Activity of Rituximab and Ofatumumab Against Mantle Cell Lymphoma (MCL) in Vitro in MCL Cell Lines and Ex Vivo in Tumor Cells Derived From Patient Tumor Samples

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4972-4972
Author(s):  
Matthew J. Barth ◽  
Gopichand Pendurty ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
John Gibbs ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Clinical Investigations ◽  
Mantle Cell ◽  
Anti Cd20

Abstract Abstract 4972 Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma (NHL) that frequently presents with advanced stage disease. The addition of rituximab, a monoclonal anti-CD20 antibody, to high dose chemotherapy regimens often followed by stem cell transplant has improved outcomes, but survival still remains low at 3–5 years. Novel agents are needed to improve outcomes in MCL. Ofatumumab is a fully human anti-CD20 monoclonal antibody directed against a novel epitope on the CD20 antigen. Ofatumumab has been shown to be more potent than rituximab against B-NHL cells in pre-clinical investigations. Ofatumumab is FDA approved for the treatment of CLL that is fludarabine and alemtuzumab refractory or with bulky disease resistant to fludarabine and is being investigated in clinical trials in NHL. In order to characterize the activity of ofatumumab against MCL, we performed pre-clinical investigations into the activity of ofatumumab against MCL cell lines and primary MCL tumor cells derived from patient tumor samples (n=2). Antibody-dependant cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in the MCL cell lines Mino, Jeko, Rec-1 and Z-138 to demonstrate sensitivity to rituximab and ofatumumab. Lymphoma cells were labeled with 51Cr prior to incubation with rituximab or ofatumumab at 10ug/mL plus human serum or effector cells (efector:target ratio of 20:1). 51Cr-release was measured and the percentage of lysis was calculated. Patient tumor cells were isolated from tumor biopsy samples by MACS sorting (negative selection). Patient tumor cells were incubated with ofatumumab or rituximab at 10ug/mL in the presence of human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Means were compared using a t-test. Expression of CD20 and the complement inhibitory proteins (CIPs) CD55 and CD59 in MCL cell lines were determined by flow cytometry and compared to the rituximab-sensitive cell line Raji and the rituximab-resistant cell line Raji 4RH. Surface density of CD20, CD55 and CD59 were determined by Imagestream analysis. Western blot was performed to measure total CD20 protein expression. Ofatumumab induced significantly higher levels of cell lysis compared to rituximab in CDC assays of all MCL cell lines tested (Mino: 65.9% vs 0.5%; JeKo 43.9% vs 13.3%; REC-1 25.4% vs 4.7%; Z-138: 56.4% vs 0.65%; all p-values <0.05). The ADCC assays showed a similar degree of lysis with ofatumumab when compared to rituximab in all cell lines tested. In primary tumor cells, ofatumumab and rituximab demonstrated similar levels of decreased cell viability following 48 hours of antibody exposure. MCL cell lines demonstrated similar expression of surface and total CD20 when compared to the rituximab-sensitive B-NHL Raji cell line. CIP expression was increased in all MCL cell lines compared to Raji cells and was similar to the rituximab-resistant Raji 4RH cell line. Our data suggest ofatumumab is more potent than rituximab against MCL cells in vitro and retains CDC activity despite high expression levels of CIPs. This increased activity was not seen in patient tumor samples; however we were limited by the number of available patient samples. In vivo experiments investigating the activity of ofatumumab in a SCID mouse MCL xenograft model and investigations into the activity of ofatumumab in MCL cells in combination with cytotoxic agents and novel small molecule inhibitors are ongoing. Disclosures: Czuczman: Genmab: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding.

Anti-CD20 Antibody GA101 Shows Higher Cytotoxicity but Is Competitively Displaced by Rituximab in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2704-2704
Author(s):  
Daniel A. Heinrich ◽  
Christian Klein ◽  
Kristina Decheva ◽  
Marc Weinkauf ◽  
Grit Hutter ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Deutschland Gmbh ◽  
Mantle Cell ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Abstract 2704 Poster Board II-680 Background: Mantle cell lymphoma (MCL) is characterized by a poor long-term prognosis with a median survival of 3–5 years. Type I anti-CD20 antibody rituximab has demonstrated a clear anti-proliferative effect in MCL and achieves increased response rates in combination with chemotherapy. GA101, a third-generation IgG1 anti-CD20 antibody displays improved ADCC and superior direct cell death induction by virtue of glycoengineering compared to rituximab and its targeting a type II epitope on CD20, respectively. Methods: Using a panel of MCL cell lines (Rec-1, HBL-2, Jeko-1, Granta-519, JVM-2 and Z-138) we determined the effect of GA101 alone as well as in combination with rituximab on cell viability and proliferation. Karpas-422 (Diffuse Large B-Cell Lymphoma) was used as a control cell line. MCL and Karpas-422 cells were treated with GA101 or rituximab at concentrations of 1 – 20μg/ml and rituximab. Cell viability was analyzed by trypan-blue exclusion tests at 0h, 24h, 48h and 72h. The panel of MCL cell lines and Karpas-422 were then treated with GA101 and rituximab each at 1 and 10 μg/ml to determine potential synergism of antibody combinations. Accordingly, a fractional product calculation was performed: synergism > 0,1; antagonism < −0,1. In addition, Western-blot and RNA-array-analyses were performed to elucidate potential intra-cellular downstream pathway mechanisms. Results: After mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (65–75% cell reduction in Granta-519 and 35–40% in Rec-1). Intermediate results were gained for Z-138, HBL-2, Jeko-1 and JVM-2 and Karpas-422 (15–20%). rituximab mono-exposure at 12,5 μg/ml showed a 25% reduction of cell count in Granta-519, 20% in HBL-2 and < 5% in Rec-1, Jeko-1 and Z-138. Combination experiments suggested the competitive binding of the two antibodies. Thus, GA101 plus rituximab combination experiments resulted in a lower cytotoxicity than GA101 alone, according to fractional product calculations. Conclusions: Although GA101 is competitively displaced by rituximab, GA101 demonstrates higher efficacy in MCL cell lines than rituximab, even at a more than 10-fold lower concentration. Currently RNA-array- and Western blot analysis are being performed to identify the critical pathways responsible for the superior cytotoxicity of GA101. Disclosures: Klein: Discovery Oncology, Roche Diagnostics GmbH: Employment. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.

PI3K Inhibition with GDC-0941 Has Greater Efficacy Compared to p110δ-Selective Inhibition with CAL-101 in Mantle Cell Lymphoma and May Be Particularly Advantageous in Multiply Relapsed Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1654-1654 ◽  
Author(s):  
Sunil Iyengar ◽  
Andrew J. Clear ◽  
Andrew Owen ◽  
Lenushka Maharaj ◽  
Janet Matthews ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Western Blotting ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Pi3k Inhibition ◽  
Mantle Cell ◽  
Morphological Variants ◽  
Gsk 3Β

Abstract Abstract 1654 Background: Mantle cell lymphoma (MCL) is an incurable, aggressive subtype of non-Hodgkin lymphoma in which there is a need for novel targeted therapies. Activation of the PI3K-Akt pathway and its role in the pathogenesis of MCL has been highlighted in a number of studies. Constitutive activation of the PI3K pathway inactivates GSK-3β, a downstream target of Akt, that can phosphorylate cyclin D1 resulting in its nuclear export. There is also evidence that cyclin D1 mRNA stability and translation is enhanced by this pathway. The class Ia PI3K p110 catalytic subunit isoforms α, β and δ are primarily implicated in oncogenesis. While the PI3K p110δ isoform is known to be enriched in lymphocytes, a gain of PIK3CA (the gene encoding PI3K p110α) copy number has been shown to be a frequent alteration in MCL. The expression and relative importance of the individual Class Ia PI3K isoforms has not been documented in this disease. With the development of isoform selective inhibitors, this is an important issue that needs to be addressed. Aims: We studied the expression of class Ia PI3K isoforms in primary MCL with relation to morphological variants and disease status. We also compared the efficacy of PI3K inhibition in MCL cell lines and primary samples using two novel inhibitors, GDC-0941(predominantly p110α/δ-selective) and CAL-101 (δ-selective), both of which are in early phase clinical trials. Methods: Tissue microarrays were constructed from triplicate 1mm cores from 144 MCL biopsies and 16 tonsil controls. The levels of p110α, p110β and p110δ isoforms were then determined by immunohistochemistry using isoform-specific antibodies. The in vitro effect of PI3K inhibitors on cell viability and apoptosis was studied in 4 MCL cell lines, (Jeko-1, Granta519, REC-1 and JVM-2), and 15 primary MCL samples. Expression of the class Ia PI3K isoforms and changes in downstream targets of PI3K were determined by western blotting. Results: P110δ was expressed at a consistently higher level in MCL samples and normal tonsil controls compared to the α and β isoforms, while p110β expression was weak and significantly lower than p110α expression. On comparing expression of isoforms at diagnosis and relapse, p110α expression was significantly increased beyond 1st relapse compared to diagnostic biopsies (p=0.04) and tonsil controls (p=0.02), an observation that was even more apparent in 6 paired samples [p=0.008, median IHC score 19.6 (5.0−53.2) at diagnosis vs. 91.5 (38.6 − 129) beyond 1st relapse]. No significant change was found in the expression of p110β or p110δ between diagnostic and relapse samples. There was no significant difference in expression levels of the 3 isoforms between blastoid and non-blastoid morphological variants. Expression of both the p110α and δ isoforms was detected by western blotting in 4 MCL cell lines, but only Jeko-1 cells were sensitive to inhibition with GDC-0941. CAL-101 produced little or no apoptosis in all 4 cell lines. In primary MCL samples, GDC-0941 was consistently more potent than CAL-101, with decrease in cell viability of 32 vs. 20% at 1μM (p=0.15), 51 vs. 25% at 5μM (p=0.02) and 67 vs. 35% at 10μM (p<0.0001) GDC-0941 and CAL-101 respectively. GDC-0941 was also able to partially overcome the stimulatory effect of sCD40L and IL4 on primary MCL samples. Western blotting showed a consistent reduction in the phosphorylation of Akt and GSK-3β in sensitive MCL cells. Conclusion: Our studies demonstrate that although p110δ is the most consistently expressed isoform, the expression of the p110α subunit increases significantly in multiply relapsed MCL. This observation, in combination with significantly greater in vitro sensitivity of MCL primary samples to GDC-0941, compared to the p110δ-selective inhibitor CAL-101, provides strong evidence for further evaluation of GDC-0941 in this disease. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria. Joel:Astra Zeneca: Research Funding; Intellikine: Research Funding.

Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 436-436 ◽  
Author(s):  
Robert Kridel ◽  
Barbara Meissner ◽  
Sanja Rogic ◽  
Merrill Boyle ◽  
Adele Telenius ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
The Other ◽  
Mantle Cell ◽  
Whole Transcriptome ◽  
Notch1 Mutations

Abstract Abstract 436 Background: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL. Methods: In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data. Results: NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p<.001). No effect of compound E was observed in Mino, the other cell line with a NOTCH1 mutation, nor in the 4 cell lines that are wild type for NOTCH1. Outcome correlation analysis showed that NOTCH1 mutations are associated with poor overall survival (1.56 versus 3.86 years respectively, p=.001), but not with significantly shortened progression-free survival (0.88 versus 1.73 years respectively, p=.07). Discussion: We have identified recurrent mutations in NOTCH1 in a subset of patients with MCL (11.6%). The frequency and the pattern of mutations are strikingly similar to what has recently been reported in chronic lymphocytic leukemia, the other major CD5 positive B-cell malignancy (Nature, 2011 Jun 5, 475:101–105 and J Exp Med, 2011 Jul 4, 208:1389–1401). NOTCH1 mutations are associated with adverse prognosis as evidenced by shortened overall survival. This latter finding, however, should ideally be validated in a larger and uniformly treated cohort. Finally, the sensitivity of the Rec-1 cell line to compound E suggests that NOTCH1 mutations could serve as the target for tailored therapy in mantle cell lymphoma. Disclosures: Sehn: Roche/Genentech: Consultancy, Honoraria, Research Funding. Connors:Roche: Research Funding.

scholarly journals The Development of a Precision Medicine Platform to Overcome Ibrutinib Resistance in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1487-1487
Author(s):  
Yang Liu ◽  
Shuangtao Zhao ◽  
Changying Jiang ◽  
Yixin Yao ◽  
Kelley Paige Murfin ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Akt Pathway ◽  
Therapeutic Resistance ◽  
Patient Sample ◽  
Mantle Cell ◽  
Btk Inhibitor ◽  
Ibrutinib Resistance

Background: While mantle cell lymphoma (MCL) initially responds to frontline therapies, this aggressive B-cell malignancy typically relapses or becomes resistant to treatment. Despite high overall response rates to the oral, covalent, first-in-class Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, most patients with relapsed/refractory MCL ultimately experience disease progression. To address the critical issue of BTK inhibitor resistance, novel therapies must be developed. Moreover, diversity in genetic alterations and the possibility that multiple pathway aberrations may contribute to disease progression and resistance makes overcoming this phenomenon with uniform treatment regimens extremely difficult, indicating that a personalized approach should be developed to overcome therapeutic resistance. In this study, molecular profiling of ibrutinib-resistant MCL patient samples has been conducted to identify targetable dysregulated signaling pathways and gene mutations associated with ibrutinib resistance. Clinical drug candidates targeting these potential pathways and their combinations with ibrutinib were analyzed in vitro to identify novel treatments that may potentially overcome ibrutinib resistance. Methods: Twenty-three agents targeting multiple pathways associated with ibrutinib resistance were deliberately chosen based on known targetable pathways in MCL to assess via high throughput drug screening. MCL tumor cells were isolated and purified from clinical apheresis and tumor excisional biopsies under established IRB-approved protocols. The same patient primary cells were subjected to gene expression analysis using a nanoString nCounter panel and whole exome sequencing (WES) to identify targetable dysregulated pathways and somatic mutations within each tumor. In vitro cell viability assays of single agents and drug combinations were tested per patient sample using the CellTiter-Glo luminescent assay (Promega), interrogating dysregulated pathways identified in each tumor. Subcutaneous, intravenous, and subrenal injections of the purified patient tumor cells were performed on NSG mice to create corresponding PDX mouse models for validation experiments. Results: nanoString nCounter analysis identified differentially expressed targetable pathways per patient sample such as BCR signaling, the PI3K/AKT pathway, NOTCH signaling, the cell cycle, and the NF-κB pathway. Correlations were identified between the WES and the nanoString nCounter analysis. For example, PTEN loss was observed in an MCL patient sample with high PI3K/AKT expression, demonstrating the potential underlying mechanism for the observed PI3K/AKT enrichment. Patients were divided into subgroups based on the identified responsive pathways in the in vitro screening. For example, PI3K/AKT pathway inhibitors were shown to be more potent against MCL samples in which the PI3K/AKT pathway was enriched. To further validate this finding, we created an MCL PDX model using a sample with enriched PI3K/AKT expression and successfully recapitulated the splenomegaly and hepatomegaly observed in the MCL patient. The MCL PDX mice were treated with the pan-PI3K inhibitor copanlisib (IP, 10 mg/kg) using a 5 on/2 off dosing schedule, which resulted in significantly reduced spleen (P &lt; 0.001) and liver size (P &lt; 0.01), as well as bone marrow involvement (P &lt; 0.05), compared with the vehicle control (Figure 1). Conclusions: Molecular matching with in vitro drug screening were utilized to develop a precision medicine platform for MCL to combat therapeutic resistance. This platform can be translated into a clinical setting to directly benefit the MCL patient population through treatment with therapies directly tailored to each patient. Disclosures Wang: Pulse Biosciences: Consultancy; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta Pharma: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Kite Pharma: Consultancy, Research Funding; Juno Therapeutics: Research Funding; Celgene: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; BioInvent: Consultancy, Research Funding; Aviara: Research Funding; BeiGene: Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding.

In Vitro and In Vivo Anti Lymphoma Effect of GX15-070 in Mantle Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4756-4756 ◽  
Author(s):  
Gwyn Bebb ◽  
Huong Muzik ◽  
Sophia Nguyen ◽  
Don Morris ◽  
Douglas A. Stewart
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cytotoxic Agents ◽  
In Vivo Experiments ◽  
Mantle Cell

Abstract Introduction Mantle cell lymphoma (MCL), an incurable B cell lymphoma, consistently over expresses bcl-2 despite not carrying the t(14;18). The attenuation of apoptosis by bcl-2 is thought to contribute to the malignant process and increase resistance to some cytotoxic agents. We recently demonstrated that GX15-070, a small molecular inhibitor of the BH3 binding groove of bcl-2, has activity against MCL cell lines in vitro. We set out to assess the effect of GX15-070 alone and in combination with Vincristine on the viability of MCL cells in vitro and in vivo. Methods 3 previously characterized bcl-2 over expressing MCL cell lines (JVM-2, Hbl-2, granta) were used. Cells were grown in standard media and exposed to a range of concentrations of GX15-070 with and without Vincristine. Dose-response was assessed by measuring viability at 48 hours using the WST-1 assay. In vivo experiments were conducted on immune deficient mice in which 5×106 cells were injected in the flank then treated IV with GX15-070 (q 2days × 5 doses), Vincristine (q4 days × 3 doses) or both starting 5 days later. Tumours were measured three times weekly. Results All three MCL cell lines over-expressed bcl-2 by western blot. Each MCL cell line showed sensitivity to GX15-070 at a range of concentrations. The addition of GX15-070 to low dose Vincristine (10−6) caused significant growth inhibition of each MCL cell line (see table 1). Discussion Our results demonstrate that using GX15-070 to target bcl-2 is an effective anti neoplastic approach against MCL cell lines in vitro. In addition, our results suggest that combining Vincristine and GX15-070 is a promising strategy in treating MCL. In vivo experiments to confirm this additive activity are still ongoing and will be presented in full. Initial impressions suggest that there is a rationale for the addition of GX15-070 to current cytotoxic regimens used to treat MCL in the setting of clinical trials. Table 1: Effect of Vincristine and GX15-070 on in vitro growth of 3 MCL cell lines Growth as % age of Control Cell Line JVM-2 HBL-2 Granta Vincristine alone (10-6 mg/ml) 92% 48% 89% GX15-070 alone (0.08 uM) 75% 76% 60% Vincristine 10-6 mg/ml and GX15-070 0.08 uM 52% 24% 52%

Targeting Oxphos Pathway Against Ibrutinib Resistance to Mantle Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 290-290 ◽  
Author(s):  
Yang Liu ◽  
Taylor Bell ◽  
Hui Zhang ◽  
Yuting Sun ◽  
Carrie J Li ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Gene Set Enrichment Analysis ◽  
Pdx Model ◽  
Mantle Cell ◽  
Ibrutinib Resistance

Abstract Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses to frontline therapy. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) has achieved 68% of overall response rate in relapsed/refractory mantle cell lymphoma (MCL) patients. However, the vast majority of MCL patients experience disease progression, demonstrating that standard-of-care approaches are failing and that a means for targeting ibrutinib resistant MCL is clinically needed. Our hypothesis is that the ibrutinib-resistant MCL may rely on the mitochondrial oxidative phosphorylation (OXPHOS) pathway to produce energy for tumor growth. In this study, we investigated the effects of IACS-010759, a small molecule mitochondrial complex I inhibitor discovered in MD Anderson Cancer Center which can block the OXPHOS pathway, to overcome ibrutinib resistance in MCL in vitro and in a patient-derived xenograft (PDX) model. Methods: The OXPHOS metabolic pathways were investigated by RNASeq in a panel of ibrutinib-sensitive and -resistant MCL samples. Cell growth inhibition assays were tested after 72-hour treatment with IACS-010759 in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, and ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, by CellTiter-Glo luminescent cell viability assay (Promega). Furthermore, an IBN-resistant MCL PDX model was established and the therapeutic effects and tolerability of IACS-010759 were investigated in the primary MCL-bearing PDX model. Results: We have done RNA sequencing (RNASeq) in 7 primary ibrutinib-resistant and 16 ibrutinib-sensitive MCL patient samples, and analyzed the data using Gene Set Enrichment Analysis (GSEA) software. The results demonstrated that the OXPHOS pathway was activated in the primary ibrutinib-resistant MCL cells but not ibrutinib-sensitive MCL cells. Based on the RNASeq data, we selected an OXPHOS inhibitor IACS-010759 to investigate its effects on both primary ibrutinib-resistant and ibrutinib-sensitive MCL cells in vitroand in PDX mice. IACS-010759 significantly inhibited cell proliferation in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, but not in ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, during a 72-hour incubation. Furthermore, the primary ibrutinib-resistant MCL PDX mice were administrated with 10 mg/kg IACS-10759 by oral gavage, for 28 days using a 5 on/2 off dosing schedule. Our data showed that IACS-010759 completely eradicated tumor growth in ibrutinib-resistant MCL PDX mice (n=5, p=0.045). All mice tolerated the treatment dose and no toxicity was found during 28 days of IACS-010759 treatment. Conclusions: The OXPHOS inhibitor IACS-010759 overcomes ibrutinib resistance both in vitro and in the PDX mouse model. The investigation of its mechanism-of-action is ongoing. IACS-010759 could have the potential for clinical use in ibrutinib-resistant relapsed/refractory MCL patients. Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Juno Therapeutics: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Dava Oncology: Honoraria; BeiGene: Research Funding; Acerta: Consultancy, Research Funding.

scholarly journals Venetoclax Resistance in Mantle Cell Lymphoma Is Mediated By BCL-XL and Can be Circumvent By Inhibiting the BH4 Domain of BCL-2

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1507-1507
Author(s):  
Daniela Steinbrecher ◽  
Felix Seyfried ◽  
Eugen Tausch ◽  
Johannes Bloehdorn ◽  
Billy Michael Chelliah Jebaraj ◽  
...  
Keyword(s):  
Cell Viability ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
High Efficiency ◽  
Cell Lymphoma ◽  
Fold Increase ◽  
Dependent Manner ◽  
Independent Manner ◽  
Mantle Cell

Apoptosis is controlled by the expression levels and interplay of pro- and anti-apoptotic BCL-2 family proteins. The specific BCL-2 inhibitor Venetoclax (VEN) showed high efficiency in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). Despite its high efficiency in CLL and MCL, refractory disease can develop. BCL-2 mutations have been described to mediate resistance in CLL cases, however these mutations are only found in a proportion of VEN resistant cases and in a fraction of cells. In order to design alternative therapeutic strategies to overcome drug resistance, a better understanding of the mechanisms mediating resistance to VEN is necessary. VEN-resistant (VEN-R) MCL cell lines (MINO and MAVER-1) were generated by chronic exposure to increasing amounts of VEN (up to 3µM). A significant and stable upregulation of BCL-XL mRNA and protein was seen in the MINO and MAVER-1 resistant cell lines (2 and 4 fold increase in mRNA and 2.6 and 4.5 fold increase in protein, respectively). We used BH3 profiling in combination with VEN treatment for 4h to investigate the differences in anti- and pro-apoptotic signaling in parental and VEN-R cell lines. Additionally, sensitivity to VEN was restored upon shRNA-mediated knockdown of BCL-XL. These results confirmed the importance of BCL-XL upregulation in mediating resistance. Furthermore, we did not detect mutations in BCL-2 upon resistance to VEN via targeted NGS, which is in contrast to results obtained in VEN-R CLL patients (Blombery et al., Cancer Discovery 2019 and Tausch et al., Hematologica 2019). However, the results obtained by dynamic BH3-profiling (VEN treatment in combination with BH3 Profiling) suggest that increase in BCL-XL is most likely not the only alteration necessary to render cells resistant to VEN. In addition, reduced activation of pro-apoptotic proteins like BAX and BAK might contribute to resistance to VEN. In order, to investigate if VEN resistance can be overcome by drug mediated inhibition of BCL-XL we used different therapeutic approaches. Combinational treatment with the BCL-XL inhibitor A-1331852 and VEN or the single treatment with Navitoclax, a combined inhibitor of BCL-2, BCL-W and BCL-XL for 48h reduced cell viability in VEN-R MINO and MAVER-1 cell lines. Furthermore, BDA-366, a BH4 domain BCL-2 inhibitor effectively reduced the cell viability after 48h of treatment in a dose dependent manner in both parental and VEN-R cell lines. The binding of BDA-366 to the anti-apoptotic BCL-2 protein leads to a conformational change into a pro-apoptotic molecule by the exposure of the BH3 domain of the protein. Despite mediating apoptosis in a TP53-independent manner, VEN treatment in CLL has been associated with inferior outcome in the presence of TP53 aberrations. In order to address the role of TP53 dysfunction in mediating resistance to VEN, we generated p53 knock out cell lines (N=2) by CRISPR/Cas9 gene editing. This significantly decreased the sensitivity to VEN compared to p53 WT cell lines. Additionally, the sensitivity to BDA-366 was significantly reduced upon knockout of p53, suggesting an interference of p53 downstream of BCL-2. Overall, VEN resistance is mediated by a permanent increase in BCL-XL mRNA and protein level in MCL. Importantly, BDA-366, which converts the anti-apoptotic BCL-2 molecule into a BAX-like death molecule, could be a potential alternative treatment strategy for BCL-2 dependent cancers even when resistant to VEN. Despite mediating apoptosis in a p53 independent manner, VEN seems to be less effective in p53 deficient cells, underlining the importance of further investigations of treatment combinations in these groups. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau. Schneider:Celgene: Other: travel grant.

scholarly journals Pirtobrutinib Overcomes Ibrutinib and Venetoclax Resistance in Mantle Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1182-1182
Author(s):  
Yang Liu ◽  
Changying Jiang ◽  
Fangfang Yan ◽  
Joseph McIntosh ◽  
Alexa A Jordan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
Mantle Cell ◽  
Btk Inhibitor

Abstract Background Mantle cell lymphoma (MCL) is a rare and aggressive B-cell lymphoma characterized by poor prognosis. Although remarkable therapeutic advances have been made by covalent Bruton's tyrosine kinase (BTK) inhibition and CAR T cell therapy, therapeutic resistance inevitably occurs and leads to dismal clinical outcome. Pirtobrutinib (LOXO-305) is a next-generation, highly selective and non-covalent BTK inhibitor. A phase 1/2 BRUIN study showed that pirtobrutinib demonstrated promising efficacy in heavily pretreated MCL patients with or without prior covalent BTK inhibition. Here, we investigated the mechanism of action of pirtobrutinib in MCL cells in vitro and proposed the potential combination therapy in a venetoclax-resistant xenograft model. Methods MCL cell proliferation was monitored by trypan blue exclusion assay after 24-, 48- and 72-hour treatment with pirtobrutinib and ibrutinib. We performed Annexin V/PI staining to measure the apoptosis inductive effects. Cell cycle analysis using propidium iodide (PI) DNA staining was conducted to compare cell cycle progression kinetics between pirtobrutinib and ibrutinib. We performed RNAseq analysis in Z138 cells to compare differentially expressed genes (DEGs) between pirtobrutinib and ibrutinib treatment. Western blotting was utilized to detect specific signaling proteins. Mino-venetoclax-R cells were inoculated subcutaneously into NSG mice and used for in vivo drug efficacy determination. Results Compared to covalent BTK inhibitor ibrutinib, the novel non-covalent BTK inhibitor pirtobrutinib was more potent in inhibiting MCL cell proliferation in a panel of MCL cell lines, especially in ibrutinib/venetoclax resistant cell lines (pirtobrutinib vs. ibrutinib, p&lt;0.01). Treatment with pirtobrutinib (10μM) for 24 hours induced higher levels of apoptosis than that by ibrutinib in all the MCL cell lines tested (p&lt;0.05), which was also confirmed at the molecular level by stronger caspase-3 activation and PARP cleavage. To understand the mechanism of action, we performed whole transcriptomic profiling by RNAseq analysis using Z138 cells treated with/without pirtobrutinib or ibrutinib. Pirtobrutinib treatment resulted in upregulation of 137 genes and downregulation of 97 genes compared to the ibrutinib treatment (adjusted p&lt;0.05). In addition to the downregulated MYC targets and PI3K/Akt pathway, gene set enrichment analysis (GSEA) revealed a significant enrichment for G2/M checkpoints and E2F targets signatures (key genes: PLK1, CDKN1A and CCNB1) in pirtobrutinib treated cells. Consistently, follow-up studies showed that γH2AX level was highly increased upon pirtobrutinib treatment. Pirtobrutinib treatment but not ibrutinib treatment resulted in G2/M cell cycle arrest. The blockade of cell cycle progression is positively correlated with decreased protein levels of critical regulators of S and G2/M phase transition such as cyclin B and CDC25C. BTK inhibitor (ibrutinib) in combination with venetoclax has shown great efficacy in preclinical models and in MCL patients. Therefore, here we assessed the in vivo efficacy of pirtobrutinib in combination with venetoclax with side-by-side comparison to ibrutinib & venetoclax in the Mino-venetoclax-R mouse model. Pirtobrutinib & venetoclax combination enhanced the efficacy of pirtobrutinib in restraining the tumor size (p&lt;0.001) in the xenograft model. Notably, this novel combinatorial treatment exerted much higher potency than ibrutinib and venetoclax combination therapy (p&lt;0.001). In addition, the pirtobrutinib & venetoclax combination was well tolerated and did not reduce overall mouse body weights compared with the vehicle treated mice. Conclusions Pirtobrutinib overcame both ibrutinib and venetoclax resistance in MCL cells in vitro and in vivo. G2/M checkpoints and E2F targets pathways were significantly enriched in both cases. Pirtobrutinib & venetoclax showed better in vivo efficacy in MCL models than combination of ibrutinib & venetoclax. Figure 1 Figure 1. Disclosures Wang: Genentech: Consultancy; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; CAHON: Honoraria; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Lilly: Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; OMI: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; Chinese Medical Association: Honoraria; Dava Oncology: Honoraria; Celgene: Research Funding; Mumbai Hematology Group: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Newbridge Pharmaceuticals: Honoraria; CStone: Consultancy; BGICS: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Scripps: Honoraria; Epizyme: Consultancy, Honoraria; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; VelosBio: Consultancy, Research Funding; Anticancer Association: Honoraria.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5374-5374 ◽  
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.

scholarly journals AZD4320 Is a Novel and Potent BCL-2/XL Dual Inhibitor in Targeting Aggressive Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-44
Author(s):  
Yijing Li ◽  
Yang Liu ◽  
Joseph McIntosh ◽  
Alexa A Jordan ◽  
Angela Leeming ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Apoptosis ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Publicly Traded ◽  
Dual Inhibitor ◽  
Car T ◽  
Mantle Cell

Introduction: Mantle cell lymphoma (MCL) is a rare subtype of B-cell non-Hodgkin's lymphoma. It is an incurable disease with frequent relapse from chemotherapies, targeted therapies, and cell therapies. Dysregulated expression of BCL-2 family members resulting in enhanced cell survival frequently occurs in many cancer types and often contributes to the development of therapeutic resistance. The BCL-2 inhibitor venetoclax has been shown to be effective in treating refractory/relapsed MCL patients. However, resistance often occurs and one of the underlying mechanisms of this resistance is the increased expression of other anti-apoptotic BCL-2 family members, such as BCL-XL and MCL-1. In this study, we assessed the in vitro and in vivo efficacy of a novel and highly potent BCL-2/XL dual inhibitor AZD4320 in preclinical models. Methods: Cell viability assay was tested after 72-hour treatment with AZD4320 in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines by CellTiter-Glo (Promega). The assay was also done after a 24-hour treatment in primary PDX cells. Cell apoptosis assay was performed to determine if AZD4320 induces cell apoptosis in MCL cell lines. Furthermore, the in vivo efficacy of AZD4320 was assessed in a CAR-T resistant MCL patient-derived xenograft (PDX) model. Results: AZD4320 significantly inhibited cell proliferation in all tested MCL cell lines, including both ibrutinib/venetoclax-sensitive and -resistant cell lines. It had an IC50 value at a low nanomolar range between 0.59 nM to 18 nM. Consistently, AZD4320 was effective in targeting primary PDX cells ex vivo. AZD4320 induced cell apoptosis in a dose-dependent manner. AZD0466, the nanomedicine formulation of AZD4320 (30mg/kg, weekly, IV), dramatically inhibited tumor growth and prolonged mouse survival in an ibrutinib-CAR-T dual-resistant PDX mouse model. All mice tolerated the treatment dose without any body weight loss. Conclusion: The novel BCL-2/XL dual inhibitor AZD4320 demonstrated excellent anti-MCL activity in both ibrutinib/venetoclax-sensitive and -resistant MCL cells in vitro. This was further validated in vivo in a ibrutinib-CAR-T dual-resistant PDX model. These findings provide evidence that dual targeting of BCL-2 and BCL-XL by AZD4320 is promising as it may overcome therapeutic resistance in relapsed/refractory MCL. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado:AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang:OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Loxo Oncology: Consultancy, Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OncLive: Honoraria; Lu Daopei Medical Group: Honoraria; Acerta Pharma: Research Funding; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; Pulse Biosciences: Consultancy; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Beijing Medical Award Foundation: Honoraria; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; Guidepoint Global: Consultancy; Targeted Oncology: Honoraria; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; InnoCare: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document