Primary Mantle Cell Lymphoma Cells Research Articles

Superior Pre-Clinical Activity of BET (Bromodomain and Extra terminal) Protein Antagonist Combined with Ibrutinib, Panobinostat or Carfilzomib Against Human Mantle Cell Lymphoma (MCL) Cells

Poor-risk Mantle Cell Lymphoma (MCL) demonstrate several genetic alterations involving cyclin D1, RB1, ATM, p53, INK4a/ARF, BMI1, HDM2, CDK4, CDKN2A, NOTCH1 & 2, c-MYC, BCL2 and BIM, as well as in the Toll like receptor (TLR), B-cell receptor (BCR) and NFkB signaling genes. Activated BCR signaling is also associated with downstream pro-growth and pro-survival activity of NFκB in the poor-risk MCL. Pre-clinical and clinical studies have shown that ibrutinib, a selective, orally bioavailable, irreversible inhibitor of BTK is active against B cell neoplasms, especially MCL. Treatment with ibrutinib induces clinical responses, but long-term durable remissions and cure remains uncertain. BET (bromodomain and extra terminal) protein family members, including BRD4, bind to acetylated lysine(s) on the histone proteins, help assemble transcriptional co-regulators at the transcriptionally active target oncogene 'super’ enhancers and promoters, and regulate the expression of important MCL-relevant oncogenes, e.g., c-MYC, BCL-2, CDK4/6 and cyclin D1. Additionally, BRD4 has been shown to bind acetylated RelA and is essential for NFκB activity. Here, we determined that treatment with the prototype BET protein antagonist JQ1 (250 to 2000 nM) or I-BET151, which disrupts the binding of BRD4 to acetylated histones, dose-dependently inhibited growth and exerted lethal activity against cultured (MO2058 and Mino, Z138 and JeKo-1) and patient-derived, primary MCL (pMCL) cells. Treatment with JQ1 reduced the occupancy of BRD4 and RNA pol II (RNAP2) on the promoters of c-MYC and BCL2, as well as attenuated the mRNA (by qPCR) and protein expression (by Western blot) of c-MYC, BCL2, MCL-1 and, CDK4/6, without affecting the levels of SOX11, but induced the levels of HEXIM1, p21, p27 and cleaved PARP in MCL cells. Importantly, JQ1 treatment reduced p-BTK, BTK and p-PLCγ2 levels in MCL cells. JQ1 treatment also attenuated the nuclear RelA (by Western and confocal immunofluorescence analyses) and inhibited the expression of several NFkB target genes, including XIAP, Iκbα, cFLIP and cIAP2 expressions. Treatment with ibrutinib (2 to 10 µM) attenuated p-BTK, p-PLCγ2, p-AKT and c-MYC levels but induced p21 levels, as well as induced apoptosis of cultured (MO2058, Mino and Z138 and JeKo-1 cells) and pMCL cells. Importantly, co-treatment with JQ1 and ibrutinib markedly inhibited p-BTK, BTK, p-PLCγ2, p-AKT and nuclear RelA levels and synergistically induced apoptosis of the cultured (JeKo-1, MO2058 and Mino) and pMCL cells (CI values < 1.0 by isobologram analysis). Further, following tail-vein infusion and engraftment of Mino cells (3 million cells/mouse) in the bone marrow and spleen of NOD/SCID mice, as compared to each agent alone, co-treatment with JQ1 (50 mg/kg/day, IP) and ibrutinib (25 mg/kg/day IP) significantly improved the survival of the NOD/SCID mice (p < 0.01). We also isolated ibrutinib-resistant Mino/IR cells ( > 20 fold resistant), following a continuous exposure of the parental Mino cells in culture to escalating levels of ibrutinib, which exhibited high levels of p-BTK, p-PLCγ2, c-MYC and CDK6. Co-treatment with JQ1 and the pan-histone deacetylase inhibitor panobinostat (PS) synergistically induced apoptosis of not only Mino but also Mino/IR cells. We also isolated carfilzomib-resistant JeKo-1/CZR cells ( > 10 fold resistant). As compared to JeKo-1, JeKo-1/CZR cells demonstrated higher protein levels of proteasome subunits and increased base-line and lesser EGCG (Epigallocatechin-3-gallate)-suppressible proteasome activity. Mutations in the PSMB5 were not detected. JeKo-1/CZR cells also displayed higher levels of c-MYC, cyclin D1, MCL-1 and CDK4. Notably, co-treatment with JQ1 and PS or ibrutinib also synergistically induced apoptosis of not only JeKo-1 but also JeKo-1/CZR cells (CI values < 1.0 by isobologram analysis). Taken together, these pre-clinical findings demonstrate synergistic preclinical activity of BET protein (BA) antagonist-based combinations with ibrutinib, panobinostat or carfilzomib against human MCL cells. Collectively, they also define potential BA-based combinations to overcome resistance to ibrutinib and carfilzomib in human MCL. DisclosuresNo relevant conflicts of interest to declare.

Inhibitors of BCR signalling interrupt the survival signal mediated by the micro-environment in mantle cell lymphoma.

Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B-cell receptor (BCR)-mediated signalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through therapies targeting BCR-associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high amount of IL-1β, IL-6, IL-8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased autocrine secretion of IL-1β, IL-6, IL-8, CCL5, IL-10, TNFα and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamatinib (R406) reversed these protective effects and decreased both basal and BCR-induced autocrine cytokine secretions associated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR-induced MCL cell adhesion to human bone marrow stromal cells (HMSCs) in short- and long-term co-culture. We demonstrated that BCR-induced survival relies on autocrine secretion of IL-1β, TNFα and CCL5 that might facilitate adhesion of MCL cells to HMSC. Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis.

Dual targeting of the thioredoxin and glutathione antioxidant systems in malignant B cells: A novel synergistic therapeutic approach

B-cell malignancies are a common type of cancer. One approach to cancer therapy is to either increase oxidative stress or inhibit the stress response systems on which cancer cells rely. In this study, we combined nontoxic concentrations of Auranofin (AUR), an inhibitor of the thioredoxin system, with nontoxic concentrations of buthionine-sulfoximine (BSO), a compound that reduces intracellular glutathione levels, and investigated the effect of this drug combination on multiple pathways critical for malignant B-cell survival. Auranofin interacted synergistically with BSO at low concentrations to trigger death in multiple malignant B-cell lines and primary mantle-cell lymphoma cells. Additionally, there was less toxicity toward normal B cells. Low AUR concentrations inhibited thioredoxin reductase (TrxR) activity, an effect significantly increased by BSO cotreatment. Overexpression of TrxR partially reversed AUR+BSO toxicity. Interestingly, the combination of AUR+BSO inhibited nuclear factor κB (NF-κB) signaling. Moreover, synergistic cell death induced by this regimen was attenuated in cells overexpressing NF-κB proteins, arguing for a functional role for NF-κB inhibition in AUR+BSO-mediated cell death. Together, these findings suggest that AUR+BSO synergistically induces malignant B-cell death, a process mediated by dual inhibition of TrxR and NF-κB, and such an approach warrants further investigation in B-cell malignancies.

Abstract 313: Essential role of ataxia telangiectasia in mitochondrial autophagy in mantle cell lymphoma

Abstract Ataxia telangiectasia mutated (ATM) is obligatory to initiate cellular responses to DNA double-strand breaks and DNA repair to preserve genomic integrity while loss of ATM leads to increased intracellular reactive oxygen species (ROS), accumulation of aberrant mitochondria, leading to abnormal mitochondrial homeostasis thereby exasperate cancer progression. ATM is frequently altered in several human cancers including 20-50% in mantle cell lymphoma (MCL). Despite ATM is known to induce global autophagy in certain cancer cells, the role of ATM in mitochondrial autophagy (mitophagy) in human cancer is imprecise. Here, we characterize a panel of MCL cell lines for their ability to trigger mitophagy induced by the mitochondrial uncoupler CCCP/FCCP. While Granta-519 lacks a functional ATM, both Jeko-1 and Mino cells exemplify as ATM proficient. Treatment with CCCP/FCCP (75µM for 4hr) elicits rapid up-regulation in ATM ser1981 phosphorylation in both Jeko-1 and Mino cells, while Granta-519 cells are mostly non-responsive. Cells were stained with TMRE (to detect mitochondrial membrane potential) or Mitotracker deep Red (to measure total mitochondrial mass) and analyzed by flow cytometry. Cells were also stained to detect both global and mitochondrial ROS (mROS) levels. CCCP/FCCP treatment resulted in rapid loss of (depolarized) membrane potential, total mitochondria mass, mROS and global ROS in both Jeko-1 and Mino cells while Granta-519 cells maintained intermediate to higher membrane potential with relatively higher total or mROS levels compared to controls. Cell fractionation analysis revealed that a fraction of resident ATM protein is located inside the mitochondrial fraction and phosphorylated upon CCCP/FCCP treatment in both Jeko-1 and Mino cells but not in Granta-519 leading to elimination of depolarized mitochondria as revealed by their basal COX4 protein levels. Immuno-precipitation analysis revealed that CCCP/FCCP treatment in both Jeko-1 and Mino cells resulted in ATM cleavage more specifically in the mitochondrial fraction. Interestingly, ATM status is unrelated to global autophagy as judged by their respective LC3I/II processing in these cell lines. Stable lentiviral knockdown of ATM in both Jeko-1 and Mino cells resulted in defective mitophagy with relatively higher COX4 expression and mROS levels. Neocarzinostatin induced DNA damage revealed loss of ATMser1981, Kap1ser824 and p53ser15 phosphorylation compared with control shRNA reconfirmed the loss of ATM function in these cells. Human primary MCL subjects with known ATM translocation [t(11;14) n=4] retain relatively higher mitochondrial mass and mROS, and are resistant to CCCP/FCCP induced mitophagy compared with ATM proficient primary MCL cells. These data underscore the possibility that ATM plays a decisive role in modulating mitophagy and contributes to the cancer-prone phenotype observed in MCL. Citation Format: Aloke Kumar Sarkar, Kumudha Balakrishnan, Mary Ayres, Sattva S. Neelapu, Varsha Gandhi. Essential role of ataxia telangiectasia in mitochondrial autophagy in mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2014-313

Dual PI3K/mTOR inhibition is required to effectively impair microenvironment survival signals in mantle cell lymphoma.

Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis and drug resistance. Antitumor activity has been observed with mTOR inhibitors. However, they have shown limited clinical efficacy in relation to drug activation of feedback loops. Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage. Here, we have performed a comparative analysis of the mTOR inhibitor everolimus, the pan-PI3K inhibitor NVP-BKM120 and the dual PI3K/mTOR inhibitor NVP-BEZ235 in primary MCL cells. We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases. Among the three drugs, NVP-BEZ235 induced the highest change in gene expression profile. Functional validation demonstrated that NVP-BEZ235 inhibited angiogenesis, migration and tumor invasiveness in MCL cells. NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL. Our findings support the use of the dual PI3K/mTOR inhibitor NVP-BEZ235 as a promising approach to interfere with the microenvironment-related processes in MCL.

Mouse models of mantle cell lymphoma, complex changes in gene expression and phenotype of engrafted MCL cells: implications for preclinical research

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22±1 to 54±3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.

Characterization of ibrutinib-sensitive and -resistant mantle lymphoma cells.

Ibrutinib inhibits Bruton tyrosine kinase (BTK), a key component of early B-cell receptor (BCR) signalling pathways. A multicentre phase 2 trial of ibrutinib in patients with relapsed/refractory mantle cell lymphoma (MCL) demonstrated a remarkable response rate. However, approximately one-third of patients have primary resistance to the drug while other patients appear to lose response and develop secondary resistance. Understanding the molecular mechanisms underlying ibrutinib sensitivity is of paramount importance. In this study, we investigated cell lines and primary MCL cells that display differential sensitivity to ibrutinib. We found that the primary cells display a higher BTK activity than normal B cells and MCL cells show differential sensitivity to BTK inhibition. Genetic knockdown of BTK inhibits the growth, survival and proliferation of ibrutinib-sensitive but not resistant MCL cell lines, suggesting that ibrutinib acts through BTK to produce its anti-tumour activities. Interestingly, inhibition of ERK1/2 and AKT, but not BTK phosphorylation per se, correlates well with cellular response to BTK inhibition in cell lines as well as in primary tumours. Our study suggests that, to prevent primary resistance or to overcome secondary resistance to BTK inhibition, a combinatory strategy that targets multiple components or multiple pathways may represent the most effective approach.

Involvement of tumor-associated macrophage activation in vitro during development of a novel mantle cell lymphoma cell line, PF-1, derived from a typical patient with relapsed disease

Human mantle cell lymphoma (MCL) cell lines are scarce and have been only sporadically described and validated, and only a few have been thoroughly molecularly or genetically characterized. We describe here the successful establishment of a new MCL line, PF-1, with typical MCL characteristics. Culturing primary MCL cells in vitro initially gave rise to an essential generative microenvironment “niche” involving macrophages required for MCL growth, and eventually produced the PF-1 MCL cell line. Our analysis revealed that PF-1 is morphologically and genotypically nearly identical to the original tumor cells. The PF-1 MCL cell line that we have developed will be useful for in vitro and in vivo studies of MCL pathogenesis and therapeutics.

Cobblestone-area forming cells derived from patients with mantle cell lymphoma are enriched for CD133+ tumor-initiating cells.

Mantle cell lymphoma (MCL) is associated with a significant risk of therapeutic failure and disease relapse, but the biological origin of relapse is poorly understood. Here, we prospectively identify subpopulations of primary MCL cells with different biologic and immunophenotypic features. Using a simple culture system, we demonstrate that a subset of primary MCL cells co-cultured with either primary human mesenchymal stromal cells (hMSC) or murine MS-5 cells form in cobblestone-areas consisting of cells with a primitive immunophenotype (CD19−CD133+) containing the chromosomal translocation t (11;14)(q13;q32) characteristic of MCL. Limiting dilution serial transplantation experiments utilizing immunodeficient mice revealed that primary MCL engraftment was only observed when either unsorted or CD19−CD133+ cells were utilized. No engraftment was seen using the CD19+CD133− subpopulation. Our results establish that primary CD19−CD133+ MCL cells are a functionally distinct subpopulation of primary MCL cells enriched for MCL-initiating activity in immunodeficient mice. This rare subpopulation of MCL-initiating cells may play an important role in the pathogenesis of MCL.

Ibrutinib Overcomes Carfilzomib Resistance In Immunoproteasome-Deficient Mantle Cell Lymphoma

The biological significance of the ubiquitin-proteasome system in the control of cellular processes has been well-recognized; however, the pathophysiological importance of the immunoproteasome (i-proteasome), the inducible form of the proteasome, has not been well-appreciated in cancer cells, particularly in mantle cell lymphoma (MCL), a clinically challenging, aggressive B-cell non-Hodgkin's lymphoma (NHL-B). The proteasome activity is regulated by standard catalytic subunits β1, β2 and β5, or inducible catalytic subunits LMP2 (β1i), MECL-1 (β2i), and LPM7 (β5i). The inducible catalytic subunits have been reported to be highly expressed in hematological malignancies. Our results indicated that inducible catalytic subunits of i-proteasome are highly expressed in both established and primary MCL cells. Interestingly, we found that the expression of i-proteasome subunits were required for the anti-MCL activity of carfilzomib, a novel second-generation proteasome inhibitor, in MCL cells. Both primary and established MCL cells with high expression of i-proteasome subunits were more responsive to carfilzomib, even at low dose treatment (IC50, between 1.2nM to 8.6nM). In contrast, MCL cells with low/no expression of i-proteasome subunits are highly resistant to carfilzomib (IC50 >200 nM). These results suggested that the i-proteasome could play an important role in determining the sensitivity of carfilzomib.To elucidate the mechanism responsible for carfilzomib sensitivity in MCL cells, we used IFN-γ to stimulate carfilzomib-resistant MCL cell line Rec-1, which lacked the expression of i-proteasome subunits especially LMP2. We found that pretreatment of Rec-1 cells with IFN-γ for 48 hours increased i-proteasome subunit LMP2 but not c-proteasome subunit b1, and led to sensitization of Rec-1 cells to carfilzomib. To provide direct evidence that upregulation of i-proteasome subunit LMP2 by IFN-γ was responsible for the observed carfilzomib sensitization, siRNA downregulation of LMP2 was applied prior to exposure to IFN-γ. After LMP2 knockdown, carfilzomib sensitization was attenuated. The data show that an intact i-proteasome, especially LMP2, appears to be necessary for its anti-MCL activity, suggesting that i-proteasome could serve as a biomarker for identifying patients who will benefit from carfilzomib.Since bruton's tyrosine kinase (Btk) is pivotal for B-cell lymphoma growth and its downstream signals are associated with NF-κB and other important survival pathways, we combined ibrutinib, a novel Btk inhibitor, with carfilzomib for treatment of MCL cells. We found that ibrutinib sensitized carfilzomib-resistant MCL cells, and overcame carfilzomib resistance in these cells. Taken together, i-proteasome could be indispensable for the action of carfilzomib in MCL cells. However, ibrutinib overcame carfilzomib resistance in MCL cells indicating ibrutinib has different therapeutic targets that are not associated with i-proteasome. Our data could demonstrate the profound potential of using an ibrutinib and carfilzomib combination in clinical trials for patients with relapsed or refractory MCL. Disclosures:No relevant conflicts of interest to declare.

Induction Of p53 Transcription and Apoptosis By XPO1 Inhibition In Mantle Cell Lymphoma

We investigated the pathophysiological significance of MDM2 and the nuclear exporter Exportin 1 (XPO1/CRM1) on p53 transcription and their therapeutic targeting in mantle cell lymphoma (MCL). MCL is currently an incurable B-cell non-Hodgkin's lymphoma. Although TP53 mutations occur rarely (i.e., in 15 to 20% of the cases) in MCL, wild-type p53 is frequently inactivated by gene amplification of BMI1, homozygous deletion of CDKN2A (INK4a/ARF, over-expression of regulatory proteins, or gene deletion. For example, tumor cells may sequester p53 in the cytoplasm via the overexpression of proteins like MDM2 and XPO1 thereby abrogating its transcriptional anti-tumor activity. The selective MDM2 inhibitor Nutlin-3a (Vassilev, 2004) has been reported to induce p53-mediated apoptosis in MCL cells, and recently it was shown that the potent, small molecule Selective Inhibitor of Nuclear Export (SINE) induces apoptosis in MCL cells (Zhang, 2013).mRNA expression levels in MCL patient samples were determined using Oncomine (Compendia Bioscience, Ann Arbor, MI). MDM2 mRNA expression levels were not significantly higher in the MCL samples (n = 8) compared to normal B-cells ( n = 5) (P = 0.27). The MDM2 levels were also not associated with overall disease survival (P = 0.12) at 5 years (n = 63). In contrast, gene expression analysis on the same dataset demonstrated increased XPO1 mRNA expression in the MCL cells versus normal B-cells (P< 0.001). Furthermore, this higher XPO1 expression was associated with poorer prognosis in MCL patients (i.e., median overall survival: 3.2 years in low expression XPO1 cases and 1.9 years in high expression CRM1 cases, P = 0.033). mRNA levels of 11 representative p53 target genes were determined in 2 MCL (i.e., Z-138 and JVM-2) cells and were compared to those of four acute leukemia (i.e., OCI-AML3, MOLM-13, REH and NALM-6) cells. Cells from all six cell lines express wild-type p53. The MDM2 inhibitor Nutlin-3a (5 µM) poorly induced p53 target genes in the Z-138 (1.9 ± 0.12-fold) and JVM-2 cells (2.1 ± 0.23-fold) compared to acute leukemia cells (13.9 ± 5.2-fold) (P< 0.001 in both MCL cells), despite their increased p53 protein levels after Nutlin-3a treatment. The data suggest that p53-mediated transcription may be impaired in MCL via a Nutlin-induced increase in cytoplasmic p53 levels. Next, we treated Z-138 with 2 or 4 mM Nutlin-3a or with 80 or 160 nM KPT-185 for 18 hours and determined mRNA changes in p53 targets. KPT-185 induced p53 target genes more potently than Nutlin-3a (i.e., 2- to 4-times). To investigate the significance of p53-mediated apoptosis, Z-138 and JVM-2 were transduced with lentivirus encoding either negative control shRNA (ShC) or p53-specific shRNA (Shp53) and stable shRNA-expressing cells were generated. The p53-specific shRNA reduced p53 basal levels by 80 to 90%, and p53 knockdown cells were ∼80% less sensitive to Nutlin-3a and ∼60% to KPT-185, indicating that MDM2 or XPO1 inhibition rely on p53 pathway to induce apoptosis. Apoptotic induction by Nutlin-3a and KPT-185 in primary MCL cells were determined in 3 samples with wild-type p53. Treatment-specific Annexin V induction percentage in 2.5 µM Nutlin-3a-treated cells was 40.7 ± 8.0%, while it was 64.5 ± 15.0% in 100 nM KPT-185 treated cells. Treatment of 1 µM KPT-185 led to more prominent apoptosis, as evidenced by 81.8 ± 7.7% annexin V staining.Our data suggest that p53 is a component of MDM2 or XPO1 inhibition-induced apoptosis, and p53 transcription is more potently activated by XPO1 inhibition rather than MDM2 inhibition. Based on these data, we propose that active modulation of p53 by SINE XPO1 antagonism and/or MDM2 inhibition transcription would be beneficial in some cancer types to enhance p53-based cancer therapy. Disclosures:Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Andreeff:Karyopharm Therapeutics Inc.: Research Funding.

Integrative Whole Transcriptome and Exome Sequencing Reveals Reprogramming Of Mantle Cell Lymphoma In Prolonged Early G1 Arrest For Clinical Response To Selective Inhibition Of CDK4 In Combination Therapy

CDK4 and CDK6, which drive cell cycle entry and progression through G1 in the presence of cyclin D, are overexpressed at a high frequency in human cancers. Targeting CDK4 with the first selective and potent CDK4/CDK6 inhibitor, palbociclib (PD 0332991), has recently achieved unprecedented clinical efficacy in both hematologic malignancies and solid tumors. Most notably, palbociclib more than tripled the progression free survival of breast cancer patients treated with letrozole. In mantle cell lymphoma (MCL), CDK4 overexpression and aberrant cyclin D1 expression leads to unrestrained cycling and proliferation that underlies disease progression. In the first phase I single-agent palbociclib clinical trial in recurrent MCL, inhibition of CDK4 by palbociclib alone resulted in a durable clinical response with tumor regression in some MCL patients, including one complete response and two partial response.However, the fundamental mechanism for differential clinical response to selective targeting of CDK4/CDK6 remains obscure. To address this question, we have developed a novel strategy that both inhibits proliferation of cancer cells and reprograms them for cytotoxic killing by reversible inhibition of CDK4/CDK6. We have shown that: 1) inhibition of CDK4/6 with palbociclib leads to early G1 arrest that is dependent on Rb, the substrate for CDK4/CDK6; 2) prolonged early G1 arrest (pG1) reprograms cancer cells for killing by diverse agents; 3) pG1 sensitization is exacerbated in synchronous S phase entry (pG1-S) upon palbociclib withdrawal. Further, pG1 sensitization appears to stem from restricted expression of genes scheduled for early G1 only, which is exacerbated in pG1-S due to incomplete restoration of cell cycle-coupled gene expression.To advance targeting CDK4 in MCL, we have implemented this strategy combining palbociclib with bortezomib at a reduced dose (1.0 mg /m2) in a phase I clinical trial (Pa-Btz) in recurrent MCL. palbociclib was administered to MCL patients on days 1-12 of a 21-day cycle to induce pG1; bortezomib was given on days 8 and 11 in pG1 and on days 15 and 18 in pG1-S. Pa-Btz was well tolerated and appeared to have a palbociclib dose-dependent durable clinical activity, with only one of 6 patients progressed at the optimal dose combination.We investigated the genes that mediate pG1 reprogramming by integrative whole exome sequencing (WES) and whole transcriptome sequencing (WTS). The dynamic changes in cell cycle-coupled gene expression were determined within individual patients in primary MCL tumor cells isolated from serial lymph node biopsies at baseline, in pG1 (day 8) and in pG1-S (day 21) in conjunction with immunohistochemistry (IHC). palbociclib inhibited CDK4 and induced pG1 in all patients initially, regardless of the clinical response, mutations in p53 or ATM, cyclin D1 3'UTR deletion, or other patient-specific deletions, amplifications and mutations. No mutations were detected in CDK4, which was expressed in primary MCL cells over CDK6. Induction of pG1 maintained the expression of cell cycle genes programmed for early G1 (CDK4, cyclin D and Rb), and prevented the expression of those scheduled for late G1 (cyclin A), S phase (Ki 67, TK) and G2/M (CDK1, cyclin B), and this was completely reversible upon release of the early G1 block.However, induction of pG1 also led to an imbalance in the expression of other cellular genes due to restricted expression of only genes programmed for early G1. Among the 868 genes that were suppressed in pG1 (not programmed for early G1) in MCL tumor of clinically-responding patients (N=4, EdgeR, FDR 0.05), 9 were conversely activated in the non-responding patients (N=4, EdgeR, FDR 0.05). These genes are involved in redox stress, metabolism and cell migration, suggesting a potential role of cell cycle-coupled metabolic imbalance in differential clinical response to targeting CDK4/6.Thus, selective inhibition of CDK4 led to Rb-dependent pG1 in tumor cells of all MCL patients despite a multitude of genomic aberrations. Integrative WES and WTS analysis of serial tumor biopsies revealed that pG1 reprograms MCL cells by inducing an imbalance in gene expression that is associated with the clinical response to the Pa-Btz therapy. Defining the functions of the candidate genes identified in the context of clinical response should shed light on the mechanism for therapeutic targeting of CDK4/CDK6 and advance genome-based patient stratification. Disclosures:Off Label Use: PD 0332991 is a CDK4/CDK6 selective inhibitor. Martin:Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau. Leonard:millennium: Consultancy.

Mantle-Cell Lymphoma (MCL) Cells Are Highly Sensitive To ABT-199 But Their Sensitivity May Be Altered By The Microenvironment Via The Up-Regulation Of Bcl-Xl and Bcl2A1

Despite improvement in the treatment of Mantle-cell lymphoma (MCL), relapse invariably occurs and innovative strategies are needed. Bcl-2 inhibitors such as ABT-737 and ABT-263 (navitoclax), which target both Bcl-2 and Bcl-xL, demonstrated antitumor activity in B-cell malignancies. However, the clinical development of navitoclax is limited by a deep thrombocytopenia, which is induced by inhibition of Bcl-xL in platelets. To overcome this toxicity, ABT-199, the first-in-class orally bioavailable Bcl-2-selective BH3 mimetic, has been developed and showed promising antitumor activity in B-cell lymphoma while sparing platelets. In the present study, the apoptotic efficiency of ABT-199 in comparison with that of ABT-737 was evaluated in seven MCL cell lines. We found two MCL cell lines sensitive to ABT-199 (LD50 of 100 and 200 nM), one intermediate (LD50 of 1000nM) and 4 resistant (LD50 from 5000 to 10000 nM). Surprisingly, LD50 values of the 2 sensitive cell lines (MINO, GRANTA-519) were slightly higher for ABT-199 than for ABT-737. We further demonstrated that the Bcl-2/Mcl-1 ratio determined by RT-PCR is a predictive biomarker for ABT-199 sensitivity. To further determine the role of Mcl-1 in ABT-199 resistance, Mcl-1 siRNA were transfected in Z-138 and JEKO-1 cells. Mcl-1 silencing sensitized these 2 cell lines to low dose ABT-199 confirming the importance of Mcl-1 in ABT-199 resistance as previously shown for ABT-737. Moreover, in Z-138 cells, which highly express Bcl-xL, we showed that Bcl-xL silencing sensitized them to ABT-199. These results show that in addition to Mcl-1, Bcl-xL might also confer resistance to ABT-199-induced apoptosis in MCL. This could explain the slight difference of sensitivity of MCL cells between ABT-199 and ABT-737. In contrast to MCL cell lines, we found so far that ABT-199 efficiency killed all tested circulating primary cells from MCL patients (n=7) with LD50 values inferior to 10 nM. Because MCL cells reside mainly in lymph nodes, we wondered whether mimicking the microenvironment could impact the sensitivity of MCL cells to BH3 mimetics like it was previously demonstrated for chronic lymphoid leukemia cells. Thus, the ABT-199 sensitive MINO and GRANTA-519 cells were cultured on CD40L-expressing fibroblasts L in order to mimic the lymph node microenvironment. Both cell lines and primary cells became resistant to ABT-199 within 24h. Investigation of the underlying mechanism revealed a strong up-regulation of both Bcl-xL and Bcl2A1 protein expression. By contrast, culture of MCL cells with parental CD40L- fibroblasts or in conditioned medium from CD40L+ L fibroblasts culture failed to induce ABT-199 resistance. These results highlight the implication of the CD40L pathway in ABT-199 resistance through the up-regulation of Bcl-xL and Bcl2A1 in MCL. In conclusion, while circulating primary MCL cells are highly sensitive to ABT-199, it would be important to address the impact of microenvironment on long-term survival of MCL cells within lymph nodes under ABT-199 treatment. Disclosures:No relevant conflicts of interest to declare.

Abstract A124: Co-treatment with BRD4 antagonist and histone deacetylase inhibitor is synergistically lethal against Mantle Cell Lymphoma (MCL) cells.

Abstract The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., Myc and BCL-2. Here we determined the effects of the BET protein inhibitor JQ1 and/or histone deacetylase (HDAC) inhibitor panobinostat (PS) on cultured (JeKo1 and MO2058) and primary human MCL cells harvested from the excised MCL involved lymph nodes. Treatment with JQ1 (100 to 2000 nM), but not its inactive enantiomer (R-JQ1), dose-dependently increased the % of cells in the G1 phase while reducing the % of S phase cells, while concomitantly inducing apoptosis in the cultured (MO2058 &amp;gt; JeKo1) MCL cells. Treatment with JQ1 was also dose-dependently lethal against primary MCL cells. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of MYC, BCL2 and CDK6 promoter in JeKo1 and MO2058 cells. Total RNA from the untreated and JQ1-treated cells was used for the quantitative PCR analysis, which showed depletion of the mRNA of c-MYC, BCL2 and CDK6 genes in JQ1-treated cells. While it had no effect on acetylated histone H3 and BRD4, JQ1 treatment dose-dependently depleted the protein levels of MYC, BCL2, CDK6 and pSer2 RNA POL II, but induced the levels of p21, p27 and cleaved PARP in MCL cells. As compared to each agent alone, co-treatment with JQ1 (but not its inactive enantiomer, R-JQ1) and panobinostat (PS) synergistically induced apoptosis of the cultured and primary MCL cells (combination indices&amp;lt; 1.0 by isobologram analyses), but not of normal CD34+ hematopoietic progenitor cells. This was associated with greater attenuation of MYC, BCL2, MCL1 and cyclin D1, but increase in the levels of p21 and cleaved PARP in cultured MCL cells. Co-treatment with JQ1 and PS was also synergistically lethal against carfilzomib-resistant JeKo1 cells (&amp;gt; 10 fold resistant), which were isolated following selection under a continuous exposure to increasing levels of carfilzomib. Following the tail vein infusion and engraftment of JeKo1 cells (5 million cells/mouse) in the bone marrow and spleen of NOD/SCID mice, co-treatment with JQ1 (50 mg/kg/day, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, administered IP) and PS (5 mg/kg, IP) versus treatment with vehicle control, or JQ1 or PS alone, resulted in significant in vivo attenuation of c-MYC, BCL-2 and cyclin D1 levels in the harvested MCL cells from the mice (p &amp;lt; 0.01). Collectively, these pre-clinical findings demonstrate that the combined treatment with BRD4 antagonist and pan-HDAC inhibitor is a synergistically effective epigenetic therapy targeted against human MCL cells, regardless of their sensitivity to proteasome inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A124. Citation Format: Bhavin Shah, Laxmi Jakkula, Warren Fiskus, Sunil Sharma, Jun Qi, John A. Valenta, Leasha J. Schaub, Melissa Rodriguez, Santhana G.T. Devaraj, James E. Bradner, Kapil N. Bhalla. Co-treatment with BRD4 antagonist and histone deacetylase inhibitor is synergistically lethal against Mantle Cell Lymphoma (MCL) cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A124.

In vitro and in vivo therapeutic efficacy of carfilzomib in mantle cell lymphoma: targeting the immunoproteasome.

Mantle cell lymphoma (MCL) remains incurable due to its inevitable pattern of relapse after treatment with current existing therapies. However, the promise of a cure for MCL lies in the burgeoning area of novel agents. In this study, we elucidated the therapeutic effect and mechanism of carfilzomib, a novel long-acting second-generation proteasome inhibitor, in MCL cells. We found that carfilzomib induced growth inhibition and apoptosis in both established MCL cell lines and freshly isolated primary MCL cells in a dose-dependent manner. In contrast, carfilzomib was less toxic to normal peripheral blood mononuclear cells from healthy individuals. The carfilzomib-induced apoptosis of MCL cells was mediated by the activation of JNK, Bcl-2, and mitochondria-related pathways. In addition, carfilzomib inhibited the growth and survival signaling pathways NF-κB and STAT3. Interestingly, we discovered that expression of immunoproteasome (i-proteasome) subunits is required for the anti-MCL activity of carfilzomib in MCL cells. In MCL-bearing SCID mice/primary MCL-bearing SCID-hu mice, intravenous administration of 5 mg/kg carfilzomib on days 1 and 2 for 5 weeks slowed/abrogated tumor growth and significantly prolonged survival. Our preclinical data show that carfilzomib is a promising, potentially less toxic treatment for MCL. Furthermore, an intact i-proteasome, especially LMP2, appears to be necessary for its anti-MCL activity, suggesting that i-proteasome could serve as a biomarker for identifying patients who will benefit from carfilzomib.

The EMT activator ZEB1 promotes tumor growth and determines differential response to chemotherapy in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a poor response to treatment and prognosis. Constitutive activation of different signaling pathways in subsets of MCLs, through genetic and/or nongenetic alterations, endows tumor cells with enhanced proliferation and reduced apoptosis. The canonical Wnt pathway (β-catenin/TCF-LEF), implicated in the pathogenesis of numerous cancers, is constitutively active in half of MCLs. Here, we show that ZEB1, a transcription factor better known for promoting metastasis in carcinomas, is expressed in primary MCLs with active Wnt signaling. ZEB1 expression in MCL cells depends on Wnt, being downregulated by β-catenin knockdown or blocking of Wnt signaling by salinomycin. Knockdown of ZEB1 reduces in vitro cell viability and proliferation in MCL cells, and, importantly, tumor growth in mouse xenograft models. ZEB1 activates proliferation-associated (HMGB2, UHRF1, CENPF, MYC, MKI67, and CCND1) and anti-apoptotic (MCL1, BCL2, and BIRC5) genes and inhibits pro-apoptotic ones (TP53, BBC3, PMAIP1, and BAX). We show that ZEB1 expression in MCL cells determines differential resistance to chemotherapy drugs and regulates transporters involved in drug influx/efflux. Downregulation of ZEB1 by salinomycin increases the sensitivity of MCL cells to the cytotoxic effect of doxorubicin, cytarabine and gemcitabine. Lastly, salinomycin and doxorubicin display a synergistic effect in established and primary MCL cells. These results identify ZEB1 in MCL where it promotes cell proliferation, enhanced tumor growth and a differential response to chemotherapy drugs. ZEB1 could thus potentially become a predictive biomarker and therapeutic target in this lymphoma.

Transcription and Translation are Primary Targets of Pim Kinase Inhibitor SGI-1776 in Mantle Cell Lymphoma

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are serine/threonine/tyrosine kinases and oncoproteins that promote tumor progression. Three isoforms of Pim kinases have been identified and are known to phosphorylate numerous substrates, with regulatory functions in transcription, translation, cell cycle, and survival pathways. These kinases are involved in production, proliferation, and survival of normal B cells and are overexpressed in B-cell malignancies such as mantle cell lymphoma (MCL). SGI-1776 is a small molecule and Pim kinase inhibitor with selectivity for Pim-1. We hypothesize that Pim kinase function can be inhibited by SGI-1776 in MCL and that inhibition of phosphorylation of downstream substrates will disrupt transcriptional, translational, and cell cycle processes and promote cell death. SGI-1776 treatment in 4 MCL cell lines resulted in apoptosis induction. Phosphorylation of transcription (c-Myc) and translation targets (4E-BP1), tested in Jeko-1 and Mino, was declined. Consistent with these data, Mcl-1 and cyclin D1 protein levels were decreased. Importantly, similar to cell line data, MCL primary cells but not normal cells showed similar inhibition of substrate phosphorylation and cytotoxicity from SGI-1776 treatment. Genetic knockdown of Pim-1/ Pim-2 affected similar proteins in MCL cell lines. Collectively these data demonstrate Pim kinases as therapeutic targets in MCL.

Induction of prolonged early G1 arrest by CDK4/CDK6 inhibition reprograms lymphoma cells for durable PI3Kδ inhibition through PIK3IP1

Phosphatidylinositol-3-kinase (PI3K) signaling is constitutive in most human cancers. Selective inhibition of PI3Kδ (p110δ) by GS-1101 has emerged as a promising therapy in chronic lymphocytic leukemia and indolent lymphomas. In aggressive non-Hodgkin lymphomas such as mantle cell lymphoma (MCL), however, efficacy has been observed, but the extent and duration of tumor control is modest. To determine if tumor killing by GS-1101 is cell cycle-dependent, we show in primary MCL cells by whole-transcriptome sequencing that, despite aberrant expression and recurrent mutations in Cyclin D1, mutations are rare in coding regions of CDK4, RB1 and other genes that control G1-S cell cycle progression or PI3K/AKT signaling. PI3Kδ is the predominant PI3K catalytic subunit expressed, and inhibition by GS-1101 transiently inhibits AKT phosphorylation but not proliferation in MCL cells. Induction of prolonged early G1-arrest (pG1) by selective inhibition of CDK4/CDK6 with PD 0332991 amplifies and sustains PI3Kδ inhibition, which leads to robust apoptosis. Accordingly, inhibition of PI3Kδ induces apoptosis of primary MCL tumor cells once they have ceased to cycle ex vivo, and this killing is enhanced by PD 0332991 inhibition of CDK4/CDK6. PIK3IP1, a negative PI3K regulator, appears to mediate pG1 sensitization to PI3K inhibition; it is markedly reduced in MCL tumor cells compared with normal peripheral B cells, profoundly induced in pG1 and required for pG1 sensitization to GS-1101. Thus, the magnitude and duration of PI3K inhibition and tumor killing by GS-1101 is pG1-dependent, suggesting induction of pG1 by CDK4/CDK6 inhibition as a strategy to sensitize proliferating lymphoma cells to PI3K inhibition.

Abstract 3265: PI3Kδ inhibitor, GS-1101, impacts transcription and translation in mantle cell lymphoma.

Abstract PI3Kα, β, γ and δ are the most ubiquitous and important PI3K family class I member isoforms that contribute to cell growth and survival processes in cancer. Among these isoforms, PI3Kα and β are broadly expressed, where PI3Kγ and δ are uniquely expressed in T- and B-cells, respectively, making them ideal candidates for targeted therapies in hematological malignancies. GS-1101, also known as CAL-101, is a potent and selective PI3Kδ inhibitor (IC50=2.5 nM) that is currently in Phase II/III clinical trials in B-cell diseases. Previous studies have elucidated disruption of survival factors and cytokine signaling mediated by PI3K/Akt pathways as major mechanism of activity in B-cell malignancies. It has been established that oncoprotein PI3Kα isoform deregulates transcription and translation processes in solid tumors, and based on the knowledge of PI3Kδ and GS-1101, we hypothesized that 1) GS-1101 treatment will inhibit gene transcription and protein translation, 2) will impact transcription and translation regulators that are downstream of PI3K/Akt pathways and 3) will reduce short-lived mRNA and proteins such as Mcl-1, c-Myc and cyclin D1, which are important in mantle cell lymphoma (MCL), a B-cell lymphoma. In JeKo-1 and Mino MCL cell lines, PI3Kδ is the most prominent isoform. In these cell lines and in primary MCL cells, the level of apoptosis was modest (less than 15%) when cells were treated with 5 μM GS-1101 for 24 hr. Consistent with Annexin/PI data, slight levels of PARP cleavage was detected. Of the two Akt phosphorylation sites, Thr308 and Ser473, phosphorylation of Akt (Thr308) showed dose-dependent decrease in both cell lines when treated by GS-1101 for 1 hr following serum starvation; however, there was no change in Akt (Ser347) levels following 24 hr of GS-1101 treatment in full serum conditions. Global RNA synthesis was substantially reduced in MCL primary samples, reaching 80% of control at 1 μM and 50% or less at 5 μM for 24 hr treatment, while only minor changes (80-90% of control) were observed in JeKo-1 and Mino. Global translation inhibition measured by leucine incorporation was observed in JeKo-1, Mino and primary MCL cells. Phosphorylation levels of translation regulators p70S6K (Ser9) and S6 (Ser235/236) showed dose-dependent decrease when treated with GS-1101 for 24 hr in JeKo-1. Meanwhile, minor decrease of 4E-BP1 (Ser65) phosphorylation level was observed in JeKo-1 under the same conditions. In Jeko-1 cells, early response genes such as short-lived MCL1 mRNA level was reduced following 24 hr of GS-1101 treatment, however, other (PIM2, MYC, CCND1) mRNA levels decreased to a lesser extent. At the protein level, both Mcl-1 and cyclin D1 expression decreased in JeKo-1 cells. In conclusion, similar to inhibition of other PI3K isoforms, PI3Kδ inhibition impacted transcription and translation, and reduced cyclin D1 and Mcl-1 levels which are important for MCL cell survival and proliferation. Citation Format: Qingshan Yang, Lisa S. Chen, Sattva S. Neelapu, Brian J. Lanutti, Varsha Gandhi. PI3Kδ inhibitor, GS-1101, impacts transcription and translation in mantle cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3265. doi:10.1158/1538-7445.AM2013-3265

Abstract 923: Ibrutinib inhibits malignant cell adhesion and migration and reduces tumor burden in lymph node and bone marrow in a murine model of mantle cell dissemination and progression.

Abstract Introduction: Ibrutnib is a potent and selective Btk inhibitor that covalently modifies Cys481 in Btk and inhibits B cell antigen receptor (BCR) signaling. Ibrutinib is currently in a Phase II study in patients with relapsed or refractory mantle cell lymphoma (MCL). In MCL patients following 1-2 weeks of ibrutinib treatment, we observed a transient increase of MCL cells in peripheral blood which was associated with a rapid reduction of tumor burden in MCL patients suggesting mobilization of malignant cells from tissues to the periphery (Wanga; Changb). At the same time, levels of plasma chemokines CCL4, CCL22, CXCL13 were significantly decreased by 40-60% (p&amp;lt;0.05) (Changb). As Btk is an essential element in BCR- and chemokine-mediated adhesion and migration in B lymphocytes, we determined effects of Btk inhibition by ibrutinib in MCL primary cells co-cultured with stromal cells, and studied its effects in vivo on MCL homing in immunocompetent C57Bl/6 mice and lymphadenopathy in a disseminated human xenograft model of MCL in SCID mice. Results and Conclusions: Ibrutinib inhibited phosphorylation of Btk, PLCγ, Erk and JNK in MCL cell lines (Mino, Jeko, HBL2) and primary MCL cells stimulated by co-culture with stromal cells or anti-IgM. Furthermore, ibrutinib inhibited BCR- and chemokine (CXCL12, CXCL13)-induced adhesion and migration of MCL cells lines, and the migration of MCL underneath bone marrow-derived stromal cells in co-culture (pseudoemperipoleisis). In addition, ibrutinib significantly inhibited the production of chemokines CCL3, CCL4, CCL22, TARC and CXCL13 by 50-70% (p&amp;lt;0.01) in human MCL co-culture or following BCR activation in MCL cell lines or primary MCL cells. Gene expression studies of both MCL cells and patients treated with ibrutinib for either 24 hrs or 8 days resulted in over 80% inhibition of NF-κB, IL-6, CCL3, CCL4 gene expression. In vivo, ibrutinib treatment at 12 mg/kg inhibited homing of MCL cells into lymphoid tissues of C57Bl/6 mice by 70%. Ibrutinib at 3 and 12 mg/kg significantly and dose-dependently suppressed tumor burden in the mandibular/cervical lymph nodes, mesenteric lymph nodes/Peyer Patches and bone marrow by over 90% in a prevention model of MCL lymphadenopathy in SCID mice. In a treatment setting of the same model where tumor injected mice were treated with vehicle for 6 weeks prior to receiving 12 mg/kg of ibrutinib for 3 weeks, a 90% and 68% reduction of tumor cells in the lymph nodes and bone marrow, respectively, was observed. These results collectively demonstrate Btk inhibition mediated by ibrutinib, suppresses adhesion and migration of malignant MCL cells in vitro and reduces tumor burden in lymph nodes and bone marrow in vivo. aWang L et al., ASH 2011. bChang BY et al., ASH 2011. Citation Format: Betty Y. Chang, Michelle Francesco, Susanne Steggerda, Stella Chang, Padmaja Magadala, Lucy Jawed, Patricia Thiemann, Joseph J. Buggy. Ibrutinib inhibits malignant cell adhesion and migration and reduces tumor burden in lymph node and bone marrow in a murine model of mantle cell dissemination and progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 923. doi:10.1158/1538-7445.AM2013-923