Activated B-cell Diffuse Large B-cell Lymphoma Cell Lines Research Articles

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled By Functional Lipoprotein Nanoparticles and the B Cell Receptor Inhibitor Ibrutinib: A Therapeutic Strategy Dependent on Cell of Origin (COO)

Introduction: Malignant cells, including lymphoma cells, have altered metabolism and, in some cases, an increased demand for cholesterol and cholesteryl esters. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative, exciting approach. Toward this end, we focused on DLBCL as a model because of differences in cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed bio-inspired high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol and cholesteryl ester uptake through binding to the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). We hypothesized that by inhibiting BCR signaling, and consequently cholesterol biosynthesis with the BTK inhibitor ibrutinib, we could overcome inherent resistance in cholesterol replete ABC DLBCL cell lines in vitro and in a xenograft model.Methods: HDL NPs were synthesized by surface-functionalizing a gold nanoparticle core as previously described (Thaxton et al JACS 2009) with the HDL defining apolipoprotein A-I and phospholipids, with the resultant HDL NPs having similar size, surface composition, charge and function as natural HDLs. However, owing to the gold nanoparticle core, HDL NPs are not an appreciable source of cholesterol or cholesteryl esters. GC DLBCL (SUDHL4) and ABC DBLCL (TMD8, HBL-1) cell lines were treated with HDL NPs as monotherapy or in combination with BCR signaling inhibitors (e.g. ibrutinib), and assayed for total cholesterol content and cell viability. Quantitative PCR was conducted to measure changes in expression of cholesterol biosynthesis genes. TMD8 tumor xenografts were initiated in SCID-beige mice, and following implantation, the mice were treated with vehicle, HDL NPs, ibrutinib, or a combination of the two treatments.Results: As we have previously reported (Yang et al PNAS 2013), GC DLBCL are exquisitely sensitive to HDL NP as monotherapy while ABC DLBCL are less sensitive. Here, our data demonstrate that HDL NPs, as a monotherapy, statistically significantly reduced total cellular cholesterol levels in SUDHL4 cells (7.96 ± 1.47 µg cholesterol/ 104 cells for 0nM HDL NP vs. 2.07 ± 2.07 µg cholesterol/ 104 cells for 100nM HDL NP; p=0.0007), resulting in profound cell death (HDL NP IC50 = 2nM). Further, HDL NP-induced cholesterol depletion resulted in upregulation of numerous cholesterol biosynthesis genes in SUDHL4 cells, including HMG-CoA synthase 1, 7-dehydrocholesterol reductase, lanosterol synthase, and others. In ABC DLBCL cells, the enhanced BCR signaling and resultant de novo cholesterol synthesis drastically diminishes the ability of HDL NPs to reduce cellular cholesterol (TMD8: 12.80 ± 5.08 µg cholesterol/ 104 cells for 0nM HDL NP vs. 16.35 ± 1.17 µg cholesterol/ 104 cells for 100nM HDL NP, p=0.84; HBL-1: 29.16 ± 5.96 µg cholesterol/ 104 cells for 0nM HDL NP vs. 23.51 ± 5.65 µg cholesterol/ 104 cells for 100nM HDL NP, p=0.75) and induce cell death (TMD8 HDL NP IC50 = 69nM; HBL-1 HDL NP IC50 = 75nM). Additionally, HDL NPs had little to no effect on the expression of the cholesterol biosynthesis genes in the ABC cell lines. To reduce cholesterol biosynthesis, which depends on BCR signaling, we combined HDL NPs with the BCR signaling inhibitor ibrutinib. We found that HDL NP synergize with ibrutinib in the ABC derived TMD8 cell line in vitro and in a xenograft model. Correspondingly, HDL NPs and ibrutinib reduced the total cellular cholesterol content of TMD8 and HBL-1 cells when administered together, but had no effect when given alone. Thus, by targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis we achieved synergistic cellular cholesterol reduction and subsequent cell death, both in vitro and in the TMD8 in vivo tumor xenograft model.Conclusion: These results in DLBCL lymphoma cell lines and tumor xenografts demonstrate that depletion of cellular cholesterol, in a specifically targeted manner dependent on the HDL receptor SCARB1 and differential BCR signaling in ABC vs GCB cells, is a powerful mechanism to induce apoptosis in lymphoma and opens a new paradigm in lymphoma biology and therapy. DisclosuresThaxton:Aurasense: Other: The HDL NPs technology was licensed by Northwestern University to a biotech company (Aurasense) founded by C. Shad Thaxton . Gordon:Janssen: Other: Data Monitoring Committee.

Abstract 5215: Inhibition of PIM kinases targets synthetic vulnerabilities and enhances antigen presentation in B cell lymphoma

Abstract The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Eμ-PIM and Eμ-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin, suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Citation Format: Patrizia Mondello, Chiara Tarantelli, Luciano Cascione, Alberto J. Arribas, Andrea Rinaldi, Anas Younes, Francesco Bertoni. Inhibition of PIM kinases targets synthetic vulnerabilities and enhances antigen presentation in B cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5215.

Inhibition of PIM Kinases Targets Synthetic Vulnerabilities and Enhances Antigen Presentation in B-Cell Lymphoma

The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Em-PIM and Em-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. (Figure 1A)The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin (Figure 1B), suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. (Figure 1C) Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. (Figure 1D) Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. (Figure 1E) In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Disclosures Younes: Xynomics: Consultancy; Biopath: Consultancy; Genentech: Research Funding; AstraZeneca: Research Funding; Syndax: Research Funding; BMS: Research Funding; HCM: Consultancy; Celgene: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Takeda: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Curis: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Abbvie: Honoraria; Pharmacyclics: Research Funding. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants.

Disrupting myddosome assembly in diffuse large B‑cell lymphoma cells using the MYD88 dimerization inhibitor ST2825.

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkins lymphoma, is classified into germinal center and activated B cell (ABC) subtypes. The myeloid differentiation primary response gene 88 (MYD88) L265P mutation is the most prevalent oncogenic mutation among patients with ABC DLBCL, the subtype that has the more inferior outcome. MYD88 oligomerization driven by the L265P mutant augments myddosome assembly and triggers the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, highlighting MYD88 oligomerization as a potential therapeutic target for this malignancy. The synthetic peptidomimetic compound ST2825, which has previously been used as an anti-inflammatory agent, has been reported to inhibit MYD88 dimerization. In the present study, the anticancer effects of ST2825 were investigated using L265P-expressing ABC DLBCL cell lines. Using confocal microscopy and high-molecular-weight fraction experiments, it was revealed that L265P-associated myddosome assembly was disrupted by ST2825. The results also revealed that disrupting myddosome assembly promoted the death of ABC DLBCL cells harboring the L265P mutation, as well as downregulating survival signals, including the inhibition of NF-κB and the suppression of IL-10 and interferon-β production. Further co-immunoprecipitation studies demonstrated that MYD88 bound to BTK in L265P-DLBCL cells, and that this binding was abrogated following ST2825 treatment. Furthermore, the combination of myddosome-assembly disruption and BTK or BCL-2 signaling inhibition led to synergistic ABC DLBCL cell death, and more robust inhibition of NF-κB activity or increased apoptosis, respectively. The results of the present study provide evidence that the synthetic peptidomimetic compound ST2825, which targets myddosome assembly, may serve as a pharmacological inhibitor. ST2825 has the potential for clinical use in patients with L265P DLBCL, and other B-cell neoplasms driven by activated MYD88 signaling.

Dasatinib Is Preferentially Active in the Activated B-Cell Subtype of Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, and at least one-third of its patients relapse after treatment with the current chemotherapy regimen, R-CHOP. By gene-expression profiling, patients with DLBCL can be categorized into two clinically relevant subtypes: activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL. Patients with the ABC subtype have a worse prognosis than those with GCB, and the subtype is defined by chronic, over-active signaling through the B-cell receptor and NF-κB pathways. We examined the effects of the Src family kinase (SFK) inhibitor dasatinib in a panel of ABC and GCB DLBCL cell lines and found that the former are much more sensitive to dasatinib than the latter. However, using multiplexed inhibitor bead coupled to mass spectrometry (MIB/MS) kinome profiling and Western blot analysis, we found that both subtypes display inhibition of the SFKs in response to dasatinib after both short- and long-term treatment. The MIB/MS analyses revealed that several cell-cycle kinases, including CDK4, CDK6, and the Aurora kinases, are down-regulated by dasatinib treatment in the ABC, but not in the GCB, subtype. The present findings have potential implications for the clinical use of dasatinib for the treatment of ABC DLBCL, either alone or in combination with other agents.

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Lenalidomide modulates gene expression in human ABC-DLBCL cells by regulating IKAROS interaction with an intronic control region of SPIB

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is associated with a poor prognosis compared with other DLBCL types and therefore represents a top priority for developing novel therapies. Lenalidomide, an immunomodulatory drug in trials for treatment of ABC-DLBCL, targets the transcription factor IKAROS for degradation by the cereblon E3 ubiquitin ligase complex. In this study, we investigated whether the gene encoding the transcription factor SPI-B is a target of IKAROS. Using cultured ABC-DLBCL cell lines, we found that high levels of SPI-B expression conferred resistance to lenalidomide. Lenalidomide treatment of ABC-DLBCL cells resulted in downregulation of SPIB at the level of transcription. SPIB was regulated directly by IKAROS through a binding site located in the first intron of the gene. Inhibition of IKAROS binding using CRISPR/Cas9-mediated transcriptional repression downregulated endogenous SPIB transcription. Finally, ectopic expression of IKAROS protected SPIB from downregulation. These results show that the mechanism of action of lenalidomide in ABC-DLBCL cells involves downregulation of SPIB transcription by cereblon-induced degradation of IKAROS. These results have implications for the design of synthetic lethal therapy for the treatment of ABC-DLBCL.

HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib

AbstractActivating mutations in MYD88 are present in ∼95% of patients with Waldenström macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88.

CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas

AbstractA hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor–κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL.

Birinapant Enhances Bendamustine-Induced Apoptosis In Activated B Cell-Diffuse Large Cell Lymphoma Cells

Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL. Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (<5% annexin V+ cells) as a single agent on any of these B cell lymphoma cell lines at ≤ 100 nM, though a low level of apoptosis (7-12% annexin V+ cells) was detectable at 10-20 µM in GC types. Addition of birinapant 30-60 minutes prior to BDM did not further enhance the already high level (>50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2, and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced <5% annexin V+ cells for U2932 and 10-15% for TMD8, addition of 100 nM birinapant 30-60 minutes prior to 10 uM BDM induced 35-40% annexin V+ cells in each of these ABC-DLBCL cell lines. This enhancement was schedule-dependent, not observed when birinapant was added after BDM. Thus, the cell lines representing FL and transformed FL are sensitive to BDM at clinically-achievable concentrations, without further enhancement by birinapant. The 3 DLBCL lines were relatively insensitive to BDM compared with FL cells, but BDM-induced apoptosis was markedly enhanced when birinapant was added before (but not after) BDM in the 2 ABC type DLBCL lines. Further explorations into the mechanism of birinapant sensitization of ABC-DLBCL to BDM, issues of dose and schedule, and role of NF-kB-dependency are ongoing. These data suggest that therapeutic trials of BDM plus birinapant would be of interest in ABC type DLBCL. Disclosures:No relevant conflicts of interest to declare.

Pharmacogenetic Approaches Identify ANTXR1 As a Potential Mediator of Response to Therapeutic NF-κB Inhibition in Diffuse Large B Cell Lymphoma

Abstract 234 Background:NF-κB is a family of transcription factors known to play an essential role in the development & survival of lymphocytes. In recent years, it has been clear that aberrant NF-κB activation is a hallmark of various lymphoid malignancies and appears to be associated with chemotherapy resistance and adverse prognosis. The canonical NF-κB pathway is frequently engaged in lymphoid malignancies wherein activated IKK phosphorylates IκB proteins inducing IκB polyubiquination and subsequent proteasomal proteolytic degradation; this allows for release and nuclear translocation of NF-κB dimers to activate target gene transcription.Gene Expression Profiling (GEP) has identified two distinct sub groups of Diffuse Large B cell Lymphoma (DLBCL). While the Activated B cell (ABC) type shows constitutive activation of NF-κB, the role for NF-κB activation in Germinal Center B Cell (GCB) DLBCL is currently unclear. Since NF-κB inhibition has been identified as a therapeutic possibility in DLBCLs, it is important to define the role of this pathway and its modulators. In this study, we sought to investigate key regulators of the NF-κB pathway that might mediate a therapeutic response to IKKβ inhibition of NF-κB in DLBCL including the GCB subtype. Through GEP and Exome Sequencing, we demonstrate that ANTXR1 is a key mediator of response to IKKβ inhibition in DLBCL. Methods/Results:We obtained a novel selective inhibitor of IKKβ, TLX-2001 that has been found to be safe in animal models. IC50 were obtained on 61 cell lines representing various lymphomas including DLBCL (N=25) using cell viability MTT assays. The drug showed efficacy in both ABC and GCB DLBCL cell lines at physiologically achievable concentrations. These results were unsurprising in ABC DLBCLs which are known to depend on NF-κB activation, but the lethality of this selective drug in GCB DLBCLs was unexpected. To better understand the role of individual genes in the response in GCB DLBCLs, gene expression profiling was performed on 61 cell lines using Human Gene 1.0 ST Array. We found that ANTXR1 expression significantly correlated with NF-κB resistance (p = 0.035).Additionally, we sequenced the exomes of DLBCL tumors (N=95) and matched normal tissue (N=34). 95 cases of DLBCLs consisted of 73 cases of primary human DLBCLs and 22 DLBCL cell lines. Whole exome sequencing was performed using the Agilent solution-based system of exon capture to sequence all protein coding exons in the CCDS database. We identified 465 recurrently somatically mutated genes in these DLBCL cases, and found that mutation status of ANTXR1 was associated with high sensitivity to IKKβ inhibition (p =0.015) of NF-κB. Cell lines with non-synonymous mutations in ANTXR1 had over 3-fold lower IC50 (mean= 2.39 μM) compared to cell lines with no mutation in ANTXR1 (mean IC50 = 8.72μM). Discussion/Conclusion:ANTXR1 is the docking receptor for bacillus anthracis toxin, and anti-tumor responses have been observed in mice injected with recombinant engineered anthrax toxin. It is also known as TEM8 and maps to chromosome 2p13.1. Increased levels of TEM8 (tumor endothelial marker 8) have been noted in various malignancies including melanoma. Our data suggest that pharmacogenetic approaches that combine gene expression profiling and whole exome sequencing are useful tools for identifying novel genes that modulate therapeutic responses in lymphoma. Disclosures:No relevant conflicts of interest to declare.