ALBERT

Description: Background: We investigated the potency, biological mechanisms of action, and described the role of MYC in cell death to the proteasome inhibitor, ixazomib (MLN2238), in TCL and HL in vitro and in vivo tumor models. In addition, we sought to identify molecular biomarkers that were associated with response/resistance to ixazomib. Methods: TCL cell lines (Jurkat, Hut78, HH) and HL cell lines (L428, L540, L1236) were treated with ixazomib for 24-72 hours, cell viability and apoptosis were analyzed by MTT and Annexin/PI by flow cytometry (FC). In vivo anti-tumor activity and survival of tumor bearing SCID mice were determined using xenografts derived from Jurkat (TCL) and L540 (HL) cell lines. Gene expression profiling (GEP) was performed using Human Affymetrix 2.0 HT array, which included Gene Set Enrichment Analysis (GSEA), for Jurkat, L540, and L428 cell lines. Results: Treatment with 20-100 nanomolar (nM) of ixazomib resulted in time- and dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines with all IC50’s

Description: Background: PTCLs are a rare, heterogeneous group of neoplasms that account for 12% of non-Hodgkin lymphomas (NHL) in Europe and USA and 〉20-25% in Asia. There is no consensus on optimal treatment and 5-year survival rates remain

Description: Background: Previous work in our lab identified that ixazomib combined with the histone deacetylase inhibitor, belinostat, or the CHK1 inhibitor, AZD7762, were synergistic in lymphoma. Our goals were to explore the biologic effects of rational combinations of ixazomib with other novel agents in TCL and HL cells. Methods: Transcriptome analysis used the Human HT 12 Genechip Illumina with RNA isolated from Jurkat cells treated with vehicle, ixazomib, belinostat, or AZD7762 as single agents, and in combinations: ixazomib + belinostat or ixazomib + AZD7762. Data were background adjusted and quantile normalized using RMAExpress, and statistically relevant genes were determined by applying LIMMA with FDR

Description: Abstract 2711 Background: Fatty acid synthase (FASN) is a key enzyme of fatty acid synthesis and is upregulated in many cancers. Increased FASN in cancer is associated with poor prognosis, while inhibition of FASN results in cancer cell death. The MEK/ERK signal transduction is one of the primary pathways that activate tumor-related FASN. Lipoprotein lipase (LPL) is also involved in fatty acid metablishm as it releases free fatty acid (FFA) from circulating lipoproteins, making them available for cellular uptake. Notably, these concepts have emerged primarily from solid tumor studies; there is a comparative paucity of data in lymphoma. We examined the functional roles of FASN and LPL in DLBCL cells and their interaction with oncogenic signal transduction pathways including MEK/ERK and an upstream target, hypoxia inducible factor-1 alpha (HIF-1a). We also investigated potential therapeutic implications of targeting fatty acid metabolism for the treatment of DLBCL. Methods: We used the DLBCL cell lines OCI-LY3, OCI-LY19, SUDHL4, and SUDHL10 in normoxic or hypoxic (0.2% O2) conditions. Cerulenin (FASN inhibitor) and Orlistat (FASN and LPL inhibitor) were utilized to examine the effect of fatty acid enzyme inhibition on cell signaling and cell death. We assessed cell viability with the MTT assay and apoptosis by flow cytometric analysis of Annexin-V/propidium iodide (PI). FASN and LPL mRNAs were quantified in DLBCL cell lines by RT-PCR as well as through gene expression profiling (GEP) analysis (by cell of origin) using the CaBIG dataset. Further, FASN and associated signaling pathways (MEK, ERK, and HIF-1a) were analyzed by Western blot. Finally, for investigation of potential interactions between FASN and HIF-1a, or MAPK signaling, we utilized short hairpin RNA interference (shRNA) to knock down (KD) pathways of interest. Results: FASN protein expression was readily detectable in all DLBCL cell lines in normoxia, while the expression of LPL was barely detectable in most cells, except in SUDHL10 and only in hypoxic conditions. RT-PCR showed that all DLBCL cell lines tested expressed high levels of FASN mRNA, while minimal levels of LPL could be detected; GEP showed that FASN was expressed more prominently in germinal center (GC) DLBCL (p=0.0006 vs GC control and p=0.0001 vs non-GC DLBCL), whereas LPL was preferentially expressed in non-GC DLBCL (p

Description: Introduction: Fatty acid (FA) metabolism is altered in many solid tumor cancers in part through increased de novo synthesis of lipids via up-regulation of FASN as well as via increased utilization of lipids via β-oxidation. The dependence of DLBCL on FA metabolism, and potential associated oncogenic signaling pathways, are largely unknown. Methods: Cerulenin (FASN inhibitor), orlistat (combined FASN and lipoprotein lipase inhibitor), and BKM120 (PI3K inhibitor) small molecules were studied in OCI-LY3, OCI-LY19, SUDHL4, SUDHL6, and SUDHL10 DLBCL cell lines as well as in primary DLBCL cells for the impact on FA inhibition signaling and the induction of cell death (by MTT, cleaved caspases, and AnnexinV/PI by flow cytometric analysis). Synergy was assessed via Calcusyn software. All effects were compared by DLBCL cell of origin (COO). In addition, global transcriptome analyses of FASN inhibition were examined on Affymetrix Human 2.0 ST Genechip. Results: FASN protein was constitutively upregulated across all DLBCL cell lines, independent of COO. Treatment with pharmacological inhibitors of FASN (i.e., cerulenin or orlistat) resulted in dose- and time-dependent reduction in cell viability in all DLBCL cell lines. To further examine the dependence of DLBCL on fatty acids, OCI-LY3, SUDHL4, and SUDHL6 grown in presence of lipoprotein-depleted serum showed exquisite sensitivity to lipid deprivation resulting in near complete cytotoxicity (i.e., 〉98%) as determined by MTT, cleaved caspase 3 and PARP, and AnnexinV/PI. Moreover, these effects were completely rescued by Very Low Density Lipoprotein (VLDL) supplementation to growth medium. These effects were markedly more prominent in primary DLBCL cells compared with control non-malignant lymphocytes. Analysis of differentially expressed gene sets comparing germinal center (GC) and non-germinal center (non-GC) B-cell tumors with normal cells showed significant over expression of genes related to PI3K and lipid metabolism in GC B-cell tumors. Furthermore, global transcriptome analysis following cerulenin exposure revealed activation of carbohydrate and oxidative stress metabolism in GC-derived SUDHL6 and SUDHL10 cells. In the non-GC cell line, OCI-LY3, there was favorable biology induced with inhibition of progression when treated with cerulenin, with downregulated significant key genes representing growth promoting cytokines (Figure 1). Analysis of key significant genes in cerulenin-treated SUDHL10 cells resulted in induction of antioxidant genes (GSR, TXNRD1, NQO1, AIFM1) and oxidative stress responsive PHB, DHX9, HSP90 and PMPCA genes, which are upstream components of PI3K signaling. Western blot analysis with prohibitin (PHB) inhibitor rocoglamide in the presence of cerulenin inhibited phosphorylation of PI3K-p85 and also prominently downregulated FASN expression of GC-derived DLBCL, suggesting the role of PHB in PI3K response to cerulenin. In addition, inhibition of PI3K with BKM120 was shown to strongly down-regulate FASN expression in OCI-LY3, SUDHL6, and SUDHL10 DLBCL cells. Furthermore, treatment with BKM120 in combination with cerulenin was strongly synergistic (CI

Description: Abstract 1581 Background It is well-recognized that de novo long chain fatty acid (FA) synthesis, driven by the key enzyme fatty acid synthase (FASN), is crucial for the growth and survival of many types of cancer cells. We and others have observed FASN protein expression in diffuse large B-cell lymphoma (DLBCL) tumors. Furthermore, we have shown that higher levels of FASN in DLBCL tumors strongly predicted inferior survival, which was independent from the international prognostic index. We also recently demonstrated that, in addition to FA synthesis, various cancer cells can acquire FA from circulating lipoproteins, using the secreted enzyme lipoprotein lipase (LPL), and that this promotes cell growth. DLBCL, however, has never been examined in this regard. In this study, we investigated the functional significance of both de novo FA synthesis via FASN and exogenous FA uptake via LPL in DLBCL. Methods Levels of FASN and LPL mRNAs in DLBCL cell lines (SUDHL4, SUDHL10, OCI-LY3, OCI-LY19) were studied using reverse transcriptase polymerase chain reaction. We determined FASN and LPL protein expression by flow cytometry using a novel anti-LPL antibody that we developed. DLBCL cell lines were cultured +/− Cerulenin (an inhibitor of FASN), Orlistat (an inhibitor of FASN and LPL), or in lipoprotein-depleted serum +/− supplementation with very low density lipoprotein (VLDL) particles. The MTT assay was used to assess cell proliferation. Results DLBCL cell lines exhibited 〉10-fold variation in levels of FASN mRNA. Cerulenin and Orlistat each caused dose-dependent inhibition of proliferation of each cell line. The cells were partially rescued by the addition of palmitic acid, the FA product of FASN. Surprisingly, flow cytometry revealed that SUDHL4 and OCI-LY3 cells, which did not secrete LPL or show detectable LPL activity, displayed the enzyme on the cell surface. Moreover, in stark contrast to several other cancer cell lines, DLBCL cells were exquisitely sensitive to withdrawal of lipoproteins from the culture media. Indeed, 75–95% of the cells underwent apoptosis after only 24 hours in lipoprotein-depleted serum. In complete serum, the provision of VLDL particles did not rescue DLBCL cells from FA synthesis inhibition using Cerulenin, suggesting that the serum contains sufficient lipoproteins to saturate the FA uptake system. This prediction was validated in experiments utilizing lipoprotein-depleted serum, in which add-back of VLDL particles completely rescued the cells from Cerulenin-induced demise in a dose-related manner, with full restoration at approximately 100–200mcg/ml of VLDL. Conclusions Our data demonstrate that DLBCL cells employ both de novo FA synthesis via FASN and exogenous FA uptake using LPL to satisfy their strict requirement for FA. Interference with either pathway, using FASN inhibitors or lipoprotein-depleted serum, is cytotoxic indicating that neither alone is sufficient to support proliferation. Further, DLBCL cells show a striking dependency on exogenous FA of dietary origin compared with all other cancer cells we have examined. The observation that the cell lines can be rescued by provision of VLDL particles strongly supports the functional significance of the exogenous FA uptake pathway for DLBCL. Our data thus demonstrate that the extracellular lipase LPL is critical for the growth and survival of DLBCL cells. Surprisingly, the cells deploy LPL to their surface, and we speculate that this promotes efficient FA acquisition from circulating lipoproteins. Recognition that DLBCL relies on both synthesis and uptake of FA will provide guidance for drug development and dietary modifications to effectively target the metabolic requirements of this tumor. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1646 Background: Darinaparsin is a novel organic arsenical compound with potent anti-neoplastic activity. Preliminary clinical studies reported encouraging activity in patients with relapsed/refractory TCL and HL. We investigated the molecular mechanisms of action of darinaparsin in HL and TCL cell lines, the degree and method of cell death, and we investigated rational drug combinations for enhancement of therapeutic activity. Methods: TCL (Jurkat, Hut78, HH) and HL cell lines (L428, L540, L1236) were treated with darinaparsin at increasing time and concentrations. MAPK, AKT/PI3K, mTOR and relevant pathways were analyzed by Western blot. Cell viability was assessed by MTT and apoptosis with Annexin V/PI flow cytometric analysis; this was confirmed by Western for caspase activation and PARP cleavage. In vivo tumor growth inhibition and survival of SCID mice was determined using xenografts derived from Jurkat (TCL) or L540 (HL) cell lines. The in vivo studies started with 7–8 mice for each control and treatment group. Once tumor volume average reached 100–250 mm3, treatment groups were injected (5 days/week) with darinaparsin SQ daily for 3 weeks. For drug combinations, varied novel agents were rationally combined with darinaparsin (BEZ-235 [PI3K/mTOR dual kinase inhibitor], PXD101 [HDAC inhibitor], olaparib [PARP inhibitor], MG132 [proteasome inhibitor], MK-2206 [pan-AKT inhibitor], SP600125 [JNK inhibitor], SB203580 [p38 MAPK inhibitor] and PD98059 (anti-MEK small molecule). We also interrogated pertinent signaling pathways with shRNA stable knock outs (KO) +/− darinaparsin. Results: Treatment with 1–5μM Darinaparsin for 72 hours resulted in time- and dose-dependent cytotoxicity in all TCL and HL cell lines. IC50 observed with 72-hour treatment in the TCL lines Jurkat, HH, and Hut87 were 2.7μM, 3.2μM, and 6.7μM, respectively, and for the HL clines L540, L1236, and L428 were 1.3μM, 2.8μM, and 7.2 μM, respectively. Darinaparsin treatment also resulted in a dose dependent increase in apoptosis, detected by Annexin-V positivity and cleavage of PARP and caspases 3, 8 and 9 in all TCL and HL cell lines. In vivo experiments with lymphoma tumor xenografts derived from Jurkat showed significant inhibition of tumor growth (P

Description: Background: TCLs are an uncommon, heterogeneous group of neoplasms with no consensus on optimal treatment and human 5-year survival rates

Description: Background: Recent clinical trials have highlighted the therapeutic benefit of BTK inhibitors in lymphoma with varying degrees of activity dependent in part on morphology and genetic origin. There appears to be a proclivity of activity in activated B-cell-like (ABC) DLBCL, while the efficacy appears more limited in germinal center (GC) DLBCL. In addition, the activity of BTK inhibitors is not clear in TCL or HL. We sought to determine the in vitro effects of the investigational BTK inhibitor CC-292 as a single agent and in combination with several targeted small molecule inhibitors in DLBCL, TCL, and HL. Methods: DLBCL cell lines (GC: SUDHL6, SUDHL10, Farage, and OCI-LY19; and ABC: OCI-LY3) were treated with increasing concentrations of CC-292 (0.1-20μM) alone and in combination with AKT inhibitor (AZD5363) and dual PI3K/mTOR inhibitor (BEZ235) for 24-72 hours. We analyzed cell viability with MTT assay and expression of NFκB, AKT, mTOR, MEK, and PARP by Western blot analysis. Calcusyn (Biosoft, Ferguson, MO) software program was used to analyze synergistic effects with drug combinations, based on isobologram and the method of Chou and Talay, a combination index (CI) value

Description: Background: The canine is a highly appealing model for cancer research and discovery in part due to comparable histopathological features with humans, a fully intact immune system, similar clinicopathologic features, a more comparable body size and pharmacokinetic properties than the mouse, varied breed-specific incidence rates as well as a shared environment with humans. We and others have shown prominent transcriptomic overlap of human and canine NHL (cNHL) (McDonald T et al. Onctogarget, 2018). PI3K/Akt signaling plays an important role in lymphomagenesis, which is also a promising therapeutic target. However, identification of predictive genetic aberrations of therapeutic efficacy remains elusive. We evaluated the clinical activity of the pan-PI3K inhibitor, buparlisib, in a pilot clinical study in cNHL. Methods :We enrolled and treated 10 dogs with buparlisibwho were diagnosed with BCL in an IRB and IACUC approved clinical study. Cases included 2 treatment naïve and 8 dogs with relapsed disease that had relapsed s/p CHOP (6), L' asparaginase (1) and VELCAP (1) treatment. Pet owners were consented and the study subjects received buparlisib9mg/kg orally for 28 consecutive days. Analysis for tumor response were evaluated on weekly basis through direct tumor measurement or use of x-rays. Post-therapy fine needle aspirates (FNA) were collected on Days 0, 7 and 21 to examine predictors of response to BKM120. RNA from fine need aspirate cells were isolated and the transcriptomic changes were evaluated using Canine Genome 2.0 Affymetrix Array, followed by unbiased systems biology assessment for biological pathways using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). We performed unbiased assessment to determine pertinent biological pathways associated with treatment response. The overall impact was used to determine the global effect on tumor progression and cancer risk based on the specific regulation of each gene. A Carcinogenic Risk Score (CRS) was calculated based on these values to determine if there is a promoted risk for cancer (positive value) or inhibitory risk for cancer (negative value) by summing the log2 fold-change values of key genes and subtracting this from the sum of log2 fold-change values of the tumor suppressors when comparing pre-treated to BKM120 treated dogs. Results: Following four weeks of BKM120 treatment, the overall response rate was 30% with 1 complete response lasting 42 days; 2 partial responses lasting 55 and 72 days; 3 stable disease; and 4 progressive disease. Mild treatment related toxicities such elevated blood glucose, thrombocytopenia and anemia, fever, nausea and lethargic symptoms, with no treatment related toxicities in 2 cases were noted. Principal Component Analysis (PCA) and hierrachical clustering analysis of differentially expressed genes show that differentially expressed genes to cluster together in all dogs during post 2 week, indicating a consistent biological activity by BKM120 in all dogs regardless of breed, prior treatment or disease status. Pathway network analysis based on differentially expressed genes predicted activation of upstream regulators associated with tumor suppression including SOX1, SOX3 and GMNN (Week 1) and CEBPA (Week 2). Analysis of "key genes" involved in multiple biological processes appeared to be associated with response of PI3K inhibitortreatment. This included down regulation of CREBBP with a Cancer Risk Score (CRS) of -0.97 and downregulation of VIM, CDH3, WNT3, WNT5B and FGFR2 with a CRS of -2.98 (Fig 1). Conclusion: Results from our pilot study in cNHL showed encouraging clinical responses with a pan-PI3K inhibitor in 3 of 10 dogs. Furthermore, our unbiased characterization of biological pathways revealed that the observed GEP changes associated with tumor suppression and they reduced the risk for cancer progression. Overall, the canine model appears to be particularly attractive model that may be leveraged for the study of clinical and biological responses to novel therapeutic oncologic agents. Disclosures Evens: Bayer: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Novartis: Consultancy; Acerta: Consultancy; Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics International DMC: Membership on an entity's Board of Directors or advisory committees; Tesaro: Research Funding; Janssen: Consultancy; Affimed: Consultancy.