Targeting Glutamine Metabolism Overcomes Resistance to Targeted Therapies in Refractory Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin's lymphoma characterized by high refractory occurrence following drug treatment. Despite the encouraging initial MCL tumor response to ibrutinib (IBN), relapse occurs only after few months of treatment due to multiple resistance mechanisms. Thus, the novel therapeutic strategies targeting resistant mechanisms are crucial. Our group has recently shown that among the highly proliferative MCL population, a subpopulation of IBN-R cells exhibits increased OXPHOS activity that is fueled by increased glutaminolysis and rely more on mitochondrial respiration for their grow and survival. The aim of this work was to uncover potential targets responsible for the upregulation of OXPHOS pathway in the refractory/relapsed (R/R) MCL by using multiple biochemical and biological strategies. We focused the present study on glutaminase (GLS), the enzyme that converts glutamine to glutamate, a precursor of α-ketoglutarate (α-KG) that links glutamate to the TCA cycle. Incorporation of α-KG into the TCA cycle is a major anaplerotic step in proliferating cells and is critical for the maintenance of TCA cycle function. To further demonstrate the reliance of OXPHOS on glutamine anaplerosis, we have further tested the combinatory effects of targeting GLS and OXPHOS using their respective inhibitors, CB-839 and IACS-010759, on tumor killing activity in R/R MCL. Methods:Primary MCL cells from patient leukapheresis or whole blood specimens, as well as established MCL cell lines were used as experimental models of MCL. Metabolomic profiling was used to determine intracellular metabolite fluxes and levels. Cell Titer Glo assay was used to measure cell proliferation/viability after treatment with inhibitors. Annexin V and propidium iodide were used to measure cell apoptosis and cell cycle arrestviaflow cytometry analysis. Magnetic microbeads-based B-cell isolation method were used for the purification of malignant B cells from patient samples. Western blot analysis was used to evaluate protein level expression. Patient-derived Xenograft (PDX) mouse model created from patients with MCL was used to evaluate the in vivo anti-tumor activity and potential clinical value of GLS and OXPHOS inhibitors. Results:Our recent metabolomic profiling studies have demonstrated that glutaminolysis and OXPHOS are upregulated in IBN-R MCL, manifested by increased glutamine uptake in the ibrutinib-resistant MCL cell lines (p=0.03).Inhibition of glutamine metabolism with the allosteric GLS1-selective inhibitor BPTES resulted in inhibition of cell viability (0.2381uM-9.98uM), indicating that MCL cells are dependent on glutamine metabolism for their proliferation. To corroborate with the above finding, we also presented evidence that GLS1 is highly increased in IBN-R and CART-R MCL patient samples and cell lines confirmed by immunoblotting. Inhibiting of GLS would lead to significant reduction in OXPHOS, mitochondria membrane potential and ATP production, as either single drug or in combination with other targeting agents. To identify a clinical actionable GLS inhibitor for the treatment of MCL, we chose a GLS1 specific inhibitor CB-839 (Selleckchem), currently under several phase II and III clinical trials investigation on solid tumors. Inhibiting GLS1 with CB-839 leads to the decreased cell viability in MCL (0.5626nM-308.4nM). Of note, the treatment with CB-839 to MCL cell lines induces S phase reduction in both Jeko-1 (17.23%) and Z-138 (14.01%), as well as induces significant apoptosis (p=0.013 and p=0.002 in Jeko-1 and Z-138 cells). GLS inhibition will be further explored in the context of mitochondria defect or hypoxia, where OXPHOS maybe deficient. Importantly, while CB-839 is continuing its validation in several solid tumor models, this is the first study providing data on its efficacy in preclinical models of MCL. Conclusion:In conclusion, we report that glutaminolysis and OXPHOS are upregulated in IBN-R MCL that could be partially due to high expression of GLS1. Our preliminary results revealed that the new GLS inhibitor, GCB-839, may present a clinical potential for a new indication and warrants more in-depth investigation. Deciphering the mechanisms involved in MCL metabolic heterogeneity and adaptability during drug resistance development would be crucial to identify key actors enabling MCL cells to escape from therapy. Disclosures Wang: Acerta Pharma:Research Funding;Molecular Templates:Research Funding;InnoCare:Consultancy;Oncternal:Consultancy, Research Funding;Celgene:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Targeted Oncology:Honoraria;MoreHealth:Consultancy;Kite Pharma:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Lu Daopei Medical Group:Honoraria;OMI:Honoraria, Other: Travel, accommodation, expenses;Verastem:Research Funding;Nobel Insights:Consultancy;BioInvent:Research Funding;Guidepoint Global:Consultancy;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;Juno:Consultancy, Research Funding;Dava Oncology:Honoraria;Loxo Oncology:Consultancy, Research Funding;Pulse Biosciences:Consultancy;OncLive:Honoraria;Beijing Medical Award Foundation:Honoraria;VelosBio:Research Funding.

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

Blood ◽

10.1182/blood.v114.22.2704.2704 ◽

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.

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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 ◽

10.1182/blood.v118.21.1654.1654 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1654-1654 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood-2019-127931 ◽

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.

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Targeting Transcription Checkpoint Using a Novel CDK9 Inhibitor in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-140865 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-29

Author(s):

Junwei Lian ◽

Yu Xue ◽

Alexa A Jordan ◽

Joseph McIntosh ◽

Yang Liu ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Mouse Models ◽

Cell Lines ◽

Cell Apoptosis ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Mantle Cell ◽

Cdk9 Inhibitor

Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that accounts for 5-8% of all non-Hodgkin lymphomas. Despite the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax (ABT-199) have proven to be effective therapeutic strategies for MCL, most patients often experience disease progression after treatment. Thus, developing a novel drug to overcome this aggressive relapsed/refractory malignancy is an urgent need. Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase belonging to the CDK family which regulates multiple cellular processes, particularly in driving and maintaining cancer cell growth. Unlike classical CDKs, CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex that mediates transcription elongation and mRNA maturation via phosphorylating RNA polymerase II (RNAP2). Previous studies demonstrated that CDK9 inhibition downregulates transcription levels of MCL-1 and MYC, which are crucial in both survival and proliferation of acute myeloid leukemia and diffuse large B-cell lymphoma. We and others found that the MYC signaling pathway was enhanced in MCL, especially in ibrutinib-resistant MCL patients. MYC is a core transcription factor driving lymphomagenesis. It does not possess enzymatic activity and has long been considered to be undruggable. MCL-1 is a key anti-apoptotic protein and is overexpressed in several hematologic malignancies. It was also found to be overexpressed in ibrutinib or venetoclax-resistant MCL cells. Thus, CDK9 is considered as a potential target that may inhibit MYC and MCL-1 pathways. Although recently it was shown that MC180295, a novel selective inhibitor of CDK9, has nanomolar levels anti-cancer potency, whether its beneficial effects extend to relapsed/refractory MCL has not yet been assessed. Methods We use three paired MCL cells sensitive/resistant to ibrutinib or venetoclax to test the efficacy of CDK9 inhibitor MC180295. Cell viability was measured by using Cell Titer Glo (Promega). Cell apoptosis assay and western blot analyses were used to identify affected pathways after MC180295 treatment. Finally, we used patient-derived xenograft (PDX) mouse models to test the therapeutic potential of MC180295 in MCL. Results First, we examined the potential efficacy of a CDK9 inhibitor MC180295 in MCL cells. MC180295 treatment results in growth inhibition of ibrutinib-resistant or venetoclax-resistant MCL cells. By assessing the caspase 3 and PARP activity, we found that MC180295 treatment induces cell death via cell apoptosis in MCL cell lines. Meanwhile, we found that RNAP2 phosphorylation at Ser2, the active form of RNAP2, is downregulated in MC180295 treated MCL cell lines. Consistent to previous studies, MC180295 treatment significantly reduces the protein level of MYC and MCL-1. In addition, we identified several other important proteins, such as cyclin D1 and BCL-XL, were also downregulated upon MCL180295 treatment. MC180295 was able to overcome ibrutinib-venetoclax dual resistance in PDX mouse models without severe side effects. To improve the efficacy of MC180295 as a single agent, we performed in vitro combinational drug screen with a number of FDA-approved or investigational clinical agents and found that MC180295 had a synergistic effect with venetoclax. We are currently investigating the underlying mechanism of action. Conclusion Taken together, our findings showed that targeting CDK9 by its specific inhibitor MC180295 is effective in targeting MCL cells, especially those with ibrutinib or venetoclax resistance and therefore supports the concept that CDK9 is a new target to overcome ibrutinib/venetoclax resistance in MCL. Disclosures Wang: MoreHealth: Consultancy; Dava Oncology: Honoraria; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Molecular Templates: Research Funding; Verastem: Research Funding; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; 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; Lu Daopei Medical Group: Honoraria; 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.

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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 ◽

10.1182/blood.v118.21.4972.4972 ◽

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.

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Inhibition of LINK-A lncRNA overcomes ibrutinib resistance in mantle cell lymphoma by regulating Akt/Bcl2 pathway

PeerJ ◽

10.7717/peerj.12571 ◽

2021 ◽

Vol 9 ◽

pp. e12571

Author(s):

Ye Zhang ◽

Peng Lu ◽

Yan Zhou ◽

Lifei Zhang

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Lines ◽

Cell Apoptosis ◽

Cell Lymphoma ◽

Akt Signaling ◽

Mantle Cell ◽

Ibrutinib Resistance ◽

Bcl2 Expression

Ibrutinib, a bruton tyrosine kinase (BTK) inhibitor which suppresses B-cell receptor signaling, has remarkably improved the outcome of patients with mantle cell lymphoma (MCL). However, approximately 33% of MCL patients have primary Ibrutinib resistance, and acquired Ibrutinib resistance is nearly universal. Long intergenic non-coding RNA for kinase activation (LINK-A) exerts oncogenic role in different types of tumors, but the role of LINK-A in intrinsic ibrutinib resistance in MCL is still unclear. Here, LINK-A expression level was first assessed using quantitative Real-time PCR (qPCR) and immunofluorescence analysis in five MCL cell lines. The effect of LINK-A on regulating MCL cells viability and apoptosis was assayed using CCK-8 and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. The association of LINK-A with AKT activation and B cell lymphoma 2 (Bcl2)expression was evaluated using qPCR and western blot analysis. We found that LINK-A level was elevated in Ibrutinib-resistant MCL cell lines (Mino, REC-1, MAVER-1, and Granta-519) compared to Ibrutinib-sensitive MCL cell lines (Jeko-1). Functionally, LINK-A overexpression in Jeko-1 cells enhanced cell viability and repressed Ibrutinib-induced cell apoptosis. LINK-A knockdown in MAVER-1 cells decreased cell viability and further accelerated Ibrutinib-induced cell apoptosis. LINK-A overexpression enhanced Bcl2 expression in Jeko-1 cells, and Bcl2 inhibition blocked the effect of LINK-A on increasing cell viability in the presence of Ibrutinib. On the contrary, LINK-A knockdown reduced Bcl2 expression in MAVER-1 cells, and Bcl2 overexpression damaged the role of LINK-A inhibition in regulating cell viability. Mechanistically, LINK-A positively regulated the activation of AKT signaling, and inhibition of AKT signaling destroyed LINK-A-induced increased of Bcl2 and resulted in a subsequent suppression of cell viability. Taken together, the current results demonstrate that LINK-A inhibition overcomes Ibrutinib resistance in MCL cells by regulating AKT/Bcl2 pathway.

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FoxM1-Mediated Signaling Promotes the Progression of Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2018-99-119349 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4176-4176

Author(s):

Hui Guo ◽

Yixin Yao ◽

Hui Zhang ◽

Elizabeth Lorence ◽

Makhdum Ahmed ◽

...

Keyword(s):

Cell Viability ◽

B Cell ◽

Board Of Directors ◽

Stromal Cells ◽

Research Funding ◽

Tumor Volume ◽

Cell Lymphoma ◽

Advisory Committees ◽

Mantle Cell

Abstract Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma subtype with elevated B-cell receptor activity. Ibrutinib (IBN), the Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have an overall response rate of 68% in relapsed or refractory MCL patients (Wang et al., NEJM, 2013). However, with the emergence of IBN resistance, novel therapies to thwart resistance are urgently needed. FoxM1 (Forkhead box M1) is a proliferation-associated transcription factor that stimulates cell proliferation and exhibits a proliferation-specific expression pattern. FoxM1 has recently been classified as a human proto-oncogene and we have previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, we investigated the prognostic significance of FoxM1 and its potential as a new MCL therapeutic target. Methods We assessed the anti-MCL effects of targeting FoxM1 in both ibrutinib-sensitive and -resistant MCL cell lines and clinical specimens. Cell viability assays were conducted targeting FoxM1 with thiostrepton, a published FoxM1 inhibitor. The drug screening was performed in a 96-well format in which MCL cells were seeded at 10,000 cells per well and were treated with the FoxM1 inhibitor thiostrepton at the following concentrations: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 uM. Cell viability was tested using the CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation period. Furthermore, we investigated the importance of FoxM1 signaling in tumor migration and adhesion in Jeko-1 WT and BTK KD (generated from Jeko-1 using CRISPR/Cas9) cells. Jeko-1 WT and two BTK KD variants were transiently transfected with control siRNA and siRNA against FoxM1 (siFoxM1) for 24 and 48 hours. Afterwards, cells were loaded into transwell migration inserts in the presence or absence of human stromal cells. Migration was evaluated by counting migrated cells and normalizing the migrated cells to migration in the absence of stromal cells. The in vivo efficacy of thiostrepton was evaluated in two cell line Jeko-1 and Jeko-1 BTK KD mouse xenograft models. Upon engraftment, thiostrepton was administered intravenously five consecutive days a week at 50 mg/kg, and tumor burden was assessed via the measurement of circulating human β2M levels and tumor volume. FoxM1 plasma levels in the xenografted mice were also evaluated using ELISA at 0, 10, 20, and 30 days. Results Inhibition of FoxM1 with thiostrepton reduced the cell viability of both ibrutinib-sensitive (Jeko-1; SP-49; PT-1; PT-2) cell lines and patient samples as well as ibrutinib-resistant MCL cells (Maver-1; Z138; Jeko-BTK KD 1 and 2; PTs 3-6) at half-maximal inhibitory concentration (IC50) in the micromolar range (IC50 = 1-3 μM) for the majority of tested cells and patient samples. siFoxM1 significantly reduced (P < 0.05) stromal cell-mediated migration of both Jeko-1 WT and BTK KD cells compared with the cells transfected with control siRNA. Moreover, siFoxM1-treated cells showed reduced levels of the migration- and adhesion-related proteins snail, vimentin, and N-cadherin compared with the control cells. Additionally, in comparison to vehicle-treated control mice, thiostrepton treatment significantly reduced tumor volume (36% and 17%, respectively) and β2M levels (71% and 79%, respectively) in Jeko-1 and Jeko-1 BTK KO xenografted mice at Day 30 of treatment regardless of BTK status. Lastly, thiostrepton treatment significantly suppressed the plasma levels of FoxM1 in both Jeko-1 (14%) and Jeko-1 BTK KO (11%) xenografted mice at Day 30 of treatment. Conclusion We have shown that FoxM1 inhibition may be a potential candidate treatment for MCL based on the results of our clinicopathological assessment and in vivo studies. Therefore, exploring the role of FoxM1 in MCL disease progression and therapeutic resistance may lead to novel therapeutic breakthroughs to improve patient clinical outcomes. Disclosures Wang: MoreHealth: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Research Funding; Kite Pharma: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Honoraria, Research Funding; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria.

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A microRNA Cluster as a Target of Genomic Amplification in B-Cell Lymphomas.

Blood ◽

10.1182/blood.v106.11.3011.3011 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3011-3011

Author(s):

Hiroyuki Tagawa ◽

Kennosuke Karbe ◽

Koichi Ohshima ◽

Yasuo Morishima ◽

Shigeo Nakamura ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Northern Blot ◽

Cell Lymphoma ◽

Lymphoma Cell ◽

Genomic Amplification ◽

T Cell Lymphomas ◽

Mantle Cell ◽

Microrna Cluster

Abstract Background: Genomic gain/amplification of 13q31-q32 is frequently observed in malignant lymphomas. C13orf25, recently established as a candidate gene in malignant lymphoma via 13q31-q32 genomic amplification, encodes two variant transcripts by alternative splicing (Ota et al, Cancer Res 2004). Seven microRNA genes (miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20 and miR-92) are clustered in C13orf25 transcript variant 2 (C13orf25 v2). Because microRNAs display dynamic temporal and spatial expression patterns, disruption of these microRNAs may be associated with tumorigenesis. Purpose: The purposes of this study are i) to reveal frequencies of the 13q gain/amplification in various lymphoma types, and ii) to examine the expression of C13orf25 v2 and seven microRNAs using various lymphoma cell lines and tumors with and without 13q gain/amplification. Experimental Design: We analyzed genomic alterations of chromosome 13 for 12 malignant lymphoma cell lines (eight B-cell and four T-cell lymphomas), and 214 cases of B-cell lymphomas (136 cases of diffuse large B-cell lymphoma (DLBCL), 27 cases of sporadic Burkitt’s lymphoma (sBL), 29 of mantle cell lymphoma (MCL), 22 of follicular lymphoma (FCL)) and 20 cases of T-cell lymphoma by using array-based comparative genomic hybridization. The expression levels of seven microRNAs using 12 lymphoma cell lines with (four) and without (eight) 13q gain/amplification were examined by Northern-blot and quantitative real-time PCR (RQ-PCR) analyses. RQ-PCR for C13orf25 v2 (microRNA cluster) was also conducted for 21 cases of DLBCL (eight cases with 13q gain/13 cases without), 10 cases of sBL (four cases with 13q gain/amp/six cases without) and 10 cases of mantle cell lymphoma. Results: Frequent (> 20%) gain/amplification of 13q were detected in DLBCL (31 cases, 23%) and Burkitt’s lymphoma (8 cases, 30%) but no gain/amplification at 13q was found in MCL, FCL and T-cell lymphomas. Genomic amplification of 13q31-q32 was observed in four cases of DLBCL and two cases of sBL, four of which were c-MYC rearranged (two cases of DLBCL and two cases of sBL). RQ-PCR and Northern blot analyses revealed that five of the seven mature microRNAs displayed overexpression in lymphoma cell lines with 13q31 genomic gain/amplification but not in those without. RQ-PCR analysis for 21 cases of DLBCL demonstrated that the cases with 13q gain/amplification (8 cases) showed significantly higher expression of C13orf25 v2 than those without (13 cases) (Mann Whitney U test, P < 0.05). Significant higher levels of the five microRNAs in sBL with 13q gain/amplification were also confirmed by Northern blot analysis. Lower expression levels of the microRNAs were found in T cell lymphoma cell lines and tumors. Conclusion: These results suggest that the microRNA cluster (C13orf25 v2) is a target of 13q/13q31 genomic gain/amplification in DLBCL and sBL.

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Combination Anti-CD74 (Milatuzumab) and CD20 (Rituximab) Monoclonal Antibody Therapy Has in Vitro and in Vivo Activity in Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v112.11.886.886 ◽

2008 ◽

Vol 112 (11) ◽

pp. 886-886 ◽

Cited By ~ 1

Author(s):

Lapo Alinari ◽

Erin Hertlein ◽

David M. Goldenberg ◽

Rosa Lapalombella ◽

Fengting Yan ◽

...

Keyword(s):

Monoclonal Antibody ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Combination Treatment ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Preclinical Model ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of Rituximab in treatment of B-cell malignancies, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), Milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since Rituximab and Milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that the MCL cell lines Mino and JeKo, and MCL patient tumor cells, express abundant surface CD74 compared to the CD74-negative cell line, Jurkat. Incubation of Mino and JeKo cells with immobilized (goat anti-human IgG) Milatuzumab (5 μg/ml) resulted in mitochondrial depolarization and significant induction of apoptosis determined by Annexin V/PI and flow cytometry (apoptosis at 8hr=38.3±0.85% and 25.4±2.6%; 24hr=73.6±3.47% and 36±3.57%; 48hr=84.9±3.91% and 50.4±4.17%, respectively, compared to Trastuzumab (control). Expression of surviving cells from anti-CD74-treated MCL cells consistently demonstrated marked induction of surface CD74 (MFI 762) compared to control (MFI 6.1). Incubation with immobilized Rituximab (10 μg/ml) resulted in 39.5±2.5% and 37.1±8.35% apoptotic events at 8hr, 58.8±3.14%, 41.2±8.27% at 24hr, and 40.1±1.3% and 45.6±3.25% at 48hr, respectively. Combination treatment of Mino and JeKo cells with Milatuzumab and Rituximab led to significant enhancement in cell death, with 77.6±3.95% and 79.6±2.62% apoptosis at 8hr in Jeko and Mino cells (P=0.0008 and P=0.00004 vs. Milatuzumab alone; P=0.00015 and P=0.001 vs. Rituximab alone); 90.4±3.53% and 76.6±4.3% at 24hr, respectively (P=0.0042 and P=0.0002 vs. Milatuzumab, P=0.0003 and P=0.0027 vs. Rituximab alone); 92.8±0.77% and 85.6±2.62% at 48hr, respectively (P= 0.026 and P=0.0002 vs. Milatuzumab alone, P=0.0000005 and P=0.00008 compared to Rituximab alone, respectively). To examine the in vivo activity of Rituximab and Milatuzumab, a preclinical model of human MCL using the SCID (cb17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, intravenous injection of 40×106 JeKo cells results in disseminated MCL 3–4 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice were treated starting at day 17 postengraftment with intraperitoneal Trastuzumab mAb control (300 μg qod), Milatuzumab (300 μg qod), Rituximab (300 μg qod), or a combination of Milatuzumab and Rituximab. The mean survival for the combination-treated group was 55 days (95%CI:41, upper limit not reached as study was terminated at day 70), compared to 33 days for Trastuzumab-treated mice (95% CI:31,34), 35.5 days for the Milatuzumab-treated mice (95% CI:33,37), and 45 days for the Rituximab-treated mice (95%CI:30,46). The combination treatment prolonged survival of this group compared to Trastuzumab control (P=0.001), Milatuzumab (P=0.0006) and Rituximab (P=0.098). No overt toxicity from Milatuzumab or the combination regimen was noted. A confirmatory study with a larger group of mice and detailed mechanistic studies are now underway. These preliminary results provide justification for further evaluation of Milatuzumab and Rituximab in combination in MCL.

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Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.436.436 ◽

2011 ◽

Vol 118 (21) ◽

pp. 436-436 ◽

Cited By ~ 3

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.

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