scholarly journals Exploring PRMT5 As a Potential Therapeutic Target in Canine Lymphomas

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4182-4182
Author(s):  
Kyle Renaldo ◽  
Shelby Sloan ◽  
JiHyun Chung ◽  
Lindsay Courtney ◽  
Konstantin Shilo ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Small Molecule ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Canine Lymphoma ◽  
Over Expression ◽  
Arginine Residues ◽  
Dose Dependent

Abstract Non-Hodgkin lymphoma (NHL) represents approximately 4 percent of all cancer diagnoses in the United States, with diffuse large B-cell lymphoma (DLBCL) accounting for approximately 40 percent of all new cases. There are numerous histologic (GC, NGC) and genetic (double hit/expressor) subtypes of DLBCL and the overall outcome of patients who receive standard therapy is heterogeneous. Lymphoma is also a common, malignancy in dogs, and while initial responses to combination chemotherapy show clinical responses, remission time is short and cures rare. Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase (PRMT) enzyme capable of driving symmetric demethylation of arginine residues on histones (H3(me2)R8; H4(me2)R3) and other proteins such as P53, and the NFkB subunit p65. PRMT5 is overexpressed and dysregulated in both solid and hematologic tumors and plays a key role in the driver activity of MYC, CYCLIND1 and NOTCH in lymphoma models. Experimental therapeutic strategies aimed at targeting PRMT5 activity have led to numerous, highly selective inhibitors that are currently being translated into the clinic with several clinical trials underway or in development. Here, we characterized patterns of PRMT5 expression and correlated these with histologic subtype in canine lymphoma tissue microarrays (TMAs, n=337 lymphoma specimens). We characterized expression of PRMT5 and its symmetric dimethylation histone marks in three canine lymphoma-derived cell lines, CLBL-1, 17-71, and OSW. Treatment of PRMT5 with a highly selective small molecule PRMT5 inhibitor (CMP220) was performed to determine the dose-dependent effects (1nM-10uM range) on proliferation (MTS assay), viability (annexin V/PI flow cytometry) and biomarkers of PRMT5 activity (SDMA, ADMA, histone symmetric demethylation). CMP220 showed PRMT5 inhibitory activity that was highly selective in a methyltransferase screening assay with 37 enzymes/complexes. TMAs of all histologic subtypes of lymphoma showed over-expression of PRMT5 in 96% of specimens. Canine DLBCL showed variable over-expression in the cytoplasmic compartment (48.8% strong, 50.0% weak, n = 165) compared to negative or weak staining in normal and hyperplastic lymph nodes (n = 40). Nuclear staining was observed primarily in lymphomas of T cell lineage (PTCL, and pre T lymphoblastic lymphomas). Primary and cell line lymphoma samples showed PRMT5 over expression by Western blot and RT-PCR. PRMT5 inhibition showed a dose-dependent decrease in symmetric dimethylarginine (SDMA) and symmetric demethylation of histone H4 arginine-3 (H4(me2s)R3) with no effect on asymmetric dimethylarginine (ADMA). Interestingly, in contrast to PRMT5 inhibition in human lymphomas, symmetric demethylation of histone H3 arginine-8 (H3(me2s)R8) showed no change with PRMT5 inhibition. The PRMT5 small molecule inhibitor CMP220 inhibited growth of CLBL-1 (IC50 of 123.2 nM at 6d) and 17-71 (IC50 of 100 nM at 6d). The OSW PTCL cell line showed profound resistance to PRMT5 inhibition with little cell death observed at 10 uM. Ex-vivo canine patient samples showed suppression of growth in a time and dose-dependent fashion. We have demonstrated that PRMT5 is expressed in canine B cell primary lymphomas and cell lines and that PRMT5 inhibition leads to growth suppression and induction of apoptosis. We are currently exploring genome-wide recruitment of PRMT5 on chromatin and examining chromatin and whole transcriptome changes that occur in canine lymphoma cell lines treated with PRMT5 inhibitors. This data provides justification for incorporating the canine lymphoma model into the preclinical development of PRMT5 inhibitors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lymphotoxin is an autocrine growth factor for Epstein-Barr virus-infected B cell lines.

1993 ◽  
Vol 177 (3) ◽  
pp. 763-774 ◽  
Author(s):  
Z Estrov ◽  
R Kurzrock ◽  
E Pocsik ◽  
S Pathak ◽  
H M Kantarjian ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Lymphoblastoid Cell Line ◽  
Autocrine Growth ◽  
Autocrine Growth Factor ◽  
Barr Virus ◽  
Epstein Barr ◽  
Dose Dependent

Because human lymphotoxin (LT) was originally isolated from a lymphoblastoid cell line, we investigated the role of this molecule in three newly established Epstein-Barr virus (EBV)-infected human B cell lines. These lines were derived from acute lymphoblastic leukemia (Z-6), myelodysplastic syndrome (Z-43), and acute myelogenous leukemia (Z-55) patients who had a prior EBV infection. Each lymphoblastoid cell line had a karyotype that was different from that of the original parent leukemic cells, and all expressed B cell, but not T cell or myeloid surface markers. In all three lines, rearranged immunoglobulin heavy chain joining region (JH) bands were found, and the presence of EBV DNA was confirmed by Southern blotting. Z-6, Z-43, and Z-55 cell lines constitutively produced 192, 48, and 78 U/ml LT, respectively, as assessed by a cytotoxicity assay and antibody neutralization. Levels of tumor necrosis factor (TNF) were undetectable. Scatchard analysis revealed that all the cell lines expressed high-affinity TNF/LT receptors with receptor densities of 4197, 1258, and 1209 sites/cell on Z-6, Z-43, and Z-55, respectively. Furthermore, labeled TNF binding could be reversed by both unlabeled TNF, as well as by LT. Studies with p60 and p80 receptor-specific antibodies revealed that the three lines expressed primarily the p80 form of the TNF receptor. When studied in a clonogenic assay, exogenous LT stimulated proliferation of all three cell lines in a dose-dependent fashion at concentrations ranging from 25 to 500 U/ml. Similar results were obtained with [3H]TdR incorporation. Monoclonal anti-LT neutralizing antibodies at concentrations of 25-500 U/ml inhibited cellular multiplication in a dose-dependent manner. It is interesting that in spite of a common receptor, TNF (1,000 U/ml) had no direct effect on Z-55 cell growth, whereas it partially reversed the stimulatory effect of exogenous LT. In addition, TNF inhibited Z-6 and Z-43 cell proliferation, and its suppressive effect was reversed by exogenous LT. Both p80 and p60 forms of soluble TNF receptors suppressed the lymphoblastoid cell line proliferation and their inhibitory effect was partially reversed by LT. Our data suggest that (a) LT is an autocrine growth factor for EBV-transformed lymphoblastoid B cell lines; and (b) anti-LT antibodies, soluble TNF/LT receptors, and TNF itself can suppress the growth of lymphoblastoid cells, probably by modulating or competing with LT.(ABSTRACT TRUNCATED AT 400 WORDS)

EBV Microrna Mir-BHRF1-2 Targets PRDM1/Blimp1: Potential Role in EBV Lymphomagenesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3547-3547
Author(s):  
Jiao Ma ◽  
Kui Nie ◽  
David Redmond ◽  
Yifang Liu ◽  
Daniel M Knowles ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Binding Site ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Rna Seq ◽  
B Cell Lymphomas ◽  
Down Regulation ◽  
Over Expression

Abstract PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). In DLBCL, PRDM1 is inactivated by mutations and deletions; however, there is also evidence that PRDM1 is down-regulated by microRNAs (miRNAs) in DLBCL and Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). A decrease in PRDM1 activity contributes to the pathogenesis of DLBCL and cHL by inhibiting plasma cell differentiation triggered by signal transduction pathways such as the NF-kB pathway. Since malignant EBV-positive B-cell lymphoproliferations are often associated with increased NF-kB activity, it is conceivable that abnormal PRDM1 down-regulation may play a role in their pathogenesis. EBV-positive B-cell lymphomas are postulated to originate from EBV-infected B-cells with latency III growth program of EBV gene expression. Thus, EBV-immmortalized lymphoblastoid cell lines (LCLs), which are of latency III type, serve as a good model to study EBV lymphomagenesis. We observed discordance in PRDM1 mRNA and protein levels in LCLs. By quantitative real-time reverse transcriptase PCR, PRDM1 mRNA levels in LCLs varied from 14.6% to 1259.7% relative to the multiple myeloma cell line U266, which expresses high levels of PRDM1. However, PRDM1 protein was discordantly low in LCLs compared to U266 based on immunohistochemistry and Western blotting assays, consistent with post-transcriptional regulation. EBV encodes 25 viral miRNAs, and we postulate that one of more of them may function to dampen PRDM1 expression. Indeed, a miRNA binding site containing seed match to bases 2-7 of EBV miR-BHRF1-2 was identified in positions 1565 to 1589 of PRDM1 3’ untranslated region. MiR-BHRF1-2 functionally targeted this specific binding site and repressed luciferase reporter activity. Mutation in the seed region of this site relieved the repression in comparison to the wild type control. MiR-BHRF1-2 was highly expressed in LCLs, while it was barely detectable in the EBV-positive Burkitt lymphoma cell line MUTU I, which has latency type I. Importantly, immunoblotting assay demonstrated an up-regulation of PRDM1 protein level in CCL156 and CCL159 LCL cells transfected with miR-BHRF1-2 inhibitor relative to those transfected with miRNA Inhibitor negative control, supporting a role of miR-BHRF1-2 in PRDM1 down-regulation in vivo. To examine the biological consequences of increased PRDM1 expression in LCL cells, PRDM1 was over-expressed in JY25 and CCL159 LCL cell lines. Enforced expression of PRDM1 induced apoptosis in both cell lines. Furthermore, bromodeoxyuridine (Brdu) incorporation study demonstrated that overexpression of PRDM1 reduced the percentage of S phase from 43.4% to 27.6% in CCL159 cells, and 39.5% to 27.9% in JY25 cells, respectively. Whole transcriptome sequencing (RNA-seq) identified a set of potential PRDM1 direct target genes whose expressions decreased in both LCL cell lines upon PRDM1 over-expression. These genes have broad functions including cell proliferation and survival, transcription and translation, mitochondrial functions, and cytoskeleton. Although no significant changes in cell cycle and apoptosis were observed upon transfection of miR-BHRF1-2 inhibitor, RNA-seq analysis of CLL159 cells transfected with miR-BHRF1-2 inhibitor revealed a small subset of repressed genes which overlapped with those identified by PRDM1 over-expression. This finding suggests that the increase in PRDM1 expression upon miR-BHRF1-2 inhibition, albeit small, is capable of repressing a subset of PRDM1 target genes with potential biological effects. In summary, our findings demonstrate that PRDM1 is a target of EBV miR-BHRF1-2. MiR-BHRF1-2 mediated PRDM1 down-regulation may contribute to the pathogenesis of EBV-associated B-cell lymphomas by inhibiting the transcription repression program of PRDM1 and limiting PRDM1-mediated cellular changes detrimental to tumor growth, including cell cycle arrest and apoptosis. Further characterization of the target genes whose expression is up-regulated by miR-BHRF1-2-mediated PRDM1 down-regulation may provide important clues to the pathogenetic function of miR-BHRF1-2 and EBV oncogenesis in general. Disclosures No relevant conflicts of interest to declare.

Antioxidant effects and in vitro cytotoxicity on human cancer cell lines of flavonoid-rich Flamboyant (Delonix regia (Bojer) Raf.) flower extract

2020 ◽  
Vol 21 ◽  
Author(s):  
Putthiporn Khongkaew ◽  
Phanphen Wattanaarsakit ◽  
Konstantinos I. Papadopoulos ◽  
Watcharaphong Chaemsawang
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Flower Extract ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Murine Leukemia Cell ◽  
Dose Dependent

Background: Cancer is a noncommunicable disease with increasing incidence and mortality rates both worldwide and in Thailand. Its apparent lack of effective treatments is posing challenging public health issues. Introduction: Encouraging research results indicating probable anti-cancer properties of the Delonix regia flower extract (DRE) have prompted us to evaluate the feasibility of developing a type of product for future cancer prevention or treatment. Methods and Results: In the present report, using High Performance Liquid Chromatography (HPLC), we demonstrate in the DRE, the presence of high concentrations of three identifiable flavonoids, namely rutin 4.15±0.30 % w/w, isoquercitrin 3.04±0.02 %w/w, and myricetin 2.61±0.01 % w/w respectively while the IC50 of DPPH and ABTS assay antioxidation activity was 66.88±6.30 µg/ml and 53.65±7.24 µg/ml respectively. Discussion: Our cancer cell line studies using the MTT assay demonstrated DREs potent and dose dependent inhibition of murine leukemia cell line (P-388: 35.28±4.07% of cell viability remaining), as well as of human breast adenocarcinoma (MCF-7), human cervical carcinoma (HeLa), human oral cavity carcinoma (KB), and human colon carcinoma (HT-29) cell lines in that order of magnitude. Conclusion: Three identifiable flavonoids (rutin, isoquercitrin and myricetin) with high antioxidation activity and potent and dose dependent inhibition of murine leukemia cell line and five other cancer cell lines were documented in the DRE. The extract’s lack of cytotoxicity in 3 normal cell lines is a rare advantage not usually seen in current antineoplastic agents. Yet another challenge of the DRE was its low dissolution rate and long-term storage stability, issues to be resolved before a future product can be formulated.

scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Abstract Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

scholarly journals Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Busacca ◽  
Qi Zhang ◽  
Annabel Sharkey ◽  
Alan G. Dawson ◽  
David A. Moore ◽  
...  
Keyword(s):  
Small Molecule ◽  
Selective Vulnerability ◽  
Dependent Manner ◽  
Histone H4 ◽  
Wild Type ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Methylthioadenosine Phosphorylase ◽  
Protein Arginine Methyltransferase 5

AbstractWe hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.

scholarly journals Proton-irradiated breast cells: molecular points of view

2019 ◽  
Vol 60 (4) ◽  
pp. 451-465 ◽  
Author(s):  
Valentina Bravatà ◽  
Francesco P Cammarata ◽  
Luigi Minafra ◽  
Pietro Pisciotta ◽  
Concetta Scazzone ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Fate ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Proton Irradiation ◽  
Expression Profiles ◽  
Treatment Plan ◽  
Mcf10a Cell ◽  
Specific Cell ◽  
Dose Dependent

Abstract Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome

scholarly journals Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

1987 ◽  
Vol 7 (1) ◽  
pp. 450-457 ◽  
Author(s):  
E H Brown ◽  
M A Iqbal ◽  
S Stuart ◽  
K S Hatton ◽  
J Valinsky ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Cluster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Dna Content ◽  
Immunoglobulin Heavy Chain ◽  
S Phase ◽  
C Value ◽  
Murine Erythroleukemia

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

scholarly journals A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Jie Wang ◽  
Katarzyna Urbanska ◽  
Prannda Sharma ◽  
Mathilde Poussin ◽  
Reza Nejati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Receptor ◽  
Brentuximab Vedotin ◽  
T Cell Lymphomas ◽  
T Cell Malignancies

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL. References 1. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). Ann Oncol Off J Eur Soc Med Oncol. 1990;1(1):45-50. 2. Horwitz SM, Advani RH, Bartlett NL, et al. Objective responses in relapsed T-cell lymphomas with single agent brentuximab vedotin. Blood. 2014;123(20):3095-3100. 3. Hughes MS, Yu YYL, Dudley ME, et al. Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions. Hum Gene Ther. 2005;16(4):457-472. Figure Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria.

scholarly journals Preclinical Activity of the MPS1 Inhibitor S81694 in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2631-2631
Author(s):  
Anna Kaci ◽  
Emilie Adiceam ◽  
Melanie Dupont ◽  
Marine Garrido ◽  
Jeannig Berrou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Solid Tumors ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Cell Cycle Distribution ◽  
Monopolar Spindle

Introduction: The dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one the main kinases of the spindle assembly checkpoint (SAC) critical for accurate segregation of sister chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. Mps1 is known to be overexpressed in several solid tumors including triple negative breast cancer. Thus, Mps1 seems to be a promising target and small molecules targeting Mps1 entered clinical trials in solid tumors. ALL originates from malignant transformation of B-and T-lineage lymphoid precursors with a variety of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development. While outcome is excellent for pediatric patients and younger adults, relapsed and refractory disease still remain a clinical challenge for elder patients. Here, we demonstrate for the first time preclinical efficacy of the small molecule Mps1 inhibitor (Mps1i) S81694 in T- and B- ALL cells including BCR-ABL1+-driven B-ALL. Materials and Methods: Expression of Mps1 was determined by RT-qPCR and WB in JURKAT, RS4-11 and BCR-ABL1+ cells (BV-173 and TOM-1). A small molecule Mps1i (S81694) was tested alone (0 to 1000nM) or in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1+ ALL cell lines. Cell viability and IC50 was assessed by MTS assays after exposure to Mps1i for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Bliss model. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of Mps1i. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation at 48h. Phosphorylation of Mps1 was detected in synchronized (by nocodazole and MG-132) cells by immunofluorescence using an anti phospho-Mps1 antibody detecting Thr33/Ser37 residues. Time-lapse microscopy was used in cell lines in presence or absence of S81694 to determine mitosis duration. Bone marrow (BM) nucleated patient cells were obtained after informed consent and incubated in methylcellulose with cytokines with or without Mps1i for 2 weeks to determine colony growth. Results: Expression of Mps1 could be detected by RT-qPCR and at the protein level by WB in all cell lines (Figure 1A and B ). IC50 after Mps1i exposure alone was 126nM in JURKAT cells, 51nM in RS4-11 cells, 75nM in BV-173 cells and 83nM in TOM-1. Significant apoptosis as detected by phosphatidylserine exposure and PI incorporation in all cell lines with BCR-ABL1+ cell lines BV-173 and TOM-1 cells being the most sensitive (80% and 60% apoptotic cells respectively)(Figure 1C). Upon Mps1i exposure we observed targeted inhibition of Mps1 phosphorylation at Thr33/Ser37 residues indicating the specific on target effect of S81694 by inhibiting Mps1 autophosphorylation (Figure 1D and E). Cell cycle profile was generally lost after treatment with S81694 in all cell lines indicating aberrant 2n/4n distribution due to SAC abrogation (Figure 1F). Furthermore, we demonstrated that S81694 exposure accelerated significantly mitosis in BV-173 cell line from 36 minutes to 19 minutes indicating effective inhibition of SAC function (Figure 1G). Interestingly, S81694 induced significant apoptosis (70%) in the imatinib resistant BV173 cell line bearing the E255K-BCR-ABL1-mutation. Combination of S81694 with TKI imatinib, dasatinib and nilotinib (but not ponatinib) was strongly synergistic in BCR-ABL1+ cells (Figure 1H). Finally, we observed inhibition of colony formation in a patient with BCR-ABL1+ B-ALL after exposure to 100nM and 250nM S81694 (reduction of 85% and 100% respectively)(Figure 1I). Conclusion: Mps1i S81694 yields significant preclinical activity in T-and B-cell ALL including BCR-ABL1+ models. Interestingly S81694 was efficacious in a TKI resistant cell line. Disclosures Kaci: Institut de Recherches Internationales Servier (IRIS): Employment. Garrido:Institut de Recherches Internationales Servier (IRIS): Employment. Burbridge:Institut de Recherches Internationales Servier (IRIS): Employment. Dombret:AGIOS: Honoraria; CELGENE: Consultancy, Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Braun:Institut de Recherches Internationales Servier (IRIS): Research Funding.

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