Resistant Lymphoma Cells Research Articles

Dual Inhibition of PKC Delta By Carfilzomib (CFZ) and Ibrutinib Results in Synergistic Activity in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Pre-Clinical Models and in Primary Tumor Cells Isolated from B-Cell Lymphoma Patients

Introduction: A better understanding in the role of the B-cell receptor-signaling pathway in lymphomagenesis led to the clinical development of ibrutinib is an orally bioavailable Bruton's tyrosine kinase inhibitor (BTKi). Ibrutinib has clinical activity in several subtypes of B-cell lymphoma (follicular lymphoma, mantle cell lymphoma and certain diffuse large B-cell lymphoma [DLBCL] subtypes). Recently, an increasing number of ibrutinib-treated patients are developing disease progression, stressing the need to optimize its therapeutic potential. To this end, we evaluated the biological effects and interactions of ibrutinib and CFZ, a non-reversible proteasome inhibitor in several lymphoma pre-clinical models including primary tumor cells isolated from lymphoma patients.Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines as well as primary tumor cells isolated from B-cell lymphoma patients (N=20) were exposed to ibrutinib and/or CFZ. Changes in cell viability were evaluated by Presto Blue and Titer Glo assays respectively. Coefficient of synergy was determined using the CalcuSyn software. To further study the mechanisms responsible for the biological effects of ibrutinib and CFZ, RSCL and RRCL were exposed to Ibrutinib+/- CFZ for 48hr. Changes in apoptosis, mitochondrial potential, and cell cycle distribution were evaluated by flow cytometry by AnnexinV-PI, DiOC6 or PI staining respectively. Subsequently, protein lysates were isolated from ibrutinib+/- CFZ exposed RSCL or RRCL and changes in downstream targets (PKCδ a member of diacylglycerol-dependent, calcium-independent serine/threonine-specific protein kinases PKC isoform know to be a substrate of caspase 3 and able to mediate apoptosis), Bcl-2 family members and cell cycle regulatory proteins were evaluated by Western blotting.Results: In vitro exposure of RSCL, RRCL, and primary tumor cells to ibrutinib and CFZ resulted in synergistic anti-tumor activity. In addition, the combination of ibrutinib and CFZ resulted in a more pronounced decrease in the mitochondrial potential, higher degree of apoptosis (as demonstrated by flow cytometry and caspase 3 or PARP cleavage) and increase in G1 cell cycle arrest when compared to single agent ibrutinib or CFZ. Perhaps related to these findings, we found that exposure of lymphoma cells to Ibrutinib and CFZ resulted in an increase in caspase 3, the active form of PKCδ and BAX levels. Using caspase inhibitor Q-VD-OPh rescued the combination killing effect of ibrutinib and CFZ, as well as abrogated induction of the active form of PKCδ. In addition, Ibrutinib in combination with CFZ decreased cyclin E and CDK2 levels.Conclusions: Together our data suggests that ibrutinib and CFZ had a strong synergistic anti-tumor activity against rituximab-chemotherapy sensitive and resistant lymphoma cells and patient samples. At the molecular level, the combination of ibrutinib and CFZ affects the balance of the apoptotic machinery and key regulatory proteins of the cell cycle that could play an important role in the synergistic effects observed. Further signaling and in vivo studies are necessary to evaluate the therapeutic role of ibrutinib in combination with carfilzomib in B-cell lymphoma (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Funds). DisclosuresCzuczman:MorphoSys: Consultancy; Celgene: Employment; Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board.

Potent Inhibition of the Growth and Induction of Apoptosis in Lymphoma By the Anthelminthic Drug Niclosamide: In Vitro Data

Background and Purpose: Niclosamide is an anthelminthic drug which has been used for the treatment of human parasitic infections for many years. Niclosamide interacts with lysosomes and induces autophagy. In recent years, it has demonstrated anti-cancer potential in leukemia, breast cancer, colon cancer, myeloma, ovarian, prostate and lung cancer models. Multiple pathways like Wnt/β-catenin, mTORC1, STAT3, NF-κB and notch signaling were reported to be involved. Only limited studies were done in lymphoma models. We hypothesized that niclosamide may also have in vitro and in vivo activities in lymphomas. Non-Hodgkin lymphomas generally respond well to chemotherapy and/ or immunotherapy, however many patients relapse and ultimately become refractory. Relapses are often caused by tumor stem cells not eliminated by cytostatic drugs. Therefore new treatment approaches and new targets are desirable.Materials and Methods: Established B lymphoma cell lines were exposed to different concentrations of niclosamide (0.1-4µM) and IC50 was calculated at 24, 48 and 72 hours. The cell concentration, viability and proliferation were assessed by CellTiter-Blue viability and trypan blue exclusion assays. Apoptosis was assessed by a combined annexin-V/ propidium iodide stain. Gene expression changes were studied using GeneChip Human Transcriptome Array 2.0 (Affymetrix) with 44 699 annotated genes. Colony forming assays were performed in methylcellulose. Ultrastructural changes were studied using a Hitachi electron microscope. As normal controls, peripheral blood mononuclear cells from individuals without active cancer were incubated with niclosamide for up to 72 hours. Samples from patients with chronic lymphocytic leukemia were also treated under the same conditions.Results: Treatment with niclosamide at doses as low as 0.1 μM resulted in time-and dose- dependent apoptosis, cytotoxicity and inhibition of proliferation in aggressive lymphoma cell lines. The 50% inhibitory concentration in a proliferation assay (mean of data at 24, 48 and 72 hours) is shown in the Table below. Niclosamide also inhibited clonal growth in semi-solid media. Electron microscopy showed that filopodia increased and lipid vacuoles developed whereas mitochondria were less numerous and had fewer cristae (when KOPN-8 was treated with 0.5 μM for 48 hours). The viability of mononuclear cells from 8 individuals without lymphoma was unchanged (or minimally decreased) when incubated with niclosamide. As far as cells from two patients with untreated chronic lymphocytic leukemia are concerned, no cytotoxicity was observed at doses between 0.5 and 5 μM. Gene expression changes were studied the cell lines Daudi and KOPN-8 treated with 2.5 μM for 3 and 6 h. 96 genes were consistently overexpressed , 59 down-regulated. Ten out of the 96 overexpressed genes involved the TNF pathway and immunoregulation including CD95. Thirteen out of the 59 down-regulated genes are involved in mitochondrial function. TableCell lineDescription of Cell TypeIC 50STDDaudiBurkitt lymphoma cell line0.37 μM± 0.12HBL-2Diffuse large B cell lymphoma cell line0.68 μM± 0.15KOPN-8B precursor ALL cell line0.6 μM± 0.08RamosBurkitt lymphoma cell line0.58 μM± 0.04RajiBurkitt lymphoma cell line0.65 μM± 0.10SU-DHL4-VRVincristine resistant lymphoma cell line0.5 μM± 0.02Conclusion: Niclosamide effectively inhibits the proliferation of B lymphoma cell lines and induces apoptosis. Preliminary data show that Niclosamide targets genes involved in the TNF pathway and interferes with mitochondrial function. Normal lymphocytes are not sensitive to niclosamide. The in-vitro activity of niclosamide is at least comparable or superior to the activity seen in other malignancies. Niclosamide may target drug-resistant lymphoma stem cells and has clinical potential. We plan to study combination treatments and perform in vivo studies.Acknowledgments: The authors thank Drs. Borje Andersson, Shile Huang, Nakle Saba, Ben Valdez and Ellen Vitetta for their kind gift of cell lines. DisclosuresNo relevant conflicts of interest to declare.

Exquisite Sensitivity of Plasma Cell Malignancies to a Novel Nucleoside Analog Is Mediated By Overexpressed Adenosine Kinase

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin, driven by infection with KSHV/HHV-8, and with notable plasmacytic differentiation. PEL carries an extremely poor prognosis with a median survival time of less than 6 months and is known to be largely resistant to conventional chemotherapy. Therefore, we conducted high throughput screens for effective inhibitors using PEL cell lines. We discovered a compound, 6-ethylthioinosine (6-ETI), a nucleoside analog with selective toxicity to PEL and multiple myeloma (MM) at nanomolar concentrations, but not to other lymphoma cell lines tested. 6-ETI also showed remarkable responses in a mouse xenograft model of PEL. To understand the molecular mechanism(s) of selectivity towards PEL, we developed and performed resistome analysis, an unbiased approach based on RNA sequencing of in vitro and in vivo generated resistant subclones. We found adenosine kinase (ADK) inactivating genomic alterations in all resistant clones as the mechanism of resistance. Concordantly, we found that plasma cells express ADK, as well as sensitive PELs and multiple myeloma cell lines, while resistant lymphoma cell lines including those with EBV infection had lower levels (if any); interestingly, the latter could be sensitized by cell crowding-induced ADK upregulation. Like other nucleoside analogues, 6-ETI induces ATP-depletion and cell death accompanied by S-phase arrest and DNA damage, but only in ADK-expressing cells. Immunohistochemistry for ADK served as a new biomarker approach to identify tumors that may be sensitive to 6-ETI, which we documented for primary specimens of PEL, multiple myeloma and other lymphoid malignancies with plasmacytic features, namely plasmablastic lymphoma. A number of nucleoside analogues have been reported to be effective in treating leukemias and B and T cell lymphomas. We performed structure-activity relationship studies and tested a number of nucleoside analogs that are in preclinical or clinical development for other hematological malignancies to identify and better understand the structural requirements for 6-ETIÕs biological activity. We successfully demonstrated that 6-ETI was more potent and selective at killing PEL and MM cell lines than other studied nucleoside analogs suggesting that this compound possesses unique and distinct features that are clinically promising. Despite the presence of treatment approaches that can greatly extended the survival of MM patients, resistance remains an issue, warranting the need for new effective agents and combinations. We identified 6-ETI as a novel and clinically viable nucleoside analog for the treatment of PEL, immunoblastic lymphoma, plasmablastic lymphoma, multiple myeloma and other ADK-expressing cancers. [Display omitted] DisclosuresNayar:Weill Cornell Medical College: Patents & Royalties: Submitted patent applicatio for 6-ETI. Cesarman:Weill Cornell Medical College: Patents & Royalties: applied for patent for 6-ETI.

Pre-clinical activity of BET protein antagonist-based combinations against ibrutinib-sensitive or -resistant human Mantle Cell Lymphoma cells

In Mantle Cell Lymphoma (MCL) cells, activated B cell receptor (BCR) and NFkB signaling exerts pro-growth and pro-survival activity. Treatment with the Bruton’s Tyrosine Kinase (BTK) inhibitor ibrutinib induces clinical responses, but long-term durable remissions and cure remains elusive. BET (bromodomain and extra-terminal) proteins, including BRD4, bind to acetylated lysine(s) on histones, assemble transcriptional co-regulators at the enhancers and promoters and increase the transcription of MCL-relevant oncogenes, e.g., MYC, BCL-2, CDK4/6, cyclin D1. Additionally, BRD4 is essential for NFkB activity. Here, we determined that BET protein antagonists JQ1 or I-BET151, which disrupt the binding of BRD4 to acetylated histones, dose-dependently inhibit the growth and induce apoptosis of cultured (MO2058 and Mino, Z138 and Jeko1) and primary MCL (pMCL) cells. Treatment with JQ1 attenuated the mRNA and protein expression of MYC, BCL2, MCL-1 and CDK4/6, while inducing the levels of HEXIM1, p21 and p27 in MCL cells. Importantly, JQ1 treatment also attenuated the nuclear RelA and inhibited the expression of several NFkB target genes, including XIAP, IkBα, cFLIP, cIAP2, as well as BTK. Compared to ibrutinib alone, co-treatment with JQ1 and ibrutinib markedly inhibited pBTK, BTK, pPLCγ2, pAKT and nuclear RelA levels and synergistically induced apoptosis of the cultured and pMCL cells. Following tail-vein infusion and engraftment of Mino cells in the bone marrow and spleen, co-treatment with JQ1 and ibrutinib, versus each agent alone, significantly improved the survival of NOD/SCID mice. By exposing parental Mino cells to escalating levels of ibrutinib, we isolated Ibrutinib-resistant Mino/IR cells (>20 fold resistant). Compared to Mino, Mino/IR cells demonstrated higher levels of BTK, AKT, XIAP, BCL-2 and CDK6, but reduced levels of NOXA, PUMA and p21. Co-treatment with JQ1 and the pan-histone deacetylase inhibitor panobinostat (PS) or the BCL-2 antagonist venetoclax (ABT199), or palbocyclib (CDK4/6 inhibitor) synergistically induced apoptosis of Mino and cultured pMCL, as well as of Mino/IR cells. These findings highlight promising BET protein (BA) antagonist-based combinations with ibrutinib, panobinostat, venetoclax or palbociclib against MCL cells. They also identify potential BA-based combinations against ibrutinib-resistant MCL cells.

Abstract 2612: BET-protein bromodomain antagonist-based combinations against ibrutinb-sensitive or resistant human Mantle Cell Lymphoma cells

Abstract Mantle Cell Lymphoma (MCL) cells demonstrate genetic alterations involving cyclin D1, CDK4, CDKN2A, MYC, BCL2, as well as in the Toll-like receptor, B-cell receptor (BCR) and NFkB signaling genes. Increased BCR signaling and transcriptional activity of NFkB is documented in MCL cells. Ibrutinib (IB) is an inhibitor of Bruton's tyrosine kinase (BTK) clinically effective against MCL, but long-term durable remissions and cure following IB treatment remains uncertain. We have previously reported that treatment with the BET protein bromodomain antagonist (BA) JQ1 disrupts the binding of the BET-protein BRD4 to the enhancers and promoters and inhibits the mRNA and protein levels of the oncogenes MYC, BCL-2 and CDK4/6 in MCL cells. BRD4 has also been shown to be essential for NFkB activity. Consistent with this, here, we determined that treatment with JQ1 (250 to 2000 nM) reduced the nuclear localization of RelA and attenuated the expression of the NFkB target genes, including TNFα, TNFAIP3 (A20), c-FLIP, cIAP2, XIAP and Bcl-xL in the cultured MCL Mino and MO2058 cells. Notably, JQ1 treatment also reduced the mRNA and protein levels of BTK, along with inhibition of the levels of p-BTK, p-PLCγ2 and p-AKT levels in the MCL cells. Concomitantly, JQ1 dose-dependently inhibited cell growth and exerted lethal activity against cultured (MO2058 and Mino, Z138 and JeKo-1) and patient-derived, primary MCL (pMCL) cells, while relatively sparing normal CD19+ B cells and CD34+ bone marrow progenitor cells. Importantly, co-treatment with JQ1 and IB also markedly inhibited the nuclear RelA levels and attenuated the levels of p-BTK and BTK and reduced NFkB target gene expressions. Co-treatment with JQ1 and IB also synergistically induced apoptosis of the cultured and pMCL cells (CI values < 1.0 by isobologram analysis). Following tail-vein infusion and engraftment of the Mino cells in the bone marrow and spleen of NOD/SCID mice, co-treatment with JQ1 (50 mg/kg/day, IP) and IB (25 mg/kg/day IP) versus treatment with each agent alone significantly improved the median survival of the mice (p < 0.01). We isolated in vitro IB-resistant Mino/IR cells (> 20-fold resistant), following continuous exposure to IB. As compared to the parental control Mino, Mino/IR cells expressed markedly higher levels of AKT, CDK6, BCL2, Bcl-xL, and XIAP. JQ1 treatment (1.0 μM) was able to inhibit the growth and induce apoptosis of Mino/IR cells. Notably, co-treatment with JQ1 and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT199 (BCL2 antagonist), or carfilzomib (CZ) (proteasome inhibitor) synergistically induced apoptosis of not only Mino but also Mino/IR cells. Taken together, these findings identify BA-based rational combinations that need to be further evaluated for their in vivo efficacy against IB-sensitive and IB-resistant MCL. Citation Format: Baohua Sun, Bhavin Shah, Warren Fiskus, Jun Qi, Santhana G. T. Devaraj, Swaminathan P. Iyer, Sunil Sharma, James E. Bradner, Youli Zu, Kapil N. Bhalla. BET-protein bromodomain antagonist-based combinations against ibrutinb-sensitive or resistant human Mantle Cell Lymphoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2612. doi:10.1158/1538-7445.AM2015-2612

Abstract 4496: Genomics-based resistome analysis revealed endogenous adenosine kinase levels as a chief determinant of specificity for a novel nucleoside analog lymphoma inhibitor

Abstract We used KSHV-infected primary effusion lymphoma (PEL) cells as a model to search for novel drugs through a combination of screening and genomic approaches. We discovered 6-ethylthioinosine (6-ETI), a nucleoside analog, as a highly effective and selective inhibitor of PEL. This compound induces necrosis accompanied by S-phase arrest without affecting known oncogenic viral proteins. To understand the selectivity towards PEL, we performed unbiased genomic analysis of 6-ETI-resistant subclones using RNASeq, which revealed that 6-ETI is activated through phosphorylation by cellular adenosine kinase (ADK). PEL cell lines have higher basal levels of ADK than resistant lymphoma cell lines, and resistant lymphoma cell lines could be sensitized by cell crowding-induced ADK upregulation. Finally, 6-ETI was shown to be highly efficacious against incipient and established tumors in a mouse model of PEL, with no apparent toxicity. Thus, we have successfully used RNASeq-based “resistome” analysis to identify the mechanism of specificity for a new and preclinically effective nucleoside analog. Citation Format: Utthara Nayar, Jonathan Reichel, Jouliana Sadek, Denise Hernandez-Hopkins, Gunkut Akar, Hufeng Zhou, Michelle A. Sahai, Peter Barelli, Ilaria Guasparri, Jennifer Totonchy, Duane Hassane, Shizuko Sei, Robert H. Shoemaker, J. David Warren, Olivier Elemento, Kenneth M. Kaye, Ethel Cesarman. Genomics-based resistome analysis revealed endogenous adenosine kinase levels as a chief determinant of specificity for a novel nucleoside analog lymphoma inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4496. doi:10.1158/1538-7445.AM2015-4496

Mechanism of TRAIL-induced Apoptotic Resistance in Lymphoma Cells

Objective:To investigate the mechanism of TRAIL-induced apoptotic resistance in lymphoma cells.Methods:U937 cells were treated with TRAIL in vitro and the cell growth index was evaluated by MTT assay.The cell cycle was detected by flow cytometry.The levels of Bax protein and mRNA were measured by Western blot and real time RT-PCR,respectively.Results:TRAIL inhibited survival of U937 cells at a concen-tration of 500 μg/L,but the inhibition rate was lower than that in the control cells(Hmy2.ciR).TRAIL arrested U937cells in G0/G1 phases,but this effect was significantly weaker when compared to that in the control cells.Further studies showed that the Bax protein level was decreased in TRAIL-treated U937 cells but no change was found in Bax mRNA expression.Conclusion:U937 cells are resistant to TRAIL-induced apoptosis.This effect may be related to a decrease in Bax protein which may be caused by degradation of Bax at the post-translational level in TRAIL-treated U937 cells.

Carfilzomib, a second-generation proteasome inhibitor, to enhance the activity of ibrutinib in rituximab-chemotherapy sensitive or resistant B-cell lymphoma cell lines and primary patient samples.

e19524 Background: Identification of critical signaling pathways required for the development, maintenance and progression of specific subtypes of B-cell lymphoma is essential in order to develop o...

Ecdysteroids sensitize MDR and non-MDR cancer cell lines to doxorubicin, paclitaxel, and vincristine but tend to protect them from cisplatin.

Ecdysteroids, analogs of the insect molting hormone, are known for their various mild, nonhormonal bioactivities in mammals. Previously, we reported that less-polar ecdysteroids can modulate the doxorubicin resistance of a multidrug resistant (MDR) mouse lymphoma cell line expressing the human ABCB1 transporter. Here, we describe the ability of 20-hydroxyecdysone (1) and its mono- (2) and diacetonide (3) derivatives to sensitize various MDR and non-MDR cancer cell lines towards doxorubicin, paclitaxel, vincristine, or cisplatin. Drug IC50 values with or without ecdysteroid were determined by MTT assay. Compound 3 significantly sensitized all cell lines to each chemotherapeutic except for cisplatin, whose activity was decreased. In order to overcome solubility and stability issues for the future in vivo administration of compound 3, liposomal formulations were developed. By means of their combination index values obtained via checkerboard microplate method, a formulation showed superior activity to that of compound 3 alone. Because ecdysteroids act also on non-ABCB1 expressing (sensitive) cell lines, our results demonstrate that they do not or not exclusively exert their adjuvant anticancer activity as ABCB1 inhibitors, but other mechanisms must be involved, and they opened the way towards their in vivo bioactivity testing against various cancer xenografts.

The Establishment of Bortezomib Resistant Myeloma Cell Line KM3/BTZ and Explore the Resistance Mechanism

Background: Multiple myeloma (MM) is an incurable B-cell malignancy resulting in significant morbidity and mortality, the incidence of second place in hematological malignancies. The proteasome inhibitor bortezomib inhibits IkappaB degradation, prevents NF-kappaB activation, and induces apoptosis in MM cells, has become first-line drug to MM. Despite its promising activity in traditional chemoresistant myeloma patients, however, some patients are resistant or become refractory to bortezomib. Chauhan D et al reported that Blockade of Hsp27 overcomes bortezomib resistance in lymphoma cells (Chauhan D et al., Cancer Re, 2003). However, bortezomib resistance mechanisms in MM remain controversial, the molecular basis of this reduced responsiveness is currently not fully understood.Objective: To establish a bortezomib-resistant cell line KM3 /BTZ of MM, then search for genes related to bortezomib resistance phenotype by analyzing the differential gene expression patterns with cDNA microarray,and explain the mechanism of bortezomib-resistant multiple myeloma.Methods: Bortezomib-resistant KM3/BTZ cell line was generated by increasing concentration of bortezomib to induce KM3 cells in vitro. MTT assay was employed to detect the cytotoxic effect on KM3 and KM3/BTZ cells. The cells apoptosis were analyzed by flow cytometry. We detect the gene expression profile changes between KM3/BTZ and its parent cell line KM3 cells by CDNA microarray. Combining of molecular annotation system MAS3.0 software and detailed analysis of documented resistance genes were used to analyze the data. Specific differentially-expressed genes were chosen for further verification using real-time RT-PCR. The expression of multi-drug resistance 1(MDR-1) gene mRNA was determined by RT-PCR.Results: The KM3/BTZ cell line was established successfully. Bortezomib resistance index values were significantly higher in KM3/BTZ (IC50 =351.2±3.51ng /ml) when compared to KM3 cells (IC50 = 17.8±1.03ng /ml),and the resistance index is 19.7 (P< 0.05). Through gene expression profiling filtering, compared with KM3,770 significantly differently expressed genes were screened out in the KM3/BTZ cell line, of which up and down regulated genes were 287 and 383 respectively, in which heat shock protein family gene HSPB2 was significantly up-regulated (Ratio=14.7455). Downregulated gene list in KM3/BTZ cell line included transcriptional regulators like ZNF family proteins (Tab 1). Upregulated genes in KM3/BTZ cell line included signal transduction related genes like MS4A family protein (Tab 1) also transcriptional regulators like ZNF family proteins (Tab 1). The expression trend of eight genes for further verification was almost consistent with the microarray, excepting gene of JUN (Fig 1). The expression of MDR-1 mRNA was not observed in either resistant or parental cells (Fig 2).Conclusion: We have successfully established bortezomib-resistant cell line KM3/BTZ. MDR-1 was not involved in bortezomib-resistant multiple myeloma cells.HSPB2, ZNF and MS4A family genes are probably related to bortezomib resistance in KM3/BTZ cells. Our results suggest that they may be new targets to overcome resistance to bortezomib in patients with MM.Table 1ZNF and MS4A family gene expression in KM3/BTZ and KM3 cellsGeneB signal*B detection*A signal*A detection*BvsA signal log ratio*BvsA change*ZNF71185.08P206.16P0.41269DZNF926.75P14.57P0.46328DZNF70410.7A26.78P0.39955DZNF4927.08P3.48P2.03448IZNF53287.93P43.54A2.01952IZNF59410.25P4.63P2.21382IZNF789269.79P97.3P2.77276IZNF506330.36P149.48P2.21006IMS4A31721.58P145.3P11.84845IMS4A4A2688.23P100.8P26.66895IMS4A6A141.96P56.47M2.513901IMS4A149.02P53.11P0.92299NC PHigh expression; A: Low expression; M:Marginally D: Decrease; I: Increase; NC: No change*: A:KM3; B: KM3/BTZ [Display omitted] [Display omitted] MDNA Marker; 1-2: K562/A cell line; 3-4: KM3cell line; 5-6: KM3/BTZ cell line DisclosuresNo relevant conflicts of interest to declare.

The SMAC Mimetic Lcl-161 Augments the in Vitro and in Vivo anti-Tumor Activity of Rituximab and Chemotherapy in Rituximab Relapse/Refractory Lymphoma Models

The addition of rituximab to front-line therapy regimens in diffuse large B-cell lymphoma (DLBCL) has greatly improved clinical outcomes, but is also associated with a disease that is more resistant to salvage chemotherapy in the second-line setting, reinforcing the need for new therapies targeted at the overlapping resistance pathways between rituximab and chemotherapy. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines which exhibited significant concurrent chemotherapy resistance. These multi-therapy resistant cell lines (TRCL) exhibit decreased expression of the pro-apoptotic Bcl-2 family proteins Bak and Bak, along with over-expression of several anti-apoptotic proteins, including the inhibitor of apoptosis proteins (IAP) survivin and livin (determined by Western blot). High IAP expression has been associated with inferior clinical outcomes in a range of hematological malignancies, and solid tumors. To determine the impact of IAP over-expression on TRCL rituximab/chemotherapy resistance we utilized a transient siRNA knockdown of both survivn and livin. TRCLs with livin knockdown had a statistically significant improvement in response to several chemotherapy agents including doxorubicin, vincristine, and the proteasome inhibitor carfilzomib (measured at 48 hours with the Cell Titer-Glo viability assay). These results support livin over-expression as a key lymphoma therapy resistance mechanism, and establish IAPs as potential therapeutic targets.Small molecule IAP inhibitors, like LCL-161 (obtained from Novartis), are chemical mimetics of the endogenous IAP antagonist termed the second mitochondrial inhibitor of caspases (SMAC). Western blot analysis indicated that TRCLs treated in vitro with LCL-161 exhibited a dose dependent decrease in the expression of several IAPs, including livin. In addition, LCL-161 increased rates of TRCL apoptosis, and produced synergistic anti-tumor activity when combined with cytarabine, gemcitabine, and carfilzomib in vitro. LCL-161 also enhanced the ex vivo anti-tumor activity of carfilzomib against primary tumor cells isolated from lymphoma patients with both de novo, and relapse/refractory disease. Cell viability and apoptosis induction were determined at 48 hours with CellTiter-Glo viability assays and flow cytometry respectively. To evaluate the anti-tumor effect of LCL-161 in vivo severe combined immunodeficiency (SCID) mice were inoculated with the TRCL Raji-4RH via tail vein injection (iv), and assigned to observation or treatment arms 7 days after inoculation. Treatments were LCL-161 alone (60mg/kg), the combination of rituximab: 10mg/kg, gemcitabine: 120mg/kg, and vinorelbine: 8mg/kg (RGV), or LCL-161 and RGV together. LCL-161 was administered on day 7 as one dose given p.o. by gavage; RGV was also administered on day 7 as a single i.v. dose given by tail vein injection. Differences in survival (measured as the time to the development of limb paralysis) were evaluated with the Log-rank, Breslow, and Tarone-Ware tests across treatment arms. As a single agent LCL-161 was ineffective in controlling Raji-4RH tumor growth in vivo. However, the combination of LCL-161 with RGV (median survival 133 days) resulted in a statistically significant (P=0.002 with each test) improvement in overall survival when compared to RGV alone (median survival 53 days).In summary, IAPs, especially livin, contribute to rituximab/chemotherapy resistance in relapse/refractory B-cell lymphoma models. However, the IAP inhibitor LCL-161 can disrupt this resistance and augment the effect of chemotherapy in resistant lymphoma cell line models, as well as relapse/refractory lymphoma patient samples. In addition, LCL-161 can improve the anti-tumor activity of the RGV chemotherapy regimen, and increase overall survival in a mouse in vivo model of human rituximab/chemotherapy resistant lymphoma. Our data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of aggressive rituximab relapse/refractory B-cell lymphomas. (Supported by a NHI SPORE Lymphoma grant: 5 P50 CA130805-04, a NIH grant R01 CA136907-01A1 and The Eugene and Connie Corasanti Lymphoma Research Fund) DisclosuresNo relevant conflicts of interest to declare.

Ofatumumab Versus Rituximab Salvage Chemoimmunotherapy in Relapsed or Refractory Diffuse Large B-Cell Lymphoma: The Orcharrd Study (OMB110928)

Ofatumumab Versus Rituximab Salvage Chemoimmunotherapy in Relapsed or Refractory Diffuse Large B-Cell Lymphoma: The Orcharrd Study (OMB110928)

Inhibition of Nuclear Factor Kappa B (NFkB)/Interferon Regulatory Factor 4 (IRF4) Signaling Pathway in Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) By Combining MLN4924, a Novel NEDD8 Activating Enzyme Inhibitor (NAE) and Ibrutinib

Abstract Identification of critical signaling pathways required for the development, maintenance and progression of specific subtypes of DLBCL is essential in order to develop novel therapeutic agents, especially for patients with relapsed/refractory disease or those not eligible for high-dose chemotherapy and autologous stem cell support (HDC-ASCT). Gene expression profiling (GEP) studies have identified three distinct subtypes of DLBCL- ABC-DLBCL, germinal center B-cell (GCB) DLBCL and primary mediastinal B-cell lymphoma (PMBL). Pre-clinical and clinical studies suggest that ABC-DLBCL is driven by an abnormally high NFκB activity that deregulates expression of Bcl-2 family proteins, and is associated with resistance to chemotherapy agents resulting in inferior clinical outcomes when compared to GCB-DLBCL or PMBL. In ABC-DLBCL, activation and maintenance of NFκB is mediated by the B-cell receptor (BCR) signaling pathway, the ubiquitin-proteasome system (UPS), oncogenic CARD11 or MYD88 mutations, and/or the effect of IRF4/SPIB on CARD11 or MYD88. Optimal NFκB targeting is an attractive therapeutic strategy in ABC-DLBCL and ongoing clinical trials are incorporating specific inhibitors (bortezomib, lenalidomide, or ibrutinib) in combination with chemo-immunotherapy in the front-line setting. On the other hand, multi-step targeting of the NFκB signaling pathway in ABC-DLBCL using investigational and currently available small molecule inhibitors could result in novel, active, and potentially less toxic regimens for ABC-DLBCL patients. To this end we studied the biological activity of MLN4924, a NAE inhibitor that selectively blocks the UPS up-stream by preventing activation of a subset of ubiquitin ligases known as cullin-RING ligases in combination with ibrutinib in lymphoma pre-clinical models. A panel of rituximab sensitive or resistant lymphoma cell lines representing ABC- and GCB-DLBCL were exposed to MLN4924. Changes in cell viability, cell cycle/NFκB activity, and expression of key regulatory proteins of the cell cycle, Bcl-2 family members, and the UPS were evaluated using the cell titer glo assay, flow cytometry and western blotting respectively. Subsequently, ABC- or GCB-DLBCL cell lines and tumor cells isolated from previously untreated or relapsed/refractory B-cell lymphoma were exposed to MLN4924 in combination with various chemotherapy agents or other available NFkB inhibitors (i.e. ibrutinib) for 24 or 48 hrs. Changes in viability were determined and coefficient of synergy was calculated using the CalcuSyn software. MLN4924 induced cell death in ABC-DLBCL cell lines and to a lesser degree in GCB-DLBCL cell lines. Anti-tumor activity plateau was seen after 48 hrs of drug exposure. In MLN4924 sensitive cells we consistently observed cell cycle arrest in G1 phase, down-regulation of Bcl-XL and PARP cleavage. MLN4924 exposure in vitro resulted in a decrease in Bcl-XL mRNA as determined by quantitative polymerase chain reaction (qPCR),due to the inhibition of NFkB activity as demonstrated in MLN4924-exposed cells by p65 co-localization studies using the imagestream technology. MLN4924 enhanced activity of cytarabine, cisplatin, doxorubicin and etoposide in ABC-, but not in the GCB-DLBCL cell lines. MLN4924 also exhibited synergistic effects when combined with ibrutinib in ABC-DLBCL cell lines at the doses tested. Our data suggests that multi-step targeting of NFκB signaling pathway in ABC-DLBCL is a viable therapeutic strategy and supports further in vivo pre-clinical studies and clinical studies in relapsed/refractory or HDC-ASCT ineligible ABC-DLBCL patients. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973.

Plumbagin modulates leukemia cell redox status.

Plumbagin is a plant naphtoquinone exerting anti-cancer properties including apoptotic cell death induction and generation of reactive oxygen species (ROS). The aim of this study was to elucidate parameters explaining the differential leukemia cell sensitivity towards this compound. Among several leukemia cell lines, U937 monocytic leukemia cells appeared more sensitive to plumbagin treatment in terms of cytotoxicity and level of apoptotic cell death compared to more resistant Raji Burkitt lymphoma cells. Moreover, U937 cells exhibited a ten-fold higher ROS production compared to Raji. Neither differential incorporation, nor efflux of plumbagin was detected. Pre-treatment with thiol-containing antioxidants prevented ROS production and subsequent induction of cell death by apoptosis whereas non-thiol-containing antioxidants remained ineffective in both cellular models. We conclude that the anticancer potential of plumbagin is driven by pro-oxidant activities related to the cellular thiolstat.

Acquired mutations in BCL2 family proteins conferring resistance to the BH3 mimetic ABT-199 in lymphoma

AbstractAcquired resistance to targeted drugs is emerging as an obstacle to successful cancer treatment. Recently, a BCL2-selective BH3 mimetic termed ABT-199 showed promising therapeutic results in BCL2-dependent tumors. Based on its high affinity for BCL2, we studied potential mechanisms conferring resistance upon ABT-199 therapy, aiming to anticipate its occurrence in the clinic. Two models of resistant lymphomas were established by continuous ABT-199 exposure. In resistant Bcl2-expressing mouse lymphoma cells, 2 missense mutations within the Bcl2 BH3 domain were identified. Both F101C and F101L mutations impeded ABT-199 binding to the BH3 domain, therefore suppressing mitochondrial apoptosis. In resistant human lymphoma cells, a missense mutation in the C-terminal transmembrane domain of proapoptotic BAX (G179E) was found, which abrogated BAX anchoring to mitochondria and blocked ABT-199–induced apoptosis both in vitro and in vivo. Importantly, G179E BAX mutation also induced partial cross-resistance to other antineoplastic drugs. Our study reveals the acquisition of mutations in BCL2 family proteins as a novel mechanism of apoptosis resistance in cancer. These results anticipate the potential development of such mutations in patients treated with ABT-199, providing a basis to preventing their occurrence and to designing drugs able to circumvent the acquired resistance.

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.

Synergy of NVP-BEZ235 and enzastaurin in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a neoplasm classified as a B-cell malignancy, that accounts for approximately 3 to 8% of Non-Hodgkin’s lymphoma (NHL) cases diagnosed annually. MCL is difficult to treat and seldom considered cured. The pathobiology of MCL is complex and includes alteration in the cell cycle, abnormalities in the DNA damage response, and constitutive activation of key antiapoptotic pathways including phosphatidyl-inositol 3-kinase (PI3K)/Akt and nuclear factor-kB. This has promoted the identification of new targeted treatments and new agents that have shown promising efficacy for future MCL therapies. The phosphatidylinositol 3-kinase (PI3K) mammalian target of rapamicin (mTOR) pathway mediates proliferation, survival, and drug resistance in lymphoma cells. NVP-BEZ235 (BEZ235) is a new, orally bioavailable inhibitor of PI3K and mTOR and a representative of a new class of anti-tumour agents.In this study, we analysed the in vitro inhibitory effects of NVP-BEZ235 on mantle lymphoma cell lines (GRANTA-519 and JeKo-1) and its effects in combination with enzastaurin, everolimus and perifosine. Our data suggest that in mantle lymphoma cell lines, BEZ235 in combination with enzastaurin elicits its antitumour effect better than combined with perifosine and everolimus. Our data reveal that the drug combination targets phosphorylation of PI3K/Akt/mTOR pathways and induces both intrinsic and extrinsic apoptosis pathways. Furthermore, inhibition of Bcl-2 anti-apoptosis family members may, in part, explain the efficacy of signalling blockade in lymphoma cells and suggests an additional therapeutic targeting strategy. Therefore, these preclinical data support the potential use of BEZ235 in patients with mantle lymphoma, and in particular provide rationale for combination with enzastaurin.

Resistance to mTOR Kinase Inhibitors in Lymphoma Cells Lacking 4EBP1

Inhibitors of the mechanistic target of rapamycin (mTOR) hold promise for treatment of hematological malignancies. Analogs of the allosteric mTOR inhibitor rapamycin are approved for mantle cell lymphoma but have limited efficacy in other blood cancers. ATP-competitive “active-site” mTOR inhibitors produce more complete mTOR inhibition and are more effective than rapamycin in preclinical models of leukemia, lymphoma and multiple myeloma. In parallel to clinical trials of active-site mTOR inhibitors, it will be important to identify resistance mechanisms that might limit drug efficacy in certain patients. From a panel of diffuse large B-cell lymphoma cell lines, we found that the VAL cell line is particularly resistant to apoptosis in the presence of active-site mTOR inhibitors. Mechanistic investigation showed that VAL does not express eukaryotic initiation factor 4E-binding protein-1 (4EBP1), a key negative regulator of translation controlled by mTOR. Although VAL cells express the related protein 4EBP2, mTOR inhibitor treatment fails to displace eukaryotic initiation factor 4G from the mRNA cap-binding complex. Knockdown of eukaryotic initiation factor 4E, or re-expression of 4EBP1, sensitizes cells to apoptosis when treated with active-site mTOR inhibitors. These findings provide a naturally occurring example of 4EBP deficiency driving lymphoma cell resistance to active-site mTOR inhibitors.

Zebularine significantly sensitises MEC1 cells to external irradiation and radiopharmaceutical therapy when administered sequentially in vitro.

Zebularine is a cytidine analogue incorporated into DNA during replication, inhibiting DNA methyltransferase 1 (DNMT1), resulting in demethylation and changes in gene expression. Such modification may improve radiosensitivity in resistant lymphoma cells. The hypothesis of this study was that zebularine and radiation would synergistically inhibit cell growth and viability. Human MEC1 malignant B cells were incubated with 0-200 µM zebularine for 48 h. Media containing zebularine was removed, and the cells were irradiated with 0-2 Gy of either external beam irradiation or (177) Lu-DOTA-TATE, a radiolabelled somatostatin analogue. Concentration and viability were measured over 48-72 h. The proportion of apoptotic cells was identified using an active Caspase 3/7 assay. Zebularine inhibited growth of cells in a dose-dependent manner during exposure. No residual growth inhibition occurred following removal of the drug. Zebularine and external irradiation inhibited cell proliferation in a dose-dependent, synergistic interaction, but the effect on viability was additive. Treatment with zebularine and (177) Lu-DOTA-TATE resulted in less inhibition of proliferation (P = 0.0135), but a synergistic decrease in viability. Apoptotic fraction was much higher in cells irradiated with (177) Lu-DOTA-TATE than external irradiation. External irradiation induces growth arrest rather than apoptosis. Apoptosis is the primary effect of radiopharmaceutical therapy on tumour cells. Treatment with the methylation inhibitor, zebularine, appears to synergistically augment these natural effects in vitro, which could be exploited clinically.