The Dual PI3K/mTOR Inhibitor PQR309 Has Synergistic Activity with Other Targeted Agents in Diffuse Large B Cell Lymphomas

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4005-4005 ◽  
Author(s):  
Chiara Tarantelli ◽  
Eugenio Gaudio ◽  
Ivo Kwee ◽  
Luciano Cascione ◽  
Elena Bernasconi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Mtor Inhibitor ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Additive Effect ◽  
Targeted Agents ◽  
Adaptive Resistance ◽  
Large B Cell

Abstract Introduction. PQR309 is a novel oral dual PI3K/mTOR inhibitor (Cmiljanovic et al, AACR 2015), being now evaluated as single agent in a phase I study in patients with relapsed or refractory lymphomas (NCT02249429). We previously reported activity of PQR309 as single agent in a large panel of lymphoma cell lines, and early combination data in two diffuse large B-cell lymphoma (DLBCL) cell lines (Tarantelli et al, ASH 2014). Here, we present extended combination data that might enhance the anti-proliferative effect of the single drug. Methods. A panel of DLBCL cell lines derived from activated B-cell like (ABC) or germinal center B (GCB) subtype, with or without BCL2 or MYC deregulation, were used: RIVA (ABC, BCL2 amplification), SU-DHL-2 (ABC), TMD8 (ABC) and U2932 (ABC, BCL2 amplification); DOHH2 (GCB, BCL2 and MYC translocations), SU-DHL-6 (GCB, BCL2 translocation), KARPAS422 (GCB, BCL2 translocation), OCI-LY18 (GCB, BCL2 and MYC translocations), SU-DHL-10 (GCB), OCI-LY1 (GCB, BCL2 translocation). Cell lines were exposed to increasing doses of PQR309 alone or in combination with increasing doses of other agents for 72h and synergism was assessed with the Chou-Talalay combination index (CI): <0.3, very strong synergism 0.3-0.9, synergism; 0.9-1.1 additive effect; >1.1, no benefit. Results. The combination of PQR309 with the BCL2 inhibitor venetoclax (ABT199) gave positive results in 6/8 cell lines, with strong synergism in three (U2932, median CI=0.1, SU-DHL-6, 0.14, Karpas422, 0.14) and synergism in the remaining three (TMD8, 0.5, RIVA, 0.56, DOHH2, 0.65). No benefit was observed in SU-DHL-2 and OCI-LY18. The combination of PQR309 with the BTK inhibitor ibrutinib was synergistic in 3/4 ABC-DLBCL (RIVA, 0.42; U2932, 0.6; TMD8, 0.57), while it was of no benefit in SU-DHL-2. The combination of PQR309 with the immunomodulator lenalidomide was of benefit in 4/4 ABC-DLBCL: synergistic in three (RIVA, 0.38; U2932, 0.4; TMD8, 0.5) and additive effect SU-DHL-2 (1.01). The combination of PQR309 with the HDAC-inhibitor Panobinostat was synergistic in 5/6 (KARPAS422, 0.21; U2932, 0.65; SU-DHL-6, 0.67; DOHH2, 0.8; SU-DHL-2, 0.83), but not in the TMD8 (1.14). In our models, synergism was observed in only 2/5 cell lines (TMD8, 0.6; DOHH2, 0.89) exposed to both PQR309 and anti-CD20 monoclonal antibody, while not in KARPAS422, SU-DHL-6, or U2932. Finally, since we had previously observed an up-regulation of the PIM1 kinase after exposure to PQR309 (Tarantelli et al, ICML 2015), potentially acting as a mechanism of adaptive resistance, we evaluated the addition of the PIM inhibitor AZD1208 to PQR309 in four DLBCL cell lines. The combination showed synergism in two (OCI-LY1, 0.29; SU-DHL-10, 0.57), an additive effect in TMD8 (0.95) and no benefit in the SU-DHL-2 (1.15). Conclusions. The novel dual PI3K/mTOR inhibitor PQR309 showed synergism when combined with additional targeted agents in different DLBCL models, including some derived from double hit lymphomas. In particular, the combination with the BCL2 inhibitor venetoclax showed very good results, especially in cell lines bearing BCL2 gene deregulation due to chromosomal translocation or genomic amplification, and the combination with a PIM inhibitor might overcome early adaptive resistance to PQR309. These data provide the basis for future pre-clinical and clinical studies. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Stathis:PIQUR Therapeutics AG: Research Funding. Fabbro:PIQUR Therapeutics AG: Employment. Wicki:PIQUR Therapeutics AG: Employment, Research Funding. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:PIQUR Therapeutics AG: Research Funding; Oncology Therapeutic Development: Research Funding.

scholarly journals Targeting NF-κB with First in Class N-Quinoline Benzenesulfonamides (NQBS) Reveals Potent Activity in Models of Diffuse Large B-Cell Lymphoma (DLBCL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4435-4435
Author(s):  
Matko Kalac ◽  
Michael Mangone ◽  
Alison Rinderspacher ◽  
Shi-Xian Deng ◽  
Luigi Scotto ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
B Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Research Funding ◽  
Nuclear Translocation ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract The first two authors contributed equally to this work Identifying pharmacologic strategies to inhibit the activation of NF-κB and its target genes has been a major research pursuit. To date, no direct inhibitors of the NF-κB subunits have been explored in the clinic. Based on the constitutive activation of NF-κB in diffuse large B-cell lymphoma (DLBCL), we used this disease model to develop drugs targeting NF-κB. Using a fluorescence-based high throughput screening (HTC) approach, a unique N-quinoline-benzenesulfonamide (NQBS) scaffold was identified as potential small molecule inhibitor of the NF-κB pathway. A confocal microscopy based HTC assay performed in human umbilical vein endothelial cells (HUVEC) identified hit compounds that contained a unique NQBS core structure. The assay screened for compounds that inhibited nuclear translocation of NF-κB subunits in TNFα-induced HUVEC cells. To date over 100 NQBS analogs have been synthesized with varying potency and cytotoxicity in inhibiting growth of DLBCL lines (OCI-Ly10, RIVA, HBL-1 and OCI-Ly3). Cytotoxicity assays demonstrated that the most potent compounds exhibit IC50s in the 0.5 to 1.5 µM range. These most potent NQBS analogs identified as CU-O42 CU-O47 and CU-O75 were also able to induce apoptosis and caspase activation. Apoptosis was preceded by exclusion of the NF-κB proteins from the nucleus. To analyze the localization of NF-κB proteins within the cell compartments before and after the treatment with CU-O42, CU-O47 and CU-O75, we used confocal microscopy, electromobility shift (EMSA) and ELISA assays. Control cells tested positive for p50/p65 both within the cytoplasm and the nucleus. Following treatment with CU-O42 NF-κB was sequestered within the cytoplasm of the cell which occurred as early as 3h after exposure. In addition, all three analogs reduced the nuclear levels of NF-κB in a concentration-dependent manner when measured by EMSA and ELISA. Furthermore, CU-O47 and CU-O75 were able to inhibit TNFα induced luciferase expression in a HEK293T cell model where luciferase is controlled by an NF-κB promoter. A KINOMEscan platform (examining the activity of over 450 different kinases) showed that no NQBS analog screened (CU-O42 and CU-O75) inhibited any of the kinases in the assay. In addition, a proteasome inhibition assay tested negative for trypsin-like and chromotrypsin-like protease activity (CU-O42, CU-O47 and CU-O75). Stabilization of the inactive trimer of p50, p65 and IκBα was hypothesized as a potential mechanism of action of CU-O42 and CU-O75 through Internal Coordinate Mechanics (ICM) software. This binding hypothesis was further corroborated by cellular thermal shift assays (CETSA) with an increase of the IκBα melting temperatures (2.5-3°C) in whole cell lysates following rapid (30min) exposure to CU-O42 and CU-O75. Using a genome-wide regulatory network perturbation analysis (DeMAND) based on the RNA-Seq data collected from OCI-Ly10 cells treated with CU-O75, we identified IκBα as one of the potential targets of the compounds. Gene set enrichment analysis demonstrated NF-κB target gene downregulation using IC20 of CU-O75 at 24h (p=0.045). In vivo experiments were conducted in two models: (1) xenografts with human DLBCL cell lines of both ABC and GC subtype; and (2) myc cherry luciferase mouse model where mice spontaneously develop aggressive lymphomas. In both models, CU-O42 was able to inhibit tumor growth. Interestingly, in the xenograft model, malignant cell growth was inhibited in both ABC (HBL-1) and GC (OCI-Ly1) cells when compared to controls (p=0.01 and p=0.02). However, overall survival of mice with ABC xenografts treated with CU-042 significantly exceeded the survival of mice with GC xenografts (p<0.01) suggesting a more sustainable response in this subtype of disease, consistent with its dependency on NF-κB. Identification of a unique NQBS scaffold has led to the chemical synthesis of over 100 structural analogs with a potent inhibition on NF-κB nuclear translocation. They display potent activity across a panel of lymphoma cell lines, producing a survival benefit in mice implanted with an ABC-subtype of lymphoma. ICM, CETSA and DeMAND suggest that this is a direct effect mediated on the proteins within the p65/p50/IκBα complex. These findings point to a novel mechanism of action and warrant further research into potential clinical translation of this class of small molecules. Disclosures Califano: Thermo Fischer Scientific: Consultancy; Ipsen pharmaceuticals: Consultancy; Cancer Genetics Inc: Consultancy; Therasis Inc: Employment. O'Connor:Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy; Bayer: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria, Research Funding; Acetylon Pharmaceuticals, INC: Consultancy.

scholarly journals Statins Potentiate the Cytotoxic Effect of ABT-199 in Diffuse Large B Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3969-3969
Author(s):  
David A. Fruman ◽  
Jong-Hoon Scott Lee ◽  
Thanh-Trang T Vo ◽  
Shruti Bhatt ◽  
Jonathan H. Schatz ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cells ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract BCL-2 is a key pro-survival protein that is highly expressed in many leukemias and lymphomas. ABT-199 (venetoclax) is a small molecule inhibitor of BCL-2 that has demonstrated impressive responses in chronic lymphocytic leukemia (CLL) leading to FDA approval for second line treatment of patients with 17p deletion. However, other hematologic malignancies are less responsive to ABT-199 as a single agent, suggesting that combinations of targeted therapies may be required to elicit more promising responses. We have investigated the potential of combining ABT-199 with HMG-CoA reductase (HMGCR) inhibitors (statins), which have known anti-cancer potential in hematologic malignancies. Using multiple chemically distinct statin compounds, we observed profound synergistic induction of apoptosis when combined with ABT-199 in both human diffuse large B cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML) cell lines. This synergy was also seen in primary murine B lymphoma cells over-expressing MYC and BCL-2. Importantly, addition of exogenous mevalonate completely rescued cells from the combination, confirming on-target efficacy of HMGCR inhibition. Using BH3 profiling, we found that simvastatin significantly primed lymphoma cells for undergoing apoptosis (termed mitochondrial priming). Notably, the degree of priming correlated with its ability to synergize with ABT-199, suggesting that BH3 profiling may be used to predict patient responses. The combination did not synergize to kill normal human peripheral blood mononuclear cells from healthy donors, suggesting that statins may selectively prime cancer cells for apoptosis. Mechanistic studies support the hypothesis that statins synergize with ABT-199 by suppressing protein prenylation, particularly protein geranylgeranylation. In support, the addition of exogenous geranylgeranyl pyrophosphate (GGPP) completely rescued cells from the effects of simvastatin. Furthermore, selective inhibition of protein geranylgeranyl transferase (GGT) increased priming and was sufficient to recapitulate the effects of simvastatin in combination with ABT-199. Statins and GGT inhibitors increased the mitochondrial abundance of a subset of BH3-only pro-apoptotic proteins. Lastly, we have identified Rap1A de-prenylation as a marker of pharmacodynamic response to statins in vivo. Thus, this project highlights a novel combination for use in aggressive lymphomas, establishes its efficacy and tolerability using preclinical models, and provides proof-of-concept to warrant investigation of its clinical potential. Disclosures Letai: AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding.

scholarly journals Voruciclib Sensitizes High Risk Diffuse Large B Cell Lymphoma to BCL2 Inhibition Mediated Cell Death and Tumor Regression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4167-4167
Author(s):  
Joyoti Dey ◽  
William Kerwin ◽  
Joseph Casalini ◽  
Angela Merrell ◽  
Marc Grenley ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Treatment Options ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Diffuse Large B Cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma in adults. Although upfront chemotherapy leads to favorable survival outcomes, relapsed or refractory patients continue to have poor prognosis with limited treatment options. In DLBCL, evasion of apoptosis - a key hallmark of cancer is mediated by functionally redundant BCL family members: BCL2, BCLxL and MCL-1. The BCL2 specific inhibitor venetoclax is approved for treating high-risk CLL, but responses in DLBCL have been limited, potentially due to compensatory upregulation of MCL-1. Currently a well-tolerated drug for inhibition of MCL-1, is unavailable in the lymphoma clinic. Voruciclib, is a novel clinical stage oral CDK inhibitor with potent activity (<10 nM) against CDKs 9, 4, 6 and 1. Multiple mechanisms for downregulation of MCL-1 activity have been described for CDK inhibitors. Arguably best characterized is transcriptional inhibition of MCL-1, a short half-life transcript, via inhibition of transcriptional regulator CDK9. We evaluated MCL-1 expression in the FFPE lymphatic tissues from 33 patients with DLBCL, and found that it was expressed in 52% of cases, of both GC (germinal center) and ABC (activated B-cell)-like type. We therefore investigated whether voruciclib could synergize with venetoclax in pre-clinical models of DLBCL via inhibition of MCL-1. In cell-based assays, exposure of DLBCL cells to voruciclib as a single agent resulted in apoptosis which was preceded by context-dependent downregulation of MCL-1. To further explore the impact of voruciclib on MCL-1 activity and DLBCL viability in vivo, we utilized Presage's CIVO tumor microinjection technology. CIVO enables investigation of multiple drugs and drug combinations simultaneously in a living tumor facilitating in vivo assessment of anti-tumor drug synergy (Klinghoffer et al. Sci. Transl Med. 2015; Dey et al. PLOS One 2016). Voruciclib was introduced as a single agent or in combination with venetoclax to DLBCL xenografts. Microinjection, resulting in localized tumor exposure to voruciclib, led to MCL-1 downregulation in vivo across multiple models of DLBCL. In contrast, tumor exposure to venetoclax led to MCL-1 upregulation. Co-exposure to voruciclib and venetoclax demonstrated that the ability of voruciclib to downregulate MCL-1 is dominant to the upregulation by venetoclax. Consistent with the hypothesis that MCL-1 compensates for loss of BCL2 function in DLBCL, synergistic cell death was observed when voruciclib was combined with venetoclax. Synergy between voruciclib and venetoclax was observed in vivo in models representing both ABC (RIVA: CI value 0.5) and GC subtypes (NUDHL1 and Toledo: CI values 0.4). Similar activity was noted when venetoclax was combined with A1210477, an investigational MCL-1 inhibitor thereby suggesting MCL-1 downregulation to play a role in the observed synergy between venetoclax and voruciclib. Consistent with these results, preliminary studies on xenografted mice have shown that systemic administration of a sub-efficacious dose of venetoclax in combination with voruciclib led to impediment of tumor growth which was greater than the effect observed with each single agent. Additional systemic studies are ongoing with venetoclax in combination with voruciclib in a panel of DLBCL models to further strengthen this observation. Based on the above findings, a Phase 1b clinical trial has been designed to evaluate the combination of voruciclib and venetoclax in patients with the goal of expediting future treatment options for relapsed/refractory DLBCL. We expect to initiate this trial at multiple centers in early 2017. Disclosures Dey: Presage Biosciences: Employment. Kerwin:Presage Biosciences: Employment. Casalini:Presage Biosciences: Employment. Merrell:Presage Biosciences: Employment. Grenley:Presage Biosciences: Employment. Ditzler:Presage Biosciences: Employment. Dixon:Presage Biosciences: Employment. Burns:Presage Biosciences: Employment. Danilov:ImmunoGen: Consultancy; GIlead Sciences: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Consultancy; Takeda: Research Funding; Dava Oncology: Honoraria; Prime Oncology: Honoraria. Klinghoffer:Presage Biosciences: Employment.

scholarly journals The Novel PI3K/mTOR Dual Inhibitor PQR309 in Pre-Clinical Lymphoma Models: Demonstration of Anti-Tumor Activity As Single Agent and in Combination and Identification of Gene Expression Signatures Associated with Response

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1782-1782
Author(s):  
Chiara Tarantelli ◽  
Eugenio Gaudio ◽  
Ivo Kwee ◽  
Andrea Rinaldi ◽  
Matteo Stifanelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phase I ◽  
B Cell ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Dual Inhibitor

Abstract Dual PI3K/mTOR inhibitors represent a promising class of anti/cancer compounds, of potential interest in lymphoid neoplasms which present activation of both targeted pathways. PQR309 is a novel, oral, member of this class of compounds and, as single agent, is currently being evaluated in a phase I for patients with solid tumors (NCT01940133). Here, we present the activity of the compound in pre-clinical models of mature lymphoid tumors, also integrating response data with genomic features. Methods. 48 cell lines [27 derived from diffuse large B-cell lymphoma (DLBCL), 10 from mantle cell lymphoma (MCL), 3 from splenic marginal zone lymphoma (SMZL), 8 from anaplastic large cell lymphoma (ALCL)] were treated with increasing doses of PQR309 and MTT assays were performed after 72 hrs exposure. A second dual PI3K/mTOR inhibitor, GDC0980, and the PI3Kdelta inhibitor Idelalisib were also used on all the cell lines. IC50, GI50, LC50, and TGI values were used to estimate the cytotoxic and cytostatic effects. PQR309-induced cytotoxic activity was tested by AnnexinV assay. Synergy was assessed by the Chou-Talalay combination index (CI) on 2 DLBCL cell lines (TMD8, U2932) exposed to increasing doses of PQR309 alone or in combination with increasing doses of other drugs for 72 hrs. Baseline gene expression profiling (GEP) was obtained on the cell lines with the Illumina HumanHT-12 Expression BeadChips and integrated with the anti-proliferative effect. Results. PQR309 showed potent anti-proliferative activity in most of the cell lines tested. The median IC50 was 242 nM (18nM-3.6 mcM), GI50 141 nM (25 nM-1.7 mcM), LC50 2.7 mcM (306 nM->10 mcM), TGI 711 nM (69 nM - >10 mcM). DLBCL (median IC50=166 nM), MCL (234 nM) and SMZL (214 nM) were all more sensitive than ALCL (664 nM) (P=0.005). Activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL subtypes were equally sensitive. Across the 48 cell lines, PQR309 and GDC0980 presented a highly correlated pattern of anti-proliferative activity (R=0.95). Idelalisib appeared significantly less active and its pattern of sensitive cell lines was less correlated with PQR309 (R=0.67) or GDC0980 (R=0.71). In DLBCL cell lines, PQR309 (1 mcM) was able to inhibit IgM-stimulation induced p-AKT(Ser 473) in 2/2 cells and the baseline p-AKT(Ser 473) levels in 1/1. PQR309 (500 nM, 72 hrs) caused apoptosis in 1/7 cell lines. Synergism or additive effected were observed in 2/2 cells combining PQR309 with the BCL2 inhibitor ABT199 (CI = 0.1 and 0.5), the immunomodulatory drug lenalidomide (0.5 and 0.4), the BTK-inhibitor ibrutinib (0.6 and 0.57) or the proteasome inhibitor bortezomib (0.9 and 0.9), and in 1/2 with the anti-CD20 monoclonal antibody rituximab (0.6), the BET inhibitor JQ1 (0.7) and the chemotherapy agent bendamustine (0.7). We then looked for baseline GEP features associated with sensitivity to PQR309, by comparing very sensitive (IC50 < 200 nM) versus less sensitive DLBCL cell lines (IC50 > 400 nM). Transcripts more expressed in sensitive cells were significantly enriched of genes involved in B-cell receptor pathway/signaling, kinases regulation, immune system. Transcripts associated with less sensitive cells were enriched of members of proteasome pathway, oxidative phosphorylation, translation initiation. Genes coding for individual proteins involved in PI3K signaling cascade were differentially expressed between the two groups of cells. Conclusions. PQR309 showed promising activity as single agent and in combination providing the basis for phase I/II studied dedicated for lymphoma patients. Baseline features associated with response were identified and are worth of being validated in the context of the next clinical trials. (CT and EG contributed equally to this work) Disclosures Hillmann: Piqur Therapeutics AG: Employment. Fabbro:Piqur Therapeutics AG: Employment. Hebeisen:Piqur Therapeutics AG: Employment. Betts:Piqur Therapeutics AG: Consultancy. Wicki:Piqur Therapeutics AG: Research Funding; University Hospital Basel: Employment. Cmiljanovic:Piqur Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:Piqur Therapeutics AG: Research Funding.

The Small Molecule CHK1/CHK2 Inhibitor PF-0477736 (Pfizer) Demonstrates Single Agent Activity and Synergizes with Chemotherapy in Diffuse Large B-Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2732-2732
Author(s):  
Enrico Derenzini ◽  
Ilaria Iacobucci ◽  
Elisa Brighenti ◽  
Federica Cattina ◽  
Richard Eric Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Cycle Arrest ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 2732 The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the signal transduction mechanims of the DNA damage response pathway. Once activated by upstream kinases [Ataxia-Telangiectasia mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases] following DNA damage, they phosphorylate downstream targets such as CDC25 phosphatases and p53, promoting G2/M cell cycle arrest, in order to facilitate DNA repair. Furthermore is now clear that the efficacy of conventional DNA-damaging anticancer drugs is limited by the activity of these protective cell cycle checkpoints. The tumor suppressor p53 is activated in normal cells following extensive DNA damage and promotes G1 cell cycle arrest and apoptosis. Cells lacking p53 activity are more resistant to genotoxic agents. It has been shown that CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, by inhibiting the response to DNA damage, preferentially in p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL harboring p53 mutations and/or CDKN2A loss have been recently shown to have a dismal outcome, being refractory to conventional antracyclin-based chemotherapy. Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a large panel of B cell lymphoma cell lines, and explore its mechanisms of action. Nine cell lines were used for in vitro viability assays: 3 Germinal center (GCB) Diffuse Large B-cell lymphoma (DLBCL) derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2. All the cell lines were screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but an homozygous deletion of CDKN2A was detected. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were first incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs of incubation, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with PF-0477736 10nM for 24 hours. The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 250–500 nM induced cell death by apoptosis in a time and dose dependent manner after 24 and 48 hours of incubation. Lower concentrations of PF-0477736 (25–50 nM) promoted a statistically significant increase in cell death only in the BJAB cells. For functional studies we characterized the two most sensitive cell lines (BJAB and U2932) and the two resistant cell lines (SUDHL-4 and KM-H2). Inhibition of cdc25c ser216 phosphorylation was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25–250 nM). A marked increase in levels of the DNA damage marker γH2AX, was detected in the BJAB, U2932, SUDHL-4 cell lines after 24 hrs. KM-H2 did not show any increase of γH2AX following treatment. All the cell lines demonstrated baseline CHK1 activation but there was no correlation with outcome. Interestingly levels of baseline pcdc25c ser216 were higher in the sensitive BJAB and U2932 cells. PF-0477736 at the fixed dose of 50 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) in the BJAB and U2932 cells at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The integrity of the p53 axis seems to be the major determinant of efficacy of PF-0477736. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome. Our study provides the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Disclosures: No relevant conflicts of interest to declare.

Therapeutic Targeting of the Bcl-6: p53 Axis with Histone Deacetylase Inhibitors in Diffuse Large B-Cell Lymphoma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3733-3733 ◽  
Author(s):  
Jennifer E Amengual ◽  
Matko Kalac ◽  
Luigi Scotto ◽  
Patrick A Sleckman ◽  
Enrica Marchi ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Hdac Inhibitors ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 3733 Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's Lymphoma. Despite advances in treatment, 1/3 of patients die from their disease. Gene expression profiling has delineated three subtypes with different genetic features known to be prognostic: the Activated B-cell (ABC), Germinal Center (GC), and grey zone types. For example, ABC DLBCL is addicted to NFkB over-expression. The oncogene, BCL6, encodes a transcription factor that functions as a transcriptional repressor within normal germinal center B-cells. Constitutive activation of Bcl-6 leads to GC-type DLBCL by turning off genes expressing cell cycle dependent kinase inhibitors, and essential tumor suppressor genes, like p53. There is a critical inverse relationship between Bcl-6 and p53, the functional status of which is linked to each transcription factor's degree of acetylation. Deacetylation of Bcl-6 is required for maintaining its effects as a transcriptional repressor. Conversely, acetylation of p53 is activating when class III histone deacetylases (HDAC), also known as sirtuins, are inhibited by drugs such as niacinamide. HDAC inhibitors are presently approved for T-cell lymphoma and may require the targeting of additional pathways to be effective in B-cell lymphomas. Trichostatin A and niacinamide modulate Bcl-6 in lymphoma cell lines. One therapeutic strategy that could favorably shift the relationship between oncogenes and tumor suppressors is the pharmacologic modification of Bcl-6 and p53 using HDAC inhibitors. Eight DLBCL cell lines were screened (4 ABC: Su-DHL2, HBL-1, OCI-Ly10, RIVA; 4 GC:OCI-Ly1, OCI-Ly7, Su-DHL6, Su-DHL4) with four class I/II HDAC inhibitors (romidepsin, vorinostat, panobinostat and belinostat) in combination with niacinamide (sirtuin inhibitor) at two dose levels each at three time points. Cell growth inhibition was measured by luminescence cell viability and apoptosis flow cytometry assays. Synergy was measured by the relative risk ratio (RRR) calculation where values <1 represent synergy. Synergy was achieved in significantly greater number and intensity in the GC versus ABC cell lines. Specifically, romidepsin in combination with niacinamide achieved the greatest synergy. To analyze mechanism of action, DLBCL cell lines were treated with combinations of class I/II HDAC inhibitors and niacinamide. Cells of both GC and ABC subtypes treated with the combination resulted in increased acetylation of p53, and increased p21 and BLIMP-1 content compared to controls. These results did not correlate with cytotoxicity as the ABC cell lines did not achieve the same synergy as the GC cells. GC cells treated with the same combinations resulted in acetylation of Bcl-6 compared with controls as measured by immunoprecipitation and Western blotting assays; ABC cells do not express Bcl-6. This finding correlated with cytotoxicity implying that a rational second pathway must be targeted to shift the balance between oncogene and tumor suppressor activity to achieve effective cell kill. p300 content was also increased suggesting that treatment with HDAC inhibitors recruit or upregulate its production and activity leading to increased acetylation. Using a novel double transgenic mouse model of aggressive spontaneous B-cell lymphoma (l-myc overexpressing crossed with CD19-tagged mCherry luciferase), in vivo effects of the drug combination were studied. These mice express equal basal amounts of Bcl-6 and p53 as GC cell lines. Mice treated with niacinamide 20 mg/kg and romidepsin 2.3mg/kg IP for 5 hours achieved increased acetylation of Bcl-6 and p53, and accumulation of p21 and BLIMP1 compared with controls. Importantly, mice treated with the combination of niacinamide 40 mg/kg and romidepsin 2.3 mg/kg IP achieved decreased tumor burden as measured by bioluminescence signal intensity compared to mice treated with each drug alone and controls. Presently, we are translating these concepts and observations in a proof-of-principle phase I trial evaluating the safety of vorinostat plus niacinamide in lymphoid malignancies. By targeting the specific pathogenetic features of DLBCL, it may be possible to tailor future treatment platforms for discrete subtypes of DLBCL. Disclosures: Off Label Use: The drugs evaluated are not approved for use in DLBCL. O'Connor:Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

scholarly journals Repurposing NAD Salvage Inhibitors for GCB Diffuse Large-B Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 789-789
Author(s):  
Claudio Scuoppo ◽  
Bowen Cai ◽  
Kenneth Ofori ◽  
Hanna Scholze ◽  
Katia Basso ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Clinical Development ◽  
Myelogenous Leukemia ◽  
Nad Synthesis ◽  
Large B Cell

Abstract Diffuse large B-cell lymphomas (DLBCLs) are a heterogeneous group of diseases in terms of cell of origin, genetics and clinical outcome. About 30% of all DLBCL patients represent an unmet clinical need as they either do not respond to the standard first line chemo-immunotherapy or recur after initial remission. DLBCL classifications based on the NMF (Non-Negative Matrix Factorization) and LymphGen algorithms have led to the identification of genetic subtypes based on the co-occurrence of specific lesions. Repositioning drugs that are approved or in active clinical development for other indications can be a powerful strategy to match these newly uncovered DLBCL subtypes to targeted therapies. We have previously shown that the screening of large panels of DLBCL cell lines representative of the genetics of the disease can facilitate the repositioning of approved drugs for biomarker-selected populations of DLBCL patients. Repositioned drugs can then be rapidly translated to the clinical use, as they have already been extensively characterized for their safety profile. As a proof of concept, we have demonstrated that Dasatinib, a Src/Abl inhibitor approved for B-cell Acute Lymphoblastic Leukemia and Chronic Myelogenous Leukemia, is highly effective in PTEN-positive DLBCLs, irrespective of their COO class (Scuoppo et al., PNAS 2019). Here we present the results of a new screening that was performed on a panel of eight cell lines (4 ABC- and 4 GCB-DLBCLs) to test the activity of 212 drugs, either approved or in advanced clinical development, for repositioning in DLBCL, followed by validation in a larger panel of 34 genetically characterized cell lines. Our results point to inhibitors of the Nicotinamide Phosphoribosyl Transferase (NAMPT) as potently active in 63% of GCB-DLBCLs. NAMPT catalyzes the conversion of Nicotinamide (NAM) to Beta-Nicotinamide Mononucleotide (Beta-NMN). This reaction is the rate-limiting step of the Nicotinamide Adenine Dinucleotide (NAD) salvage pathway, the major metabolic route of NAD regeneration in mammalian cells. We validated the on-target activity of NAMPT inhibitors by multiple genetic and pharmacological approaches. First, we found that the activities of three chemically distinct drugs (FK-866, STF-118804 and KPT-9274) were highly correlated across the 34 lines DLBCL panel. Second, we were able to abolish the activity of all three NAMPT inhibitors by supplementing cells with Beta-NMN. Third, we showed that transduction of the drug-resistant mutant NAMPT H191R offsets the activity of the drugs. Dose-response assays on the full DLBCL cell line panel confirmed ABC-DLBCL resistance and also highlighted the presence of sensitive (GCB-S) and resistant (GCB-R) GCB subsets that can be separated by a subnanomolar IC50 threshold. To generate biomarkers capable of predicting GCB-S patients, we examined the RNA-seq profiles and genetic make-ups of the DLBCL cell lines collection and observed that the GCB-S subset was associated to the LymphGen EZB subtype, characterized by the presence of EZH2 mutations and BCL2 translocations. Conversely, the GCB-R subtype was linked to a 5-gene expression signature for the Kynurenine De Novo pathway, an alternative route for NAD synthesis. Accordingly, expression of each of the five De Novo Kynurenine pathway genes induced resistance to NAMPT inhibitors. These results were validated in xenotransplants of luciferized DLBCL lines and Patient Derived Xenotransplant models (PDXs) that were transcriptionally classified for the status of the Kynurenine De Novo signature. Together, these data support the repositioning of NAMPT inhibitors as a therapeutically relevant strategy for EZB-type GCB-DLBCLs. Disclosures Pasqualucci: Astra Zeneca: Research Funding; Sanofi: Research Funding.

scholarly journals A Phase I Trial of Brentuximab Vedotin in Combination with Lenalidomide in Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3988-3988 ◽  
Author(s):  
Jeffrey P. Ward ◽  
Jessica Thein ◽  
Jingqin Luo ◽  
Nina D. Wagner-Johnston ◽  
Amanda F. Cashen ◽  
...  
Keyword(s):  
Phase I ◽  
B Cell ◽  
Research Funding ◽  
De Novo ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Brentuximab Vedotin ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: The addition of rituximab to CHOP has improved the overall survival of patients with diffuse large B-cell lymphoma (DLBCL); however, approximately 30% of patients will relapse. Stem cell transplantation (SCT) provides a second chance at cure, but the prognosis for patients who are not candidates for SCT or who have refractory disease is poor, and new treatments with novel agents are needed. Brentuximab vedotin (BV), an antibody-drug conjugate that combines an anti-CD30 monoclonal antibody and the microtubule disrupting agent MMAE, has a single agent response rate (RR) of 44% (CR 17%) in CD30 (+) (Jacobsen, Blood 2015) and 27% (CR 3.7%) in CD30 (-) relapsed/refractory (rel/ref) DLBCL (Bartlett, ASH 2014). Lenalidomide (Len), an immunomodulator with multiple described mechanisms of action, has a single agent RR of 28% (CR 7%) in rel/ref DLBCL (Witzig, Ann Oncol 2011). Notably, the Len RR was 52.9% in the subset of patients with non-germinal center-like (non-GCB) DLBCL, compared to 8.7% in GCB DLBCL (Hernandez-Ilizaliturri, Cancer 2011). Given that both compounds have single agent activity in DLBCL and favorable, non-overlapping toxicity profiles, we hypothesized that the combination of BV and Len would be an effective and tolerable regimen in rel/ref DLBCL. Methods: This investigator initiated, phase I/dose expansion trial is ongoing to identify the safety and maximum tolerated dose (MTD) of the combination of BV and Len (Clinical Trials.gov NCT02086604). Eligible patients have rel/ref de novo or transformed DLBCL after at least one prior systemic therapy and have previously received or are ineligible for SCT. Response assessments are performed after cycles 2, 4, 6, 9 and then every six months for two years by PET/CT scan and response determined per the Revised International Working Group Response Criteria for Malignant Lymphoma 2007. The study is in two parts, a dose-escalation portion using a 3+3 design to determine the MTD, followed by a dose-expansion cohort enrolling patients with either CD30 (+) or CD30 (-) DLBCL assessed by visual assessment using routine IHC per local laboratory. BV is administered every 21 days and Len is dosed continuously for a maximum of 16 cycles until disease progression or unacceptable toxicity. Results: Eighteen patients have been enrolled to date. The median age is 61 years (range 51-79), with 83% having an ECOG performance status of 0-1. Median number of prior therapies is 2 (range 1-6), with 39% undergoing a previous autoSCT, and one patient a previous alloSCT. 72% of patients were refractory to their last regimen. 13 patients have CD30 (-) and 5 CD30 (+) DLBCL. Treatment-related adverse events (AEs) occurring in >20% of patients include anemia (50%), elevated ALT (28%), hypocalcemia (22%), peripheral neuropathy (22%), neutropenia (28%), thrombocytopenia (33%), and hypokalemia (28%). Anemia, febrile neutropenia, thrombocytopenia, and hypokalemia were the only grade 3/4 related AEs observed in >10% of patients. Growth factors were not given during cycle 1 but were administered in 11 patients with subsequent cycles. One patient came off study for thrombocytopenia after completing 8 cycles, while in a CR. 47% have required at least one dose reduction. The DLTs per dose cohort are summarized in the table. The MTD of the combination is 1.2 mg/kg of BV Q21d with 20 mg Len given continuously. At the time of this analysis, 17 patients (1 too early) have had restaging evaluations; 7 CR (41%), 2 PR, 3 SD, and 5 PD, for an overall RR of 53%. Five CRs occurred after C2, 1 after C6 and 1 after C8. All responses are ongoing with a range of 5 to 35 wks. Among the 7 CRs, 2 patients have CD30 (+) and 5 patients CD30 (-) DLBCL, 4 pts were GCB and 3 non-GCB. Of the four patients with CD30 (-) disease categorized as GCB, two achieved a CR. Conclusions: This Phase I study of BV plus Len identified the MTD of the combination at BV 1.2 mg/kg Q21d with Len 20 mg/d continuously. Dose expansion cohorts of 15 patients each for CD30 (-) and CD30(+) DLBCL are currently accruing. The predominant toxicity of the study regimen is related to cytopenias, consistent with prior experience. Although patient numbers are small, the high CR rate is intriguing. Updated results will be presented at the meeting. Table 1. # Patients Assigned BV Dose Assigned Len Dose # of DLT DLT Toxicity 9 1.2mg/kg 20mg 1 Neutropenia 6 1.2mg/kg 25mg 2 Neutropenia, DVT 3 1.8mg/kg 25mg 2 Fatigue, Neutropenia Disclosures Ward: Boehringer Ingelheim: Consultancy. Wagner-Johnston:Celgene: Research Funding; Gilead: Consultancy. Fehniger:Celgene: Research Funding. Bartlett:Gilead: Consultancy, Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Millennium: Research Funding; Colgene: Research Funding; Medimmune: Research Funding; Kite: Research Funding; Insight: Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding.

scholarly journals The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37 Targeted Radioimmunotherapy in Activated B Cell like Diffuse Large B Cell Lymphoma Cell Lines

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2574-2574
Author(s):  
Gro Elise Rødland ◽  
Katrine Melhus ◽  
Roman Generalov ◽  
Sania Gilani ◽  
Francesco Bertoni ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Employment Equity ◽  
Large B Cell Lymphoma ◽  
Equity Ownership ◽  
Large B Cell

The CD37 targeting radioimmunoconjugate 177Lu-lilotomab satetraxetan (Betalutin®) is currently being evaluated as monotherapy in a clinical phase 2b trial for patients with follicular lymphoma (FL) and in a phase 1 trial for patients with diffuse large B-cell lymphoma (DLBCL), as well as in a phase 1b trial in combination with rituximab for patients with relapsed/refractory FL. Herein we have investigated the effect of 177Lu-lilotomab satetraxetan in seven activated B-cell like (ABC) DLBCL cell lines. Although the radioimmunoconjugate showed anti-tumor activity, primary resistance was observed in a subset of cell lines: U-2932 and RIVA. Both cell lines are representative for TP53 deficient Double Expressor (DE) DLBCL. Importantly, resistance was not a consequence of reduced binding of the radioimmunoconjugate to cell surface expressed CD37. Thus, we set out to identify drugs able to overcome the resistance to 177Lu-lilotomab satetraxetan in both resistant ABC-DLBCL cell lines. We performed a viability-based screen combining 177Lu-lilotomab satetraxetan with the 384-compound Cambridge Cancer Compound Library. Drug combinations were scored using Bliss and Chou-Talalay algorithms. We identified and characterized the dual-specific CDK1/2 and AURA/B kinase inhibitor JNJ-7706621 as compound able to revert the resistance to radioimmunotherapy (RIT), alongside topoisomerase and histone deacetylases (HDAC) inhibitors. Kinetic studies of the effect of mono- and combination therapy of U-2932 and RIVA cells with JNJ-7706621 and 177Lu-lilotomab satetraxetan are suggestive of a model in which radiation damage induced G2-arrested lymphoma cells eventually enter mitosis (repair or escape) and mitotic entry, progression and exit are impaired by JNJ-7706621 mediated inhibition of CDK1/2 and AURKA/B. Extended residence-time of cells in mitosis due to chromosome condensation and congression defects as well as spindle and mid-spindle assembly failure is likely pivotal for the increased sensitivity to persistent 177Lu-lilotomab satetraxetan deposited DNA damage, ultimately promoting cytokinesis failure (multinucleation, aneuploidy, increased cell size) and cell death. In conclusion, CD37-targeting 177Lu-lilotomab satetraxetan RIT showed activity in several ABC-DLBCL lymphoma cell lines. CD37-independent RIT-resistance was identified in two cell lines representative of aggressive DE ABC-DLBCLs with inactive TP53, and reversed by subsequent inhibition of CDK1/2 and AURKA/B by JNJ-7706621. These findings may be of potential relevance for ongoing clinical trials of 177Lu-lilotomab satetraxetan in relapsed, ASCT-non-eligible DLBCL, and may also be more generally applicable to other 177Lu-based RITs and alternative radionuclide utilizing targeted therapies. Future pre-clinical investigations are required to elucidate the potential application of CDK1/2 and AURKA/B inhibitors as a strategy to revert RIT resistance in TP53 deficient cancers. Disclosures Rødland: Nordic Nanovector ASA: Patents & Royalties, Research Funding. Melhus:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Generalov:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Bertoni:Nordic Nanovector ASA: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants; NEOMED Therapeutics 1: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Syljuåsen:Nordic Nanovector ASA: Patents & Royalties, Research Funding. Patzke:Nordic Nanovector ASA: Employment, Patents & Royalties.

scholarly journals BET Bromodomain Inhibitor OTX015 Affects the Expression of Micrornas Involved in the Pathogenesis of Diffuse Large B-Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4495-4495 ◽  
Author(s):  
Luciano Cascione ◽  
Eugenio Gaudio ◽  
Elena Bernasconi ◽  
Chiara Tarantelli ◽  
Andrea Rinaldi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tumor Models ◽  
Expression Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, accounting for 30%-40% of all cases. Despite a major improvement in the cure rate, a large number of DLBCL patients lack therapeutic options. Aberrant changes in histone modifications, DNA methylation and expression levels of non-coding RNA, including microRNA (miRNA), contribute to DLBCL pathogenesis and represent potential therapeutic targets. OTX015 targets bromodomain and extra-terminal (BET) proteins, which are epigenetic readers contributing to gene transcription. It has shown preclinical activity in hematologic and solid tumor models (Gaudio et al, AACR 2014; Noel et al, EORTC-NCI-AACR 2013) and promising early results in an ongoing phase I study (Herait et al, AACR 2014; NCT01713582). To better understand the mechanism of action of OTX015, we studied molecular changes induced by this compound in DLBCL cell lines. Methods. Total RNA was extracted from 2 DLBCL cell lines, the germinal center B-cell (GCB) type DOHH2 and activated B-cell-like (ABC)-type SU-DHL-2, following treatment with 500 nM OTX015 or DMSO for 4h or 8h. RNA samples were labeled with cyanine-3 dye using the Agilent microRNA Complete Labeling System & Hyb Kit and hybridized to the Agilent Human microRNA microarray v.3. Raw expression values were obtained with Agilent Feature Extraction Software, log-transformed and normalized by the quantile method. Data were filtered to exclude relatively invariant features and those below the detection threshold. Limma (Linear Models for Microarray data analysis) was employed using R/Bioconductor and the filtered dataset. Baseline miRNA profiling was obtained from 22 DLBCL cell lines with the Nanostring nCounter Human v2 miRNA Expression Assay kit. Baseline gene expression profiling (GEP) was obtained in these cell lines with the Illumina HumanHT-12 v4 Expression BeadChip. Selected miRNA changes were validated by real-time PCR. Validated miRNA targets were retrieved using the miRWalk database (Dweep et al, 2011). Gene Set Enrichment Analysis (GSEA) software was used to assess enrichment of miRNA targets in the GEP datasets. Results. miRNA profiling of the GCB and ABC DLBCL cell lines exposed to OTX015 identified four downregulated miRNAs and eight which were upregulated. Among them, the oncomirs miR-92a-1-5p (log2 FC, -2.01; P=0.004) and miR-21-3p (log2 FC, -0.37; P=0.0045) were downregulated, while the tumor suppressor miR-96-5p (log2 FC, 0.39; P=0.041) was upregulated. Interestingly, changes of these miRNAs matched GEP variations of validated target genes (e.g., miR-92a-1-5p: CDKN1A, log2 FC, 0.81, CDKN2A, log2 FC, 0.81; miR-96-5p: MYC, log2 FC, -0.57, MYD88, log2 FC, -0.35). We then evaluated if these three miRNAs play a role in OTX015-sensitivity by obtaining baseline miRNA and GEP profiling data in 22 DLBCL cell lines. Compared to 8 cell lines with lower sensitivity to OTX015 (IC50 >500 nM), the 14 sensitive cell lines (IC50 <500 nM) presented lower miR-96-5p expression levels (log ratio, 2.12; P=0.026) and their GEPs were significantly enriched for validated miR-96-5p targets (normalized enrichment score, 1.4; P=0.026), suggesting miR-96-5p levels may predict response to OTX015. Conclusions. Changes in the expression levels of biologically relevant miRNAs may contribute to response to OTX015. miR-92a-1-5p, the oncomir which was most strongly downregulated by OTX015, is a member of the MYC target MIR17HG (mir-17-92 cluster), involved in the pathogenesis and chemo-resistance of lymphomas, mainly contributing to PI3K/AKT/mTOR pathway activation. Since the cell cycle transcriptional regulator E2F1 is targeted by mir-17-92, OTX015 may contribute to cell cycle arrest and to downregulation of the E2F1 target gene reported with BRD inhibitors in DLBCL cell lines. miR-21-3p, also downregulated by OTX015, is a well-known oncomir, and forced miR-21-3p expression in transgenic mice results in the development of leukemias and lymphomas. miR-96-5p, upregulated by OTX015, targets oncogenes such as RAS or MYC, and low expression has been reported in mantle cell lymphoma. Interestingly, low miR-96-5p baseline levels were associated with higher sensitivity to OTX015, an observation meriting validation in other tumor models and evaluation in clinical studies. Disclosures Stathis: Oncoethix SA: Consultancy, Research Funding. Riveiro:Oncoethix SA: Consultancy, Research Funding; Oncology Therapeutic Development: Employment. Bertoni:Oncoethix SA: Research Funding.

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