclear p53, p63 and p73 accumulated as multiple phosphorylated isoforms and truncated derivatives. The association of p63 with mitochondria in human cells is novel. Comprehensive information on the subcellular distributions and responses of p53, p63 and p73 to 2-FaraA provides additional insight into mechanisms for induction of apoptosis in the treatment of B-lymphoproliferative disorders with fludarabine.