Deletion of Murine Rhoh induces More Aggressive Diffuse Large B Cell Lymphoma (DLBCL) Via Interaction with Kaiso and Regulation of BCL-6 Expression

Abstract RHOH encodes a GTPase-deficient, hematopoietic-specific small GTPase first identified as a hypermutable gene in DLBCL (Pasqualucci et al. 2001). RhoH is critical for T cell receptor signaling and Rhoh-deficient (RhohKO) mice have T cell lymphopenia (Gu et al., 2006) and loss of function mutations of RHOH are associated with Epidermodysplasia Verruciformis (Crequer et al., 2012). However, the role of RhoH in the biology of DLBCL is still unknown and its role in B lymphoid development is incompletely studied. We investigated the role of RhoH in normal germinal center formation and in a murine model of DLBCL by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice (Cattoretti G, et al., 2005). In young RhohKOmice, deficient development of CXCR5+ follicular T helper (Tfh) cells results in defective germinal center (GC) formation and impaired immunoglobulin switching in vivo. In spite of this defect in GC formation, RhohKO; Bcl-6Tg (KOTg) mouse demonstrated accelerated lymphoma progression associated with larger spleens and significantly earlier death (Log-rank test p<0.01, Figure 1). Immunohistochemistry data suggested increased expression of IRF-4 and enhanced expression of BCL-6 in KOTg mice, findings confirmed by immunoblot and consistent with an activated B-cell (ABC)-DLBCL phenotype. To analyze the mechanism underlying these results, B cell lymphoma cell lines from KOTg lymphoma mice were established. Multiple attempts to establish RhohWT lymphoma cell lines failed, although we also successfully established a lymphoma cell line from RhohKO; Bcl-6(ntg) (KONtg) mice. Re-expression of RhoH in these lines via retrovirus mediated gene transfer led to significantly decreased proliferation (5.9x106±9.6x105 cells vs 8.6x106±9.6x105 cells after 5-days culture; KOTg vs KOTg-RhoH, mean±SEM, p<0.05) that was associated with clear reduction in BCL-6 expression. These data suggest that BCL-6 is a direct or an indirect transcriptional target of RhoH. Our laboratory previously reported that KAISO, a dual-specific, Broad complex, Trantrak, Bric-a-brac/Pox virus, Zinc finger (POZ-ZF) transcription factor interacts and colocalizes with RhoH in the nucleus, whereas knockdown of RhoH inhibits the nuclear localization of KAISO in Jurkat cells (Mino A, et al., 2016). In addition, Kaiso has been shown to be a key regulator of spleen germinal center formation by repressing Bcl-6 expression in splenocytes (Koh D, et al., 2013). We hypothesized that the deletion of Rhoh may lead to the decreased nuclear localization of KAISO and result in increased the expression of Bcl-6. We first confirmed that RhoH bound KAISO in RhoH-transduced KO lymphoma cells by co-immunoprecipitation. Further immunoblot analysis and quantitative PCR (qPCR) demonstrated decreased BCL-6 expression in lymphoma cells in which RhoH was re-expressed (KOTg-RhoH and KONtg-RhoH) compared with empty vector-transduced lymphoma cell lines. Interestingly, p53 a BCL-6 target was increased in RhoH-transduced lymphoma cell lines. These data indicate that RhoH affects BCL-6 expression in B cell lymphoma cell lines and suggest that RhoH may be involved in DLBCL development by co-regulating BCL-6 expression affecting downstream targets via interaction with KAISO. Figure. Figure. Disclosures Williams: Bluebird Bio: Research Funding.

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Anti-CD45 Mediated Cytolysis of Human T-Cell Lymphoma Cells.

Blood ◽

10.1182/blood.v104.11.4637.4637 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4637-4637

Author(s):

Gerald G. Wulf ◽

Anita Boehnke ◽

Bertram Glass ◽

Lorenz Truemper

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cytolytic Activity ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Lymphoma Cells ◽

Human T Cell

Abstract Anti-CD45 mediated cytoreduction is an effective means for T-cell depletion in rodents and humans. In man, the CD45-specific rat monoclonal antibodies YTH24 and YTH54 are IgG2b subclass, exert a predominantly complement-dependent cytolytic activity against normal T-lymphocytes, and have been safely given to patients as part of conditioning therapies for allogeneic stem cell transplantation. The efficacy of such antibodies against human lymphoma is unknown. Therefore, we evaluated the cytolytic activity of YTH24 and YTH54 by complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), as well as by direct apoptotic and antiproliferative effects, against a panel of Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL) cell lines, and against primary specimens. Significant CDC activity (>50% cytolysis) of the antibodies YTH54 and YTH24 was observed against three of five T-cell lymphoma lines, but against only one of nine B-cell lymphoma lines and none of four HD cell lines. The combination of YTH54 and YTH24 induced ADCC in all T-cell lymphoma cell lines and three primary leukemic T-cell lymphoma specimens, but were ineffective in B-cell lymphoma and HD cell lines.There were only minor effects of either antibody or the combination on lymphoma cell apoptosis or cell cycle arrest. In summary, anti-CD45 mediated CDC and ADCC via the antibodies YTH24 and YTH54 are primarily effective against lymphoma cells with T-cell phenotype, and may be an immunotherapeutic tool for the treatment of human T-cell lymphoma.

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Identification of Anti-Lymphoma Biomarkers of Response to the Anti-CD37 Antibody Drug Conjugate (ADC) IMGN529

Blood ◽

10.1182/blood.v128.22.4187.4187 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4187-4187 ◽

Cited By ~ 1

Author(s):

Eugenio Gaudio ◽

Chiara Tarantelli ◽

Alberto Arribas ◽

Luciano Cascione ◽

Ivo Kwee ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Cell Of Origin ◽

Antibody Drug Conjugate ◽

Induced Apoptosis

Abstract Background IMGN529 is an antibody drug conjugate (ADC) consisting of an anti-CD37 antibody with direct anti-tumor activity conjugated via a thioether linker to the cytotoxic maytansinoid antimicrotubule agent DM1. IMGN529 has shown pre-clinical (Deckert et al, Blood 2013) and clinical activity in lymphoma (Stathis et al, ASH 2014; NCT01534715). Here, we assessed the anti-tumor activity of IMGN529 on a large panel of B cell and T cell human lymphomas to identify potential biomarkers of response. Methods Fifty-four lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), n.=27; mantle cell lymphoma (MCL), n.=10; anaplastic large T-cell lymphoma, n.=5; marginal zone lymphomas, n=6, others, n=6] were exposed to increasing doses of IMGN529 or to the unconjugated DM1 for 72h. Cell proliferation was measured using the MTT. Apoptosis induction was defined by at least 1.5-fold increase in caspase 3/7 signal activation with respect to controls using the Promega ApoTox-Glo Triplex Assay. CD37 surface expression was assessed by cytofluorimetry. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down). Results. The IMGN529 median IC50 in the 54 cell lines was 780pM (95%C.I., 263pm-11.45nM). Activity was stronger (P<0.001) in B cell lymphoma cell lines (n= 46; median IC50=450pM; 95%C.I., 150-800pM) than in T cell lymphoma cell lines (n=8; median IC50=22.5nM; 95%C.I., 14-40nM). The median IC50 for DM1 was 30pM (C.I.95%, 20-40pM) with no differences between B and T cell lymphoma origin. IMGN529 induced apoptosis in 33/54 (61%) lymphoma cell lines. Surface CD37 expression was higher in cell lines derived from B than from T cells (P< 0.0001): IMGN529 IC50 values, but not of DM1, were negatively correlated with surface CD37 expression across all cell lines (R=-0.39; P= 0.018), but not within the individual B or T cell subgroups. Among B cell lines, DLBCL cell of origin, TP53 status or the presence of BCL2 translocation did not affect the sensitivity to IMGN529, while IC50s were higher in the presence of MYC translocation (P= 0.043). No association was seen between IMGN529-induced apoptosis or the sensitivity to DM1 with DLBCL cell of origin, TP53 status or the presence of BCL2 or MYC translocations. We then compared the baseline gene expression profiling of DLBCL cell lines that were highly sensitive to IMGN529 (IC50< 800pM; "S") versus less sensitive/resistant DLBCL cell lines (IC50>10nM, "R"), separately for germinal center B cell type (GCB) (S, n=11; R, n=8) and for activated B cell like (ABC) (S, n=4; R, n=3). In both DLBCL groups, MYC targets, genes involved in unfolded protein response, glycolysis and DNA repair were enriched in transcripts more expressed in R than S cell lines. Transcripts associated with low sensitivity included CD44, VIM, ANXA2, BCL2, ANXA2P1, HSP90B1, NFKBIZ, CDK6, BIRC5 in GCB and HSPA1B, HSP90AA1, CADM1, CD86, TUBB2A, TUBG1, NOTCH1 in ABC cell lines. HEBP1, PHB, PSME3, RNU6-15, RPL13 were more expressed in both GCB and ABC R. Genes involved in PI3K/AKT/mTOR, hypoxia, INF-gamma, TNFA signaling via NFKB and in complement were more expressed in S than in R cell lines. Genes associated with sensitivity to IMGN529 comprised: CD37 (IMGN529 target), CD79A, CHI3L2, FAM117B, LPAR5, NFATC1, PTPN22, RBM38, SGPP1, SLC6A16 in both GCB and ABC cell lines; BASP1, CXCR5, BIK, LY86, TLR10, CD86, LCK, CD22, PTPN22, BCL6, PIK3IP1, CDKN2A in GCB; AFF3, PIM1, MGMT, PDE4B, NFKBIE, SYK, FOXO1in ABC. Conclusions. IMGN529 showed a very strong anti-tumoral activity in pre-clinical lymphoma models. High expression of CD37 and mostly genes involved in BCR signalling were associated with sensitivity to IMGN529. Conversely, the presence of MYC translocation, a high expression of MYC targets and of genes known to be involved in drug resistance (BCL2, BIRC5, CDK6, heat-shock proteins, annexins, proteasome and tubulin components) appeared to negatively affect the response to the ADC but also represent therapeutic targets for novel combinations to be explored. Disclosures Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Sloss:Immunogen Inc: Employment.

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A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma

Blood ◽

10.1182/blood-2021-151245 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 711-711

Author(s):

Anagh Anant Sahasrabuddhe ◽

Xiaofei Chen ◽

Kaiyu Ma ◽

Rui Wu ◽

Richa Kapoor ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Germinal Center ◽

De Novo ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

In Vivo Studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

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B Cell Lymphomas Are Resistant towards Bone Morphogenetic Protein (BMP) Induced Growth Inhibition.

Blood ◽

10.1182/blood.v108.11.2381.2381 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2381-2381

Author(s):

Kanutte Huse ◽

Marianne B. Eide ◽

Christian Kersten ◽

Erlend B. Smeland ◽

June H. Myklebust

Keyword(s):

Growth Inhibition ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Lymphoma Cells ◽

Bmp Receptors ◽

Induced Apoptosis

Abstract Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily, and mediate their effects mainly through the Smad signalling pathway. Whereas TGF-β is well established as one of the most potent negative regulators in hematopoietic cells, the role of BMPs remains more elusive. We have previously shown that BMP-6 inhibits the growth of naïve and memory human B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that resistance towards BMP-induced growth inhibition is a possible mechanism for lymphomagenesis. In the current study, 7 B cell lymphoma cell lines (representing Burkitt lymphoma (BL) and DLBCL) and tumour material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We analyzed the expression of BMP receptors by FACS analysis, and found variable expression of the BMP receptor type I (Alk2, Alk3 and Alk6) and type II (BMP RII, Activin RIIA and RIIB) among the cell lines and in primary lymphoma cells, suggesting variable binding of BMPs. We next investigated the effect of BMP-2, BMP-4, BMP-6 and BMP-7 on proliferation and survival of B lymphoma cell lines, and found 2 of 7 cell lines to be resistant towards BMP-2 and BMP-4 induced growth inhibition. In contrast, 4 of 7 and 7 of 7 cell lines were resistant to BMP-6 and BMP-7 induced growth inhibition, respectively. In Sudhl6 cells that were highly sensitive to BMP-2 and BMP-6 induced apoptosis and inhibition of proliferation, we demonstrated that the cytokines IL-10, CD40 Ligand and BLyS were able to counteract the negative effects induced by BMPs, while IL-2 and IL-4 were not. On the contrary, both BMP-2 and BMP-6 greatly increased anti-IgM activation induced apoptosis. In resistant lymphoma cells, the BMPs were not able to induce detectable levels or induced low levels of phosphorylated SMAD1/5/8 compared to sensitive cell lines. Low or no increase in phosphorylation of SMAD1/5/8 induced by BMPs could only partly be explained by low/ undetectable expression of BMP receptors. Hence, upregulation of inhibitory Smads (Smad6, Smad7) or mutations in receptors or Smads represent other possible mechanisms for resistance to BMPs in lymphomas, and this is currently under investigation. We also investigated if the lymphoma cells produced BMPs themselves and found that 5 of 7 cell lines and 3 of 5 primary lymphomas produced significant amounts of BMP-7. Some lymphoma cells also had detectable levels of BMP-4 and BMP-6. Our findings that lymphoma cells are resistant towards BMP-7 and to some degree BMP-6 induced growth inhibition, whereas they produce these cytokines, suggest that resistance towards BMP induced signalling in B cell lymphomas can contribute to increased tumour growth.

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Combination of Enzastaurin and HDAC Inhibitors Synergically Induce Apoptosis in Lymphoma Cells.

Blood ◽

10.1182/blood.v114.22.4783.4783 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4783-4783

Author(s):

Juraj Bodo ◽

Jan Sedlak ◽

Jaroslaw P. Maciejewski ◽

Eric D. Hsi

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Parp Cleavage ◽

Primary Lymphoma ◽

Low Concentrations

Abstract Abstract 4783 Introduction Histone deacetylase inhibitors (HDACis) are approved for use in the setting of cutaneous T-cell lymphoma with modest benefit. Enzastaurin is an investigational PKCβ inhibitor that has growth inhibitory and pro-apoptotic effects in both B and T-cell lymphoma. Specifically, enzastaurin-induced inhibition of PKC leads to rapid accumulation of β-catenin that triggers c-Jun dependent induction of p73, followed by apoptosis. We investigated the cytotoxicity and mechanisms of cell death of combination enzastaurin and low concentrations of HDACis in B-cell lymphoma and T-cell lymphoma cell lines and primary lymphoma/leukemia cells. Experimental design Apoptosis was measured by flow cytometry and PARP cleavage. Phospho-GSK3β (S9), pS6, phospho-c-jun (S73) and β-catenin were analyzed by Western blot or quantum-dot immunoflourescence as measures of PKCβ inhibition. Cytotoxicity was determined by WST-1 proliferation assay and colony forming cell (CFC) assays. Results As expected, enzastaurin induced dephosphorylation of GSK3β and S6RP associated with increased β-catenin expression followed by phosphorylation of c-jun (S73) and PARP cleavage in SU-DHL-6 (diffuse large B-cell lymphoma line) cells. Treatment with low concentrations of suberoylanilide hydroxamic acid (SAHA) showed slight or no changes in studied proteins. Combined enzastaurin/SAHA treatment resulted in strong synergistic apoptosis in two treated germinal center B-cell-like and two activated B-cell-like lymphoma cell lines, two T-cell lymphoma cell lines and four different primary lymphoma/leukemia samples. Similarly, combined enzastaurin/ valproic acid treatment induced synergistic apoptosis in SU-DHL-6 cell line, suggesting the synergy is generalizable to other HDACis. In comparison to the single agent treatment, combined enzastaurin/ SAHA treatment resulted in activation of proapoptotic MAPK, c-jun N-terminal kinase, further increase of phospho c-jun (S73) levels, increased FasL levels, and amplification of PARP cleavage. Quantitative immunofluorescence assay showed a more rapid increase of β-catenin levels with the combination than either agent alone. Furthermore, compared to the low dose SAHA treatment alone, hyperacetylation of histone H3 was detected in samples when enzastaurin was added in combination with low dose SAHA, likely the consequence of displacement of HDAC by β-catenin. In addition, no change in CFC output in normal bone marrow exposed to this combination was observed. Conclusion Enzastaurin/ HDACi therapy can synergistically inhibit growth and induce apoptosis in lymphoid malignancy through increased biochemical effects attributed to each agent. These data support further investigation of addition of PKCβ inhibitors to HDACi in order to increase their anti-lymphoma effects. Disclosures: No relevant conflicts of interest to declare.

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Targeting MYC-Driven B-Cell Lymphoma By Inhibition of the Histone Methyltransferase DOT1L

Blood ◽

10.1182/blood-2018-99-115475 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2839-2839 ◽

Cited By ~ 2

Author(s):

Anagha Deshpande ◽

Benson Chen ◽

Parham Ramezani-Rad ◽

Alessandro Pastore ◽

Luyi Zhao ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Cell Lines ◽

Target Genes ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Advisory Committees ◽

Genome Wide

Abstract Aberrant activation of the MYC proto-oncogene is a recurrent feature in human B-cell lymphomas of diverse sub-types, correlating with adverse prognosis and therapy resistance. Direct pharmacological MYC-targeting has proved difficult, but recent studies have shown that targeting chromatin regulators critical for MYC-driven oncogenesis may provide alternative avenues for therapeutic intervention. Recently, it has been demonstrated that MYC-driven oncogenesis in certain solid tumors is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L. We hypothesized that B-cell lymphomas with hyperactive MYC-signaling might be responsive to DOT1L inhibition. In order to test this hypothesis, we tested the effect of the DOT1L inhibitor Pinometostat (EPZ-5676) on a panel of human B-cell lymphoma cell lines featuring elevated MYC. Pinometostat treatment reduced global H3K79 methylation levels, accompanied by a time and dose-dependent decrease in proliferation of several Burkitt's lymphoma cell lines including P493-6, Daudi and Raji. We observed that key MYC-target genes including CDK4, PPAT and NPM1 were significantly downregulated upon Pinometostat treatment, suggesting that DOT1L is required for the transcriptional activation of MYC-target genes in these cells. Pinometostat-treated B-lymphoma cells showed a significant decrease of cells in S-phase compared to controls as assessed by BrdU-labeling assays. Similar results were also obtained in a panel of B-cell lymphoma cell lines with MYC-rearrangements including mantle cell lymphoma (MCL) cell lines Jeko-1, JVM2, Mino-1 and Maver-1 and the diffused large B-cell lymphoma (DLBCL) cell line Karpas 422. Next, we sought to investigate whether the DOT1L-dependence of MYC-driven B-cell lymphoma could be reproduced in a well-defined model of MYC-driven B-cell lymphoma. Towards this end, we utilized a mouse model in which expression of the Cre recombinase from a B cell specific promoter leads to ectopic expression of a transgenic human MYC allele and concomitant deletion of the tumor suppressor Pten in B cells. Similar to our in vitro studies, Pinometostat treatment led to a significant reduction in proliferation of B-cell lymphoma cells from these mice with an IC50 of 0.5 µM. Furthermore, we sought to ascertain whether these findings reflected on-target effects related to DOT1L inhibition. Therefore, we deleted DOT1L using CRISPR/Cas9 in B-cell lymphoma cell lines and assessed the effect on proliferation using competitive-proliferation assays. We observed that DOT1L-deletion progressively diminished the relative growth of anti-DOT1L sgRNA-expressing P493-6 and Jeko1 cells compared to non-targeted cells invitro. In order to test the requirement for DOT1L in lymphoma propagation in vivo, we performed intravenous injections of equal number of Jeko-1 cells with either anti-DOT1L or anti-Renilla control sgRNAs into sub-lethally irradiated non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID) mice. Mice injected with control anti-Renilla sgRNAs succumbed to disease with a median latency of 34 days while the latency of disease in the anti-DOT1L sgRNA cohort was 45 days. In summary, DOT1L depletion significantly delayed disease latency in this invivo disseminated model of B-cell lymphoma (P=0.02). We then performed transcriptomic analyses of Pinometostat-treated B-cell lymphoma cell lines compared to DMSO-treated counterparts using RNA-seq. Gene-set enrichment analysis (GSEA) of RNA-seq data from three different B-cell lymphoma cell lines demonstrated that Pinometostat treatment significantly decreased the expression of MYC-target genes. In order to investigate the intriguing role of DOT1L in regulating MYC-target gene expression, we used ChIP-seq to assess the genome-wide occupancy of MYC following DOT1L inhibitor treatment. Strikingly, our studies demonstrated that DOT1L inhibition significantly reduced the chromatin occupancy of MYC. Taken together, our experiments demonstrate the role of DOT1L in MYC-driven B-cell lymphoma pathogenesis invitro and invivo. Furthermore, our genome-wide studies demonstrate the importance of DOT1L for genomic MYC occupancy. Based on these findings, we propose that therapeutic DOT1L targeting may be a viable strategy in MYC-driven B-cell lymphoma. Disclosures Weigert: Roche: Research Funding; Novartis: Research Funding. Rickert:Pfizer: Employment. Ren:Elli Lilly: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

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Schedule-Dependent Synergy Between Belinostat and Pralatrexate in T-and B-Cell Lymphoma Cells in Vitro

Blood ◽

10.1182/blood.v126.23.5128.5128 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5128-5128

Author(s):

Godefridus Peters ◽

FHA van Gemert ◽

I Kathmann ◽

Saskia AGM Cillessen ◽

G Jansen ◽

...

Keyword(s):

Folic Acid ◽

T Cell ◽

B Cell ◽

Cell Lines ◽

Drug Exposure ◽

Cell Lymphoma ◽

Fixed Ratio ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Substrate Affinity

Abstract Pralatrexate (Folotyn; FOL) and belinostat (Beleodaq; BEL) are two new agents that have recently been registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and have also shown promising activity in other types of lymphoma. FOL is a folate analogue and a potent inhibitor of dihydrofolate reductase (DHFR), designed to accumulate in cancer cells preferentially via the reduced folate carrier (RFC) and retained therein via efficient polyglutamylation. Inhibition of DHFR leads to an imbalance of deoxynucleotides (e.g. depletion of dTTP and an increase in dUTP) resulting in DNA strand breaks and inhibition of DNA synthesis. BEL is a hydroxamic acid-based pan-histone deacetylase (HDAC) inhibitor that broadly inhibits all of the zinc-dependent HDAC enzymes, with high affinity for the Class I, II and IV isozymes. HDAC inhibition results in an alteration in the degree of histone and non-histone protein acetylation, which in turn affects transcription of genes essential in cellular proliferation, cell cycle and DNA repair. We investigated whether folate transporters other than RFC, i.e. folate receptor α (FRα) and the proton-coupled folate transporter (PCFT) could contribute to the efficacy of FOL. Moreover, we explored whether the toxicity of FOL can be controlled by levo-leucovorin (Fusilev), the natural stereoisomer of leucovorin, and whether in combination experiments BEL had the ability to potentiate the cytotoxicity of FOL. A panel of lymphoma cell lines was used for the combination studies including: the B-cell lymphoma cell lines SU-DHL-4, SU-DHL-5, HT, Jeko-1 and T-cell lymphoma cell lines Karpas-299 and Hut-78. RFC-mediated uptake efficiency of FOL was determined in competition uptake experiments with [3H] Methotrexate (MTX), revealing a 6-fold better RFC substrate affinity for FOL, and 2-fold better than levo-leucovorin. FOL displayed very poor substrate binding affinity for FRα (>100-fold lower than folic acid and > 10 lower than levo-leucovorin). FOL had a low substrate affinity for PCFT (>10-fold lower than folic acid and levo-leucovorin in [3H] leucovorin uptake competition experiments). Levo-leucovorin could completely protect toxicity by FOL, but had no effect on BEL toxicity. Sensitivity of lymphoma cell lines (IC50 concentrations after 72 hrs drug exposure) to FOL drug varied from 2.8 nM (Hut-78), 5.5 nM (SU-DHL4 and 5), 7.4 nM (HT) to 20 nM (Karpas-299 and Jeko-1) while IC50 values for BEL were in the range of 100 nM (SU-DLH-4 and 5, Jeko-1 and Hut-78) to 200 nM (Karpas-299 and HT). The interaction between BEL and FOL was studied using the median-drug effect analysis with Calcusyn software. At a fixed ratio between the drugs based on the IC50 concentration the average combination index (CI) for all the lymphoma cell lines revealed an additive effect (CI: all around 1.0). In two selected cell lines (SU-DHL-4 and HT) sequential exposure to the drugs (24 hr pretreatment with either BEL or FOL) followed by 48 hr to the combination, did not improve the results with CI values varying between 0.9 and 1.4. As an alternative approach a non-fixed ratio was used by exposing SU-DHL-4 and HT cells to IC25 concentrations of either BEL or FOL in combination with the other drug. Exposure to IC25 concentrations of FOL did not decrease the IC50 for BEL (CI around 1.2), but exposure to IC25 concentrations of BEL markedly increased the sensitivity to FOL as reflected by the low CIs varying from 0.40 to 0.66. Mechanistic studies focused on induction of apoptosis, showed cleavage of caspase 8 and 9 in HT and SU-DHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest. In conclusion, the combination of FOL and BEL showed additive activity in various lymphoma cell lines, while a schedule-dependent synergism was observed. Based on these data, proficient inhibition of HDAC activity by BEL holds promise in sensitization of tumor cells to FOL. Furthermore, toxicity of FOL could be completely protected by levo-leucovorin. Disclosures Reddy: spectrum: Employment, Equity Ownership.

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