Darinaparsin in T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL) Cells Lines and SCID Xenograft Mouse Models: Single-Agent Activity and Synergistic Cell Death Combined with a PI3K/mTOR Inhibitor

Abstract Abstract 1646 Background: Darinaparsin is a novel organic arsenical compound with potent anti-neoplastic activity. Preliminary clinical studies reported encouraging activity in patients with relapsed/refractory TCL and HL. We investigated the molecular mechanisms of action of darinaparsin in HL and TCL cell lines, the degree and method of cell death, and we investigated rational drug combinations for enhancement of therapeutic activity. Methods: TCL (Jurkat, Hut78, HH) and HL cell lines (L428, L540, L1236) were treated with darinaparsin at increasing time and concentrations. MAPK, AKT/PI3K, mTOR and relevant pathways were analyzed by Western blot. Cell viability was assessed by MTT and apoptosis with Annexin V/PI flow cytometric analysis; this was confirmed by Western for caspase activation and PARP cleavage. In vivo tumor growth inhibition and survival of SCID mice was determined using xenografts derived from Jurkat (TCL) or L540 (HL) cell lines. The in vivo studies started with 7–8 mice for each control and treatment group. Once tumor volume average reached 100–250 mm3, treatment groups were injected (5 days/week) with darinaparsin SQ daily for 3 weeks. For drug combinations, varied novel agents were rationally combined with darinaparsin (BEZ-235 [PI3K/mTOR dual kinase inhibitor], PXD101 [HDAC inhibitor], olaparib [PARP inhibitor], MG132 [proteasome inhibitor], MK-2206 [pan-AKT inhibitor], SP600125 [JNK inhibitor], SB203580 [p38 MAPK inhibitor] and PD98059 (anti-MEK small molecule). We also interrogated pertinent signaling pathways with shRNA stable knock outs (KO) +/− darinaparsin. Results: Treatment with 1–5μM Darinaparsin for 72 hours resulted in time- and dose-dependent cytotoxicity in all TCL and HL cell lines. IC50 observed with 72-hour treatment in the TCL lines Jurkat, HH, and Hut87 were 2.7μM, 3.2μM, and 6.7μM, respectively, and for the HL clines L540, L1236, and L428 were 1.3μM, 2.8μM, and 7.2 μM, respectively. Darinaparsin treatment also resulted in a dose dependent increase in apoptosis, detected by Annexin-V positivity and cleavage of PARP and caspases 3, 8 and 9 in all TCL and HL cell lines. In vivo experiments with lymphoma tumor xenografts derived from Jurkat showed significant inhibition of tumor growth (P<0.001) and improved survival (P<0.001) in darinaparsin-treated SCID mice compared with untreated control (see Figure); identical tumor reduction and survival data was noted in L540 xenografts. Next, we investigated relevant signaling pathways that were up- or down-regulated following darinaparsin exposure. We identified activation of the PI3K, MAPK, and mTOR pathways in all TCL and HD lines, expect for lack of MAPK response in L540; activation of pAKT was observed only in TCL cell lines (Jurkat and HH). Additionally, a concomitant decrease in levels of the inhibitory phosphatases (PTEN, SHP1) associated with AKT and MEK/ERK pathways were apparent in all TCL and HL cell lines treated with darinaparsin. Next, we tested cytotoxicity of darinaparsin (by MTT) in MEK and ERK KO using stably transfected shRNA in L540, Hut78 and Jurkat lines. There was minimal effect of these KOs in Jurkat, while there was increased cytotoxic effect with ERK shRNA in L540 and MEK shRNA in Hut78. Finally, we performed combination studies in L540 and Jurkat cell lines using the aforementioned novel targeted agents combined with darinaparsin in order to identify potential syngerism. Interestingly, the most notable combination was the PI3K/mTOR dual kinase inhibitor, BEZ-235, combined with darinaparsin; this resulted in synergistic cell death in Jurkat TCL cells (combination indices [CI]=0.5) and L428 HL cells (CI=0.5) cells as confirmed by MTT, Annexin-V and cleavage of PARP and caspases. Conclusions: Collectively, these data show that the novel organic arsenical, darinparsin, induced significant cell death in TLC and HL cell lines and in lymphoma SCID xenograft models. Cell death with darinaparsin appeared to be dependent in part on the MAPK pathway, however this was cell line dependent. Additionally, we identified synergistic cell death in TCL and HL cell lines when darinaparsin was combined with a PI3K/mTOR dual inhibitor. Continued study of darinaparsin in TCL and HL is warranted either as a single agent or through co-targeting with PI3K/mTOR-based therapy. Disclosures: Off Label Use: Darinaparsin for the treatment of T cell lymphoma and Hodgkin's disease.

Download Full-text

Targeting the PI3K/mTOR Pathway in Lymphoma with PQR309 and PQR620: Single Agent Activity and Synergism with the BCL2 Inhibitor Venetoclax

Blood ◽

10.1182/blood.v128.22.3017.3017 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3017-3017

Author(s):

Chiara Tarantelli ◽

Eugenio Gaudio ◽

Petra Hillmann ◽

Filippo Spriano ◽

Ivo Kwee ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Mtor Inhibitor ◽

Cell Lymphoma ◽

Single Agent ◽

Apoptosis Induction ◽

In Vivo Experiments

Abstract Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e13569 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e13569-e13569

Author(s):

Enrica Marchi ◽

Matko Kalac ◽

Danielle Bongero ◽

Christine McIntosh ◽

Laura K Fogli ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Cell Lines ◽

Molecular Level ◽

Cell Lymphoma ◽

Single Agent ◽

T Cell Lymphoma ◽

Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

Download Full-text

Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Blood ◽

10.1182/blood-2021-152176 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3986-3986

Author(s):

George A. Ward ◽

Simone Jueliger ◽

Martin Sims ◽

Matthew Davis ◽

Adam Boxall ◽

...

Keyword(s):

Cell Death ◽

T Cell ◽

Cell Lines ◽

Western Blotting ◽

Inflammatory Mediators ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Immunogenic Cell Death

Abstract Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL). Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand). Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays. Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability. Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo. By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro. Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds. Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. Disclosures Sims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.

Download Full-text

Novel Bruton's Tyrosine Kinase Inhibitor Bgb-3111 Demonstrates Potent Activity in Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.5374.5374 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5374-5374 ◽

Cited By ~ 2

Author(s):

Carrie J Li ◽

Yang Liu ◽

Taylor Bell ◽

Jack Wang ◽

Hui Guo ◽

...

Keyword(s):

Tumor Growth ◽

Cell Viability ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

Mantle Cell ◽

Btk Inhibitor ◽

Dose Dependent

Abstract Background: Aberrant B-cell receptor signaling is an important contributor to lymphomagenesis in mantle cell lymphoma (MCL). Bruton's Tyrosine Kinase (BTK), a component of the BCR signaling axis, has been validated as a clinically relevant target, and BTK inhibitor ibrutinib received FDA approval for treatment of MCL in 2013. Growing concerns that single agent ibrutinib exerts off-target effects that interfere with other treatments such as rituximab-induced antibody-dependent cell cytotoxicity limit its utility in combination treatments. In this study, we assessed the in vitro and in vivo effects of BGB-3111in MCL models. Methods: We performed cell viability assays with BGB-3111 treated MCL cell lines to determine inhibition of cellular proliferation. The same assays were conducted on primary human MCL cells and patient-derived xenograft (PDX) tumor samples. Dose-dependent inhibition of BTK auto-phosphorylation and inhibition of downstream targets such as PLC-γ were determined by phospho-protein immunoblotting and immunoprecipitation. A reverse-phase protein assay (RPPA) was conducted on BGB-3111-treated Mino cells to evaluate changes in MCL oncogenic signaling. Induction of apoptosis in MCL cells treated with increasing doses of BGB-3111 was quantified using flow cytometry. For in vivo experiments, an ibrutinib-sensitive MCL PDX mouse model was treated with 50 mg/kg/day BGB-3111 and monitored for mean tumor burden and survival. Results: BGB-3111 potently inhibited cell viability in a panel of MCL cell lines, with an activity range of 1-10 uM, and induced apoptosis in a dose-dependent manner in several MCL cell lines.BGB-3111 treatment of MCL cells demonstrated a dose-dependent decrease in p-BTK (Y223) and inhibition of downstream effectors without impacting total protein levels, while RPPA revealed upregulation of the PI3K-Akt signaling axes. In addition, BGB-3111 treatment did not impact phosphorylation of off-target kinases affected by ibrutinib treatment. In vivo, BGB-3111 suppressed tumor growth and prolonged tumor survival in BGB-3111 treated mice. Conclusion: The second generation BTK inhibitor BGB-3111 demonstrates selectivity for BTK in vitro and BTK inhibition in vivo. BGB-3111-treated PDX mouse models examining survival, tumor growth, and other factors point to BGB-3111 as an effective single agent BGB-3111 is being investigated in Phase I clinical trials. Disclosures Wang: Beigene: Employment. Wang:Asana BioSciences: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria; Acerta: Consultancy, Research Funding; Kite Pharma: Research Funding; BeiGene: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Juno Therapeutics: Research Funding.

Download Full-text

The Novel HDAC Inhibitor Chidamide Synergizes with Rituximab to Inhibit DLBCL Tumor Growth in Vitro and In Vivo By up-Regulating CD20 Expression

Blood ◽

10.1182/blood-2018-99-114598 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2947-2947

Author(s):

Xu-Wen Guan ◽

Wang Hua-Qing ◽

Li Jia ◽

Feng-Ting Liu

Keyword(s):

Cell Death ◽

Tumor Growth ◽

Cell Lines ◽

Cell Lymphoma ◽

Combined Treatment ◽

B Cell Lymphoma ◽

Surface Expression ◽

Cd20 Expression

Abstract Background: Histone deacetylases (HDACs) are crucial proteins for supporting tumorigenesis. HDACs reverse chromatin acetylation and alter transcription of oncogenes and tumor suppressor genes by removing acetyl groups from histones. HDAC inhibitors are considered as promising anti-cancer drugs, particularly in combination with other standard treatment regimens. Chidamide is the world first oral HDAC inhibitor which selectively inhibits class I HDAC1, HDAC2, and HDAC3 as well as class IIb HDAC10. Chidamide has been approved by China FDA in 2015 for the treatment of relapsed or refractory peripheral T-cell lymphoma. Diffuse large B-cell lymphoma (DLBCL) is the most aggressive form of B-cell lymphoma. Treatment with R-CHOP i.e. Rituximab (the anti-CD20 monoclonal antibody) plus CHOP (Cyclophosphamide, doxorubicin, vincristine, and prednisone) has significantly improved clinical outcome for DLBCL patients. However, treatment-induced deacetylation of CD20 gene and consequently down-regulation of CD20 protein expression causes an acquired resistance to further treatment with R-CHOP. We hypothesize that inhibition of HDACs by Chidamide could overcome Rituximab-mediated down-regulation of CD20 and facilitate Rituximab-induced DLBCL tumor growth inhibition. The aim of this study is to determine the synergistic effect of Chidamide and Rituximab in the treatment of DLBCL in vitro and in vivo. Methods: The levels of CD20 (MS4A1) mRNA expression and clinical outcomes in patients with DLBCL treated either with R-CHOP or CHOP were obtained from the Gene Expression Omnibus (GEO) repository (NCBI GSE 10846). The association of CD20 expression with overall survival (OS) was analyzed by Cox regression analysis and the cut-off point was calculated by the X-tile software. CD20 protein surface expression and Rituximab-induced cell death were analyzed by flow cytometry. The IC50s of Chidamide and the synergisms with Rituximab (10 µg/ml) on five DLBCB cell lines (OCI-LY3, OCI-LY7, Su-DHL6, Su-DHL8, and Su-DLH10) were determined by MTT test after cells were treated with a range of concentrations of Chidamide with or without Rituximab for 24 hours. The synergism was calculated using ComboSyn software to obtain the combination index (CI). For in vivo experiments, the human DLBCL cell line OCI-LY7 were injected to 6 weeks BALB/C nude mice to develop xenograft DLBCL mice models. After tumors were palpable, mice were divided into four groups and injected with NaCl (control), Rituximab, Chidamide and Rituximab plus Chidamide daily for three weeks. The tumor volumes were monitored frequently during the treatment. Results: In R-CHOP treated cohort (n=233), higher expression of CD20 expression (n=137) is significantly associated with superior clinical outcomes compared with lower CD20 expression (n=96) with P=0.0038, HR=0.4753, 95% CI=0.274-0.779. However, the levels of CD20 have no effect on clinical outcome in DLBCL patients treated with CHOP (n=183). The levels of CD20 protein surface expression on five DLBCL cell lines were significantly and positively correlated with the sensitivities of cells to Rituximab-induced cell death (P=0.0018, R=0.88). HDAC1, HDCA2 and HDCA3 proteins were detected in these DLBCL cell lines. Treatment with Rituximab significantly reduced CD20 surface expression but treatment with Chidamide significantly increased CD20 surface expression in DLBCL cells. The CI numbers for combined treatment with Chidamide and Rituximab were either <0.01 (very strong synergism) or <0.3 (strong synergism), indicating that Chidamide significantly synergized Rituximab-induced cell death. For in vivo assay, treatment with either Rituximab or Chidamide alone slightly but not significantly reduced tumor volume. Combination with Chidamide and Rituximab significantly inhibited tumor growth in DLBCL xenograft mice (P<0.0001). Mice with combined treatment showed significantly prolonged survival compared with other groups. Conclusions: our data demonstrate for the first time that inhibition of HDACs by Chidamide significantly synergized Rituximab-induced tumor growth inhibition in vitro and in vivo. We propose that CD20 surface expression should be used clinically to evaluate treatment response in patients with DLBCL. Chidamide is a promising sensitizer for the treatment of DLBCL with R-CHOP. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Overcoming CAR T Resistance with Non-Covalent BTK Inhibitor Loxo-305 in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-137645 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-10

Author(s):

Yang Liu ◽

Vivian Changying Jiang ◽

Joseph McIntosh ◽

Alexa A Jordan ◽

Yijing Li ◽

...

Keyword(s):

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

Annexin V ◽

Car T ◽

Mantle Cell ◽

Btk Inhibitor

Background: Mantle cell lymphoma (MCL) is a distinctive B-cell non-Hodgkin's lymphoma characterized by poor prognosis. Despite clinical success of the covalent Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, a subset of patients need to discontinue ibrutinib therapy due to treatment related adverse events, which are primarily caused by off-target effects. Furthermore, primary or acquired resistance to ibrutinib continues to emerge and often leads to dismal clinical outcomes. Therefore, exploration of more target-specific BTK inhibitors is crucial to minimize the adverse events and provide clinical benefit. CAR T therapy has achieved unprecedented response in patients with relapsed or refractory MCL. However, the development of resistant phenotypes is a new emerging medical challenge in MCL patients with unknown mechanisms. Here, we characterize the therapeutic efficacy of LOXO-305, a next generation non-covalent small molecule inhibitor with high selectivity for BTK. Preclinical efficacy of LOXO-305 alone or in combination with venetoclax (ABT199), a selective Bcl-2 inhibitor, was evaluated in MCL using in vitro and in vivo CAR T-resistant PDX models. Methods : In vitro cell viability was measured after 72 hour treatment with LOXO-305 alone and in combination with ABT-199 in MCL cell lines using Cell Titer Glo luminescent cell viability assay (Promega). To determine whether LOXO-305 induces cell death through cell apoptosis, we used annexin V/PI staining followed by flow cytometry analysis. To evaulate in vivo drug efficacy we used patient-derived xenograft (PDX) models established from primary patient samples. Results: LOXO-305 treatment, as a single agent, resulted in effective MCL cell growth inhibition in a panel of MCL cell lines including ibrutinib and/or ABT-199-resistant cell lines (IC50=6.6-24.4μM), except for JeKo BTK KD cells with BTK knockdown (KD) via CRISPR/Cas9 technology (IC50>30 μM). To improve the efficacy, we decided to investigate the potential of LOXO-305 in combination with ABT199, since the combo of ibrutinib and ABT199 is clinically beneficial in MCL patients. Indeed, LOXO-305 significantly improved the inhibitory effect of ABT-199 in the ABT-199 resistant Mino-R and JeKo BTK KD cells, suggesting that this combination could be further explored in overcoming ABT-199 resistance in MCL. The compelling synergistic effect was further confirmed by annexin V/PI apoptosis assay. Next, we assessed the in vivo efficacy of LOXO-305 in an ibrutinib-CAR T dual-resistant PDX model. LOXO-305 effectively reduced tumor size after 40 days of treatment as a single agent. Moreover, LOXO-305 treatment showed significant anti-tumor effects in an ibrutinib-ABT199-CAR T triple-resistant PDX model that recapitulates the most aggressive human MCL variants invivo. In this model, LOXO-305 treatment effectively decreased the tumor load in mice spleen and liver (p<0.05) as well as in bone marrow and peripheral blood, compared to vehicle-treated mice (p<0.001). Conclusions: By using various in vitro and in vivo multiple resistant MCL models we determined that LOXO-305 holds great promise for an effective single agent or combined treatment of the most eggressive forms of MCL, and that a continued investigation of the rationale for a combined therapy with ABT-199 is imperative to understand its role in overcoming ibrutinib-ABT199-CAR T triple resistance. Disclosures Wang: OMI: Honoraria, Other: Travel, accommodation, expenses; MoreHealth: Consultancy; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Loxo Oncology: Consultancy, Research Funding; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; Oncternal: Consultancy, Research Funding; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Targeted Oncology: Honoraria.

Download Full-text

Synergistic Effects of a Novel Water-Soluble Small Molecule, ON 013105, and Rituximab on Mantle Cell Lymphoma In Vitro and In Vivo

Blood ◽

10.1182/blood.v116.21.771.771 ◽

2010 ◽

Vol 116 (21) ◽

pp. 771-771

Author(s):

Anil Prasad ◽

Ashutosh Shrivastava ◽

Ramana Reddy ◽

Amanda M. Gillum ◽

E. Premkumar Reddy ◽

...

Keyword(s):

Cell Death ◽

Tumor Volume ◽

Cell Lymphoma ◽

Single Agent ◽

Dependent Manner ◽

Mantle Cell ◽

Dose Dependent ◽

Efficacy Studies

Abstract Abstract 771 Mantle cell lymphoma (MCL) is a well-defined subtype of B-cell non-Hodgkin's lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation, and associated with constitutive over-expression of cyclin D1. MCL generally has poor clinical outcome marked by relapse. There is considerable need for novel and more effective agents against MCL. ON 013105 belongs to the styryl benzylsulfones, a novel family of non-ATP competitive kinase inhibitors with potent antitumor activity. Here, we report that ON 013105 induced cell death in a dose-dependent manner in two well-characterized MCL cell lines, Granta 519 and Z138C. In vitro cell death was preceded by the activation of caspases 3 and 9 and cleavage of PARP, indicating induction of apoptosis. In addition, ON 013105-treated cells exhibited reduced expression of cyclin D1 and c-myc. These effects on expression and apoptosis were not evident in cells treated with ON 013101, an inactive (non-cytotoxic) isomer of ON 013105. Since it is common clinical practice to combine Rituximab (RTX) with chemotherapy regimens in treating CD20+ B cell-lymphoma, we studied ON 013105 combined with rituximab, and found ON 013105-induced apoptosis more efficiently than when employed as a single agent. The combination effect on cell death was synergistic in nature. To further study this activity, we focused on Mcl-1, a member of the anti-apoptotic Bcl-2 family known to inhibit apoptosis induced by cytotoxic stimuli through antagonizing pro-apoptotic Bcl-2 family members. We observed a dramatic decrease in Mcl-1 expression in cells treated with ON 013105 (but not with ON 013101) in combination with RTX, compared to ON 013105 alone. We also evaluated the effects of ON 013105 in combination with Doxorubicin or Vincristine and found that both these compounds also significantly enhanced the cytotoxic effects of ON 013105. In vivo pharmacokinetics studies in a mouse model system revealed that plasma concentrations up to 50 μM could be safely achieved by administering ON 013105 at 100 mg/kg via i.v or i.p routes. Significant levels of ON 013100 (30-40% of the peak levels of ON 013105), an active metabolite, were also detected in the circulation, presumably due to the in vivo dephosphorylation of ON 013105 by phosphatase action. ON 013105 was well tolerated in mice, both as a single agent and when used in combination with rituximab, and there were no systemic toxic effects to the host and no loss in body weight. In vivo efficacy studies in mouse xenograft models employing transplanted MCL cells demonstrated that ON 013105 effectively inhibited tumor growth in a dose-dependent manner. ON 013015 at 25 mg/kg (Q2D) and 75mg/kg (Q7D) induced 46% and 80 % reduction of tumor volume, respectively, compared to controls, over 4 weeks of treatment. Moreover, ON 013105 at 25 mg/kg (Q2D) in a combination regimen with RTX (2.5 mg/kg, Q3D) induced over 85% reduction of tumor volume. Though in vivo efficacy studies of ON013015 (25 mg/kg, Q2D) in combination with Doxorubicin (3.5mg/kg, Q7D) or Vincristine (0.3mg/kg, Q2D) showed drastic decrease in tumor growth in mouse models, this effect was accompanied by severe side effects to the host, including mortality. In sum, ON 013105, alone and in combination with RTX may be a potent therapeutic agent against MCL. A Phase I dose escalation trial of ON 013105 as a single agent is underway in patients with relapsed/refractory lymphoma including MCL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Combination of Epigenetic Agents Synergistically Reverse the Malignant Phenotype in Models of T-Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.2727.2727 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2727-2727 ◽

Cited By ~ 3

Author(s):

Enrica Marchi ◽

Matko Kalac ◽

Danielle C Bongero ◽

Christine M McIntosh ◽

Laura K Fogli ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Research Funding ◽

Molecular Level ◽

Cell Lymphoma ◽

Single Agent ◽

T Cell Lymphoma ◽

Combination Group ◽

T Cell Lymphomas

Abstract Abstract 2727 CHOP and CHOP-like chemotherapy remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL) and peripheral T-cell lymphomas (PTCL) given their marked single agent activity in these diseases. The interaction between the HDACIs (depsipeptide (R) and belinostat (B)) and a DNMT inhibitor (decitabine (D)) was investigated in vitro, in vivo and at the molecular level in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (P12, PF-382). For all cytotoxicity assays, a luminescence based cell viability assay was used (CellTiter-Glo™) followed by acquisition on a Biotek Synergy HT. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR<1 are defining synergism). Apoptosis was assessed by staining with Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition and analyzed using FlowJo. The IC50s for B, R, vorinostat (V), panobinostat (P), D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours in all the cell lines. For the combination experiment we selected the most active DNMTI, decitabine. In the cytotoxicity assays, the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines studied. The RRRs for all the combinations were between 0.0007 and 0.9. When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 1 displays the range of apoptosis induction for B, R ± D and the RRR value for the most significant data.Table 1:BDB + DRRR(% Apoptotic + Dead Cells)H9100 nM (22.9%)500 nM (17.9%)51.5%0.7HH100 nM (42.9%)1 uM (46.9%)61.3%0.8P 12150 nM (16%)1 uM (42.7%)80.1%0.4PF 382100 nM (8.3%)1 uM (27.9%)40.1%0.8RDR + DH92 nM (22.2%)500 nM (17.9%)63.6%0.5HH2 nM (80%)1 uM (46.9%)89.7%0.6P 122 nM (9.9%)10 uM (58.7%)98%0.03PF 3822 nM (54.5%)500 nM (17.9%)88.7%0.2 An in vivo xenograft study in 6–8 weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells was performed. Mice were separated into different cohorts and treated i.p. for 3 cycles with D or B or their combination according to the following schedules: D at 1.5 mg/kg on days 1, 3, 5; B at 40 mg/kg/day for 10 days (I cycle); D at 1.5mg/kg on days 15,17,19,21; B at 65 mg/kg/day for 10 days (II cycle); D at 1.5 mg/kg on days 29,31,33,35,37,39,41,43; B at 100mg/kg for 19 days (III cycle). Statistically significantly tumor growth inhibition was observed in the combination cohort compared to all the other cohorts (analysis on day 42, 45). We analyzed the molecular basis for this synergistic effect by evaluating gene expression patterns using the Illumina Human HT-12 v4 Expression BeadChip microarrays. These analyses revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. As shown in Figure 1, a set of genes (A) is down-regulated by both drugs. Other genes (B) are up-regulated by D and the effect is maintained in the combination. Other genes (C+E) are slightly up-regulated by R, though not significantly modified by D, and more strongly up-regulated in the combination group. Similarly, genes to some extent up-regulated by D but not by R (D+F) appeared to be more significantly affected by the combination. As shown in Figure 2, the effects of the two drugs are largely different (only 39 genes modified in common by all the treatment groups). Most of the effects induced by the single agent treatment are maintained in the combination group (174 genes out of 191 for romidepsin and 211 genes out of 221 for decitabine). Interestingly, an additional 944 genes appeared to be modulated uniquely by the combination treatment strongly supporting the hypothesis of synergism also at the molecular level. Collectively, the data suggest that the combination of a DNMTI and HDACIs is synergistic in in vitro and in vivo model of T-cell lymphoma and is able to synergistically reverse the malignant signature at the molecular level. These data may constitute the basis for future phase I-II clinical trials. Disclosures: O'Connor: celgene: Consultancy, Research Funding; merck: Research Funding; Novartis: Research Funding; spectrum: Research Funding.

Download Full-text

The Bruton’s Tyrosine Kinase Inhibitor CC-292 in Diffuse Large B-Cell Lymphoma (DLBCL), T-Cell Lymphoma (TCL), and Hodgkin Lymphoma (HL): Induction of Cell Death and Examination of Rational Novel/Novel Therapeutic Combinations

Blood ◽

10.1182/blood.v124.21.1772.1772 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1772-1772

Author(s):

Robert A. Cerulli ◽

Ravi Dashnamoorthy ◽

Andrew M. Evens

Keyword(s):

Cell Death ◽

T Cell ◽

Cell Viability ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Mtt Assay ◽

Mtor Inhibitor ◽

Cell Lymphoma ◽

Single Agent ◽

Akt Inhibitor

Abstract Background: Recent clinical trials have highlighted the therapeutic benefit of BTK inhibitors in lymphoma with varying degrees of activity dependent in part on morphology and genetic origin. There appears to be a proclivity of activity in activated B-cell-like (ABC) DLBCL, while the efficacy appears more limited in germinal center (GC) DLBCL. In addition, the activity of BTK inhibitors is not clear in TCL or HL. We sought to determine the in vitro effects of the investigational BTK inhibitor CC-292 as a single agent and in combination with several targeted small molecule inhibitors in DLBCL, TCL, and HL. Methods: DLBCL cell lines (GC: SUDHL6, SUDHL10, Farage, and OCI-LY19; and ABC: OCI-LY3) were treated with increasing concentrations of CC-292 (0.1-20μM) alone and in combination with AKT inhibitor (AZD5363) and dual PI3K/mTOR inhibitor (BEZ235) for 24-72 hours. We analyzed cell viability with MTT assay and expression of NFκB, AKT, mTOR, MEK, and PARP by Western blot analysis. Calcusyn (Biosoft, Ferguson, MO) software program was used to analyze synergistic effects with drug combinations, based on isobologram and the method of Chou and Talay, a combination index (CI) value <1 indicates synergism. We also examined the efficacy (cytotoxicity by MTT) of CC-292 (0.1-10μM) in L428 HL and in Jurkat and HH TCL cell lines. Results: Treatment with CC-292 resulted in a dose dependent decrease in cell viability in all DLBCL lines. The 50% inhibitory concentration (IC50) for Farage, OCI-LY19, SUDHL6, and SUDHL10 were 2.2μM, 6.3μM, 7.2μM, and 3.3μM respectively. Furthermore, treatment with 2.5 to 10μM of CC-292 effectively reduced phosphorylation of mTOR, AKT, and MEK, while it increased phosphorylation of NFκB (p65) and cleaved PARP in Farage, OCI-LY19, and SUDHL10. Treatment with CC-292 in combination with BEZ235 (PI3K/mTOR inhibitor) showed synergistic cell death in Farage cells (CI value at IC50, 75, and 90: 0.842, 0.731, and 0.661, respectively). Further, CC-292 treatment in combination with AZD5363 (AKT inhibitor) also resulted in synergistic cell death in Farage cells (CI value at IC50, 75, 90: 0.772, 0.771, 0.698) (Figure 1). In addition, CC-292 in combination with AZD5363 (AKT inhibitor) significantly down regulated phosphorylation of AKT and mTOR downstream targets, p70 S6K, and eIF4E by Western blotting compared with either drug as a single agent. Finally, in HL and TCL cells, CC-292 was highly effective in reducing cell viability with the following IC50 values in the presence of CC-292: 2.8μM and 2.2 μM for HH and Jurkat (TCL), respectively, and 7.1μM for L428 (HL) cells (Figure 2). Conclusion: In summary, we demonstrated in this preclinical investigation the efficacy of the BTK inhibitor, CC-292, as a single agent as well as in synergistic combinations with an AKT small molecule inhibitor and a dual PI3K/mTOR inhibitor at clinically relevant doses in ABC-DLBCL and multiple GC-DLBCL cell lines. Furthermore, these synergistic combinations decreased mTOR and AKT dependent phosphorylation to a significant extent. Additionally, we demonstrated the cytotoxic effect of CC-292 in HL and TCL cell lines. Collectively, these results suggests that CC-292 is active as a single agent in TCL and HL and novel/novel combinations with PI3K/mTOR or AKT inhibitors may have potential therapeutic value in the treatment of DLBCL. Figure 1: Farage lymphoma cells treated with CC-292, AKT inhibitor (AZD5363), or both and cell viability measured with MTT assay (72 hours). Figure 1:. Farage lymphoma cells treated with CC-292, AKT inhibitor (AZD5363), or both and cell viability measured with MTT assay (72 hours). Figure 2: T-Cell Lymphoma (HH and Jurkat) and Hodgkin Lymphoma (L428) cell lines treated with CC-292 and cell viability measured by MTT assay (72 hours). Figure 2:. T-Cell Lymphoma (HH and Jurkat) and Hodgkin Lymphoma (L428) cell lines treated with CC-292 and cell viability measured by MTT assay (72 hours). Disclosures No relevant conflicts of interest to declare.

Download Full-text

BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽

10.1136/esmoopen-2018-000387 ◽

2018 ◽

Vol 3 (6) ◽

pp. e000387 ◽

Cited By ~ 10

Author(s):

Chiara Tarantelli ◽

Elena Bernasconi ◽

Eugenio Gaudio ◽

Luciano Cascione ◽

Valentina Restelli ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Antitumour Activity ◽

Single Agent ◽

Treatment Strategies ◽

Bet Inhibitor ◽

Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

Download Full-text