Raji 4RH Cells Research Articles

Venetoclax Pre-Treatment of CLL/SLL Patients Enhances Autologous CAR T-Cells Efficacy

Introduction Venetoclax is an oral targeted therapy drug that is FDA-approved for the treatment of chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL) in both the frontline and relapsed/refractory (R/R) settings. While Venetoclax's dominant mechanism of action is the inhibition of Bcl-2 and thus, priming malignant CLL/SLL cells for apoptosis, there are emerging data highlighting the effects of Venetoclax on T-cell fitness/function. T-cell populations in CLL/SLL are known to be functionally impaired, in part due to abnormal expansion of T-regulatory cell (Treg) populations that blunt normal anti-tumoral cellular immune responses by CD8 + T-cells, Th1 Helper T-cells, and NK-cells and allows CLL/SLL cells to proliferate. Treg cell frequency has also been shown to directly correlate with CLL/SLL progression, and treatment of CLL/SLL results in lower circulating Treg populations. The unfavorable and generally anti-inflammatory tumoral microenvironment fostered by CLL/SLL cells is a major obstacle to the development of effective chimeric antigen receptor (CAR) T-cell options for this disease, for which there are currently no FDA-approved products despite more than a decade of investigation. Objective: In our current work, we studied the impact of Venetoclax on T-cell composition and function in serially collected specimens from patients with CLL/SLL; and evaluated the function of CAR T-cells manufactured from Venetoclax treated patients. Methods Peripheral blood mononuclear cells (PMBCs) and plasma was collected from CLL/SLL patients (N=5) who were treated with Venetoclax using the standard ramp up dosing-protocol. All procedures were approved by our IRB under BDR 164122. Samples were collected at pre-treatment (baseline) and several post-Venetoclax dosing timepoints (Day +1 day, day +7, and day +30). B-cells were isolated by negative selection using magnetic beads. CLL cells were the B-cells were exposed Venetoclax alone or in combination with several chemotherapy agents. Cell viability was determined by Cell Titer glo after 48hrs of drug exposure. In addition, PBMCs samples were analyzed using multicolor parameter flow cytometry to determine T-cell subsets composition including memory phenotype/stemness and others at each time point. Thorough serum cytokine (IL-15, IL-6, Il-2 and IL-10) analysis was conducted at each time point. Using baseline and 1-week post Venetoclax PBMCs samples, T-cells were activated, and autologous second generation CD19-directed CAR T-cells were manufactured. The efficacy of CARs were then tested at a 1:1 effector to target ratio against Raji and Raji 4RH cells for 24 hours and analyzed for cytotoxicity via flow cytometry. Results As expected Venetoclax was active in all samples tested. In addition, synergistic activity was observed when combined with chemotherapy agents or targeted agents (i.e. BRD-4 inhibitors). Regarding T-cell function, flow cytometry analysis at day +30 of Venetoclax therapy demonstrated that T-helper and T-cytotoxic T-cells displayed less exhaustion markers when compared to pre-Venetoclax samples (Baseline). Of interest, autologous αCD19 CAR T-cells manufactured 1 week following Venetoclax therapy induced higher cytotoxicity against Raji (rituximab chemotherapy sensitive cell lines) or Raji 4RH (rituximab-chemotherapy resistant cell lines) cells when compared to CAR T-cells generated from Venetoclax pre-treatment T-cells. Conclusion Our data suggests that Venetoclax has anti-tumor activities beyond direct CLL cytotoxicity. Venetoclax therapy appears to repair T-cell subsets in CLL patients. Perhaps by decreasing exhaustion markers in CD4/CD8 T-cells, Venetoclax potentiate the cytotoxicity of anti-CD19 CAR T cells against B-cell lymphoma. Further work is needed to fully elucidate the mechanism(s) by which Venetoclax modifies the fitness and function of T-cell subsets. These efforts may guide the development of combination strategies using BH-3 mimetics and T-cell engagers based therapeutic options for patients with CLL/SLL.

Optimizing Ex-Vivo Expanded NK Cell- Mediated Cellular Cytotoxicity By Obinutuzumab Combined with NKTR-255 in Burkitt Lymphoma (BL)

Background: We have previously reported the success of treating children with Burkitt lymphoma (BL) resulting in a ≥90% long-term EFS utilizing short intensive rituximab containing chemoimmunotherapy (Cairo et al, Blood, 2007; Goldman /Cairo et al, Leukemia, 2012, Goldman /Cairo et al, Br J Haematol., 2014; Goldman /Cairo et al, Leukemia, 2021). However, the prognosis is dismal in children who are refractory or progress after chemoimmunotherapy (Cairo et al Br J Haematol. 2018). The mechanisms associated with this poor prognosis are mainly due to chemoradioimmunotherapy resistance and an immunosuppressive BL microenvironment (TME) (Cairo et al Br J Haematol. 2016; Cairo et al Br J Haematol. 2019) and represent an urgent unmet need. The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully ex-vivo expanded functional and active peripheral blood NK cells (PBNK) with irradiated feeder cells APC (K562-IL21-41BBL) (Chu/Cairo, et al, JITC 2020). We previously demonstrated that obinutuzumab has significantly enhanced ex-vivo expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and NK cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, which may lead to sustained anti-tumor immune response. Objective: To investigate the anti-BL effects of NKTR-255 on the ADCC of ex-vivo expanded NK cells with obinutuzumab against rituximab-resistant BL in vitro and in vivo using human BL xenografted NSG mice. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Lee, et al, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFN-g, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, Front. Immunol, 2022). Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Luciferase expression Raji-4RH cells were xenografted to NSG mice as we previously described (Chu/Cairo, et al, JITC 2021). Results: NKTR-255 significantly enhanced the proliferation of ex-vivo expanded NK cells (p<0.0001) at day 7. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab against rituximab-resistant BL cells like Raji-2R (E:T=3:1, p <0.01), and Raji-4RH (E:T=3:1, p<0.01) as compared to the control groups. NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN- g: p<0.001, granzyme: p<0.01 and perforin: p<0.01) as compared to controls. Our in vivo study demonstrated that the combination of NKTR-255, Obinutuzumab and expanded NK cells significantly improved the survival of mice xenografted with Raji-4RH compared to controls (Fig.1). Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with Obinutuzumab against rituximab-resistant BL cells in vitro with enhanced IFN- g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and Obinutuzumab against rituximab-resistant BL cells using humanized NSG models are very promising. Mechanisms studies of BL relapsed from the combination therapy are under investigation. (Supported by HHOW and St Baldrick grants).

Trastuzumab regulates phosphoinositide 3 kinase/protein kinase B/mammalian target of rapamycin pathway to improve chemotherapy resistance in lymphoma cells

This study assessed the effect of trastuzumab on PI3K/AKT/mTOR (phosphatidylinositol-3-kinase/Protein Kinase B/Mechanistic Target of Rapamycin) pathway and chemotherapy resistance of lymphoma cells. The expressions of p-AKT (Rat phosphorylated AKT protein) and p-mTOR (Mouse p-mammalian target of rapamyein) were measured by immunohistochemical analysis, immunoblotting, real-time qPCR (quantitative polymerase chain reaction) and Western blot, along with analysis of drug resistance rate, cell viability, anti-tumor activity and apoptosis. p-AKT (Rat phosphorylated AKT protein) and p-mTOR (Mouse p-mammalian target of rapamyein) were abundantly expressed in the cytoplasm, while p-S6 was only abundantly present near the nucleus, and p-4E-BP1 was abundantly expressed at both sites. All the proteins were highly expressed in PCNSL (primary central nervoussy stemlymphoma) except for p-AKT (P < 0.05). In addition, the proliferation rate of drug-resistant cells was rapid in the presence of drugs. The p-AKT level of trastuzumab chemo resistant Raji cells was significantly lower than Raji 2R and Raji 4RH cell lines. Moreover, Trastuzumab sensitivity was significantly increased in cells after inhibitory expression and overexpression of PIK3R2. MK-2206 inhibited Akt and decreased cell proliferation. p-AKT knockdown decreased Raji cell activity after exposure to doxorubicin. MK2206 and doxorubicin jointly promoted PARP (poly ADP-ribose polymerase) cleavage, and cells were exposed to MK2206 and either adriamycin or dexamethasone, both had a synergistic effect on reducing cell viability. Idelalisib exposure resulted in altered expression of Bcl-2. p-AKT and p-mTOR were significantly elevated in PCNSL specimens, along with higher expressions of p-S6 and p-4E-BP1. Drug-resistant cells proliferated faster than parental sensitive cells in the presence of drugs, and Raji cells sensitive to trastuzumab chemotherapy had low p-AKT expression. Finally, MK-2206 inhibited Akt and reduced viability of BL cell lines. p-AKT inhibition resulted in enhanced response to cytotoxic chemotherapy and promotes apoptosis after cytotoxic chemotherapy.

Contribution of Bcl-2 Inhibitor Venetoclax Toward Anti-CD19 CAR T Cell Efficacy in Relapsed/Refractory Diffuse Large B Cell Lymphoma

IntroductionClinical outcomes of anti-CD19 chimeric antigen receptor T-cell (CAR-T CD19) therapy for the treatment of relapsed/refractory Diffuse Large B Cell lymphoma (r/r DLBCL) must be improved, as patients undergoing CAR-T CD19 therapy can expect a cure rate of only 41% and a median survival of just 6.3 months if unresponsive. Of patients presenting with naïve DLBCL, 35% will advance to third-line treatment with CAR-T CD19 therapy. Poor clinical responses to CAR-T CD19 therapy and a small window of survival for unresponsive patients highlights the critical need for improvement of CAR-T CD19 therapy in the setting of r/r DLBCL. Overexpression of anti-apoptotic Bcl-2 protein has been characterized in r/r DLBCL, and contributes to the disease's aggressive phenotype. Aberrant expression of the Bcl-2 protein proves targetable by an FDA-approved small molecule inhibitor which mimics the role of pro-apoptotic BH3 proteins, known as Venetoclax. Here, we investigate the effect of Venetoclax on CAR-T CD19 cell immunophenotype, effector function, and cytotoxicity upon co-culture with a lab-generated model of immortalized rituximab/chemo-resistant B cell lymphomas (Raji4RH) which recapitulate the resistant phenotype of r/r DLBCL seen in patients.MethodsPBMCs were isolated and activated from apheresis samples from consenting adults (healthy donors) using the Miltenyi Biotec TransACT human CD3/CD28 Kit. Cells were transduced with lentiviral vectors encoding for 2nd generation CAR constructs 24 hours post-activation. Transduced cells were cultured in RPMI media supplemented with hIL-2 (1ng/mL) and hIL-7 (10ng/mL). At day 14, cells were cultured with Raji4RH and Venetoclax in either combined or solitary cultures. Co-culture cytotoxicity assays were performed by pre-labeling target cells with permeant dye (Thermo Fisher Scientific CellTrace Blue Proliferation Kit), then incubating with CAR-T CD19 cells (GFP labeled) at various effector:target ratios and Venetoclax concentrations for 24 hours. Sample wells were stained with viability dye and acquired on BD LSR II Cytometer. CAR-T CD19 cell immunophenotype was identified following 24-hour exposure of Venetoclax to CAR T cell populations at various concentrations. Following exposure, samples were stained with viability dye, CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD25, and CD95. Frequencies of T REG, T Central Memory, T naive, and T Stem Cell Memory populations were calculated. CAR-T CD19 cell effector function was elucidated by activating (24 hours), then exposing CAR-T CD19 cells to concentrations of Venetoclax for 24 hours. Brefeldin A was added to culture for the remaining 4 hours of incubation. Cells were collected and stained intracellularly for IFNγ, Perforin, and Granzyme B; and extracellularly for CD3, CD4, and CD8. Sample acquisition for effector function assay was performed on BD LSRII Cytometer.ResultsConcurrent in vitro CAR-T CD19 administration and Venetoclax exposure significantly increases Raji4RH cell death as opposed to single agent cohorts. CAR-T CD19 cell viability is unaffected by Venetoclax at concentrations which confer synergy, and a predominantly CD8+ population is favored. Finally, Venetoclax appears to significantly increase the frequency of T N/T SCM and T CM within exposed cultures, while affording a 21% decrease in T REG frequency. Additionally, the notable increase in T N/T SCM and T CM phenotypes following exposure to Venetoclax did not arise as a consequence of decreased cellular viability.ConclusionThe combination of Venetoclax exposure and αCD19 CAR T cell administration demonstrates synergy when employed in the context of rituximab-resistant lymphoma cells, which may be explained by the effect of the BH3 mimetic's exposure on CAR-T CD19 cell immunophenotype. The ability to manipulate Bcl-2 protein expression within CAR-T CD19 cells affords insight into the role that the Bcl-2 family pathway plays within CAR-T CD19 cell biology. In addition to answering fundamental questions of CAR T cell biology, the combination of Venetoclax and CAR-T CD19 therapy may provide a solution to the observed clinical gap, in which CAR-T CD19 therapy alone cures less than half of all patients who advance to this third line treatment regimen. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

XIAP Knockdown Can Increase the Activity of Caspase 3 and 7, and Restore the Apoptotic Response in Cell Line Models of Rituximab Relapse/Refractory B-Cell Lymphoma

Rituximab-chemotherapy relapse/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) patients have lower response rates to second-line chemotherapy, and have a poor overall prognosis. Pre-clinical models of rituximab resistance demonstrated that the acquirement of resistance to anti-CD20 antibodies leads to concomitant resistance to chemotherapy drugs, suggesting the existence of share pathways of resistance between rituximab and chemotherapy agents. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines (RRCL), which also display significant resistance to a wide range of chemotherapy agents. RRCLs exhibit a deregulation in the apoptotic response pathway at several levels. Previous work by our group demonstrated that the X linked inhibitor of apoptosis (XIAP) is a key regulator of chemotherapy resistance in RRCLs. Moreover, we have established that knocking down XIAP in RRCLs can enhance the anti-tumor cytotoxic effect of chemotherapy, but a clear mechanistic role for XIAP in RRCLs has yet to be established. To investigate how XIAP regulates chemotherapy response in resistant lymphoma models we first looked at expression levels of XIAP and its binding partners in RRCLs and rituximab sensitive cell lines (RSCL). Although we observed no change in XIAP expression between the RRCL Raji 4RH and RSCL Raji, we did observe changes in several of the XIAP interacting proteins. Protein levels for both of the primary apoptotic effector caspases (caspase 3 and caspase 7) were substantially decreased in the Raji 4RH cell line compared to Raji cells. In addition, when we exposed Raji and Raji 4RH cells to gemcitabine (50uM) for 28 hours we observed substantially less release of cytochrome C from the mitochondria in Raji 4RH compared to Raji. Cytochrome C is an essential part of the apoptosome, which functions as a primary activator of caspases 3 and 7. Both of these observations support the hypothesis that impaired effector caspase activation and signaling contributes to chemotherapy resistance in RRCLs. To determine if targeting XIAP could restore caspase activation in RRCLs we used a transient siRNA knockdown of XIAP in the Raji 4RH cell line. Cells were treated with either the XIAP targeting siRNA, or a scramble sequence control, and then exposed to chemotherapy for 36 hours. Cellular viability was measured with the Promega CellTiter-Glo ATP assay, while caspase 3 and 7 activity was determined with the Caspase 3/7-Glo assay, also purchased from Promega. XIAP knockdown increased caspase activity by more than 500% compared to controls when cells were exposed to etoposide (20uM). This correlated with a 60% decrease in cellular viability in cells with an XIAP knockdown. In addition, the addition of the caspase inhibitor QVD-OPh along with the RIP kinase inhibitor necrostatin-1 (both at 10uM) was enough to restore cellular viability to the same levels observed in scramble treated controls exposed to the same dose of etoposide. Additional experiments looking at activation of caspases 2 and 6 in Raji 4RH cells following XIAP knockdown did not show any difference compared to scramble controls. In summary, XIAP appears to directly inhibit the caspase dependent apoptotic response in RRCLs. In addition, targeting XIAP can restore the apoptotic response in cell line models of rituximab resistant disease. These results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against rituximab rel/ref B-cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Effects of a Novel Fusion of ALT-803, an IL-15 Superagonist, with 4 Single-Chains of Anti-CD20 Antibody (2B8T2M) in Combination with Expanded Natural Killer Cells Against Rituximab Sensitive and Resistant Burkitt Lymphoma (BL)

Background:The prognosis of patients with relapsed or progressed B-cell (CD20+) non-Hodgkin lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemo-radiotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, BJH, 2016). Rituximab has been widely used in frontline treatment of B-NHL, however, some patients retreated with rituximab relapse which limit patient treatment options (Goldman/Cairo, Leukemia, 2013). Several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune cells with chimeric antigen receptors (Chu/Cairo et al, Can Imm Res 2015), as well as second-generation engineered anti-CD20 antibodies (Tiwari/Cairo et al BJH 2015). Nature Killer (NK) cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors. A variety of activating and inhibitory receptors on the NK cell surface are engaged to regulate NK cell activities. However, NK therapy is limited by several factors, including small numbers of active NK cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of NK cell immunosurveillance. Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 (Altor BioScience Corporation) is a superagonist of an IL-15 variant bound to an IL-15RαSu-Fc fusion with enhanced IL-15 biological activity (Zhu et al . 2009 J Immunol), longer half-life and increased potency (Han, et al . Cytokine . 2011). Recently, a new therapeutic molecule 2B8T2M was generated by fusing ALT-803 to four single-chains of the tumor-targeting monoclonal antibody, rituximab (Liu/Wong, et al, JBC, 2016). 2B8T2M displayed tri-specific binding activity through its recognition of the CD20 molecule on tumor cells, activated NK cells to enhance antibody-dependent cellular cytotoxicity (ADCC), and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016).Objective: To examine if 2B8T2M significantly enhances the cytotoxicity of exPBNK against rituximab-sensitive and -resistant BL cells.Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Denman/Dean Lee, PLoS One, 2012). CD56+CD3- exPBNK cells were isolated using Miltenyi NK cell isolation kit. ALT-803 and 2B8T2M were generously provided by Altor BioScience Corporation. NK receptors expression and cytotoxicity were examined as we previous described (Chu/Cairo, et al, Can Imm Res 2015). IFN-γ and granzyme B levels were examined by standard enzyme-linked immunosorbent assays. Equal doses of Rituximab, ALT-803, Rituximab+ALT-803, obinutuzumab (obinu) were used for comparison. IgG was used as controls. Rituximab-sensitive and -resistant BL cells Raji, Raji-2R and Raji-4RH, were used as target cells.Results: Both ALT-803 and 2B8T2M at equal doses significantly enhanced NK activating receptors such as NKG2D, NKp30, NKp44, and CD16 on exPBNK cells compared to IgG controls.2B8T2M significantly enhanced exPBNK cytotoxicity against rituximab-sensitive Raji cells compared to the controls IgG, Rituximab, ALT-803, Rituximab+ALT-803, obinu (2B8T2M vs IgG vs ALT803 vs Rituximab vs ALT803+Rituximab vs obinu=98.34%+0.24% vs 56.5+1.83% vs 62.8+1.1% vs 69.52+1.79% vs 81.85+1.83% vs 81.23+1.8%, p<0.001, E:T=1:1). 2B8T2M also significantly enhanced exPBNK cytotoxicity against rituximab-resistant Raji-2R cells (2B8T2M vs IgG vs ALT803 vs Rituximab vs ALT803+Rituximab vs obinu=90.7%+1.36% vs 20.47+7.7% vs 48.18+8.07% vs 37.44+3.63% vs 51.02+1.42% vs 65.51+3.03%, p<0.001, E:T=1:1) and resistant Raji-4RH cells (p<0.001, E:T=1:1) (Fig.1). Furthermore, we confirmed the enhanced cytotoxicity by measuring IFN-γ and granzyme B production. 2B8T2M significantly enhanced IFN-γ and granzyme B production from exPBNK against rituximab-sensitive Raji and -resistant Raji-2R and Raji-4RH compared to IgG (p<0.001), Rituximab (p<0.001), ALT-803 (p<0.001), Rituximab+ALT-803 (p<0.001), and obinutuzumab (p<0.001).Conclusions: 2B8T2M significantly enhanced exPBNK activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant BL cells. The in vivo functions of 2B8T2M with exPBNK against rituximab-sensitive and -resistant BL cells using humanized NSG models are under investigation. [Display omitted] DisclosuresAlter:Altor BioScience: Employment, Equity Ownership. Jeng:Altor Bioscience: Employment, Equity Ownership. Rhode:Altor BioScience: Employment, Equity Ownership. Lee:Intellia Therapeutics, Inc: Consultancy; Courier Therapeutics, Inc: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director. Wong:Altor BioScience: Employment, Equity Ownership. Cairo:Jazz Pharmaceuticals: Speakers Bureau.

Potentiate Immune Activation and Function By Targeting Inhibitor of Apoptosis Proteins (IAPs) in Relapse/Refractory DLBCL

Background: The prognosis of relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) that had progressed after or are ineligible for high dose chemotherapy and autologous stem cell support (HDC-ASCS) is extremely poor (median overall survival of 6.3 months). Cellular based immunotherapy with Chimeric Antigen Receptor T-cell against CD19 (CART-19) has proven high effective in r/r DLBCL with an overall response rate of 52-83% and an overall survival rate of 40%. While r/r DLBCL benefits from CART-19 therapy, a significant number of progress after initial response stresses the need to improve the efficacy of this novel approach. A significant amount of work is being done in improving the manufacturing or activation of CART cells. However, there is few data on lymphoma associated factors that could influence response to CART-19 therapy. Previously we reported an imbalance and dysfunction of pro- and anti-apoptotic proteins, including Bak/Bax, Mcl-1/BCLxL/Survivin and upregulation of inhibitor of apoptosis (IAP) family proteins in rituximab+chemotherapy (R-chemo) resistant DLBCL. Targeting IAPs by small inhibitor LCL161 in lymphoma cells significantly overcame chemotherapy resistance in vitro and in vivo. LCL161 potentiated CART-19 cell cytotoxicity mediated in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma pre-clinical models. Here, we investigate the mechanism of action to enhance immune activation by targeting IAPs in r/r DLBCL. Methods: Primary T-cells were isolated and activated from peripheral blood apheresis samples from consenting adults (healthy human donors or patients with DLBCL) using Pan T-Cell Isolation Kit (MACS) and activated by T-cell Dynabeads human activator CD3/CD28 kit. Cells were expanded in RPMI media supplemented with IL2 for 2 weeks. At day 14, cells phenotype analysis was performed (CD3, CD4, CD8) by flow cytometry. T-cell number was calculated by trypan blue exclusion assay and proliferation was measured by presto blue assay after exposure to LCL161 or control for 48hrs. After exposure, CD4 and CD8 ratios were determined by cellular surface staining. T-cell function assay was determined by intracellular staining with perforin and granzyme B. Regulatory T-cells were determined as CD25+ and FOXP3+ staining cells. T-cell cytolytic activity was accessed by 51Cr release assay using 51Cr labeled rituximab-chemotherapy sensitive (Raji and RL cells) or resistant (Raji 4RH and RL 4RH) cell lines as target cells (effector/target ratio 10/1). To confirm the role of IAP proteins in the effects of LCL161 in T-cell activation, we conducted cytotoxicity assays using XIAP knock out lymphoma cell lines as target cells. Stable XIAP knock out cell lines were generated by CRISPR-Cas9 gene editing system. Lentiviral Cas9 nuclease particles were transduced into Raji and Raji 4RH cells followed by lentiviral XIAP sgRNA transduction. Lentiviral sgRNA non-targeting control was also used as negative control. Result: In vitro exposure of T-cells to LCL161 did not affect cell viability. On the other hand, it led to an increase in CD8+ T-cells and in the CD8:CD4 ratio. In addition, the percentage of regulatory T cells (CD25+ and FOXP3+) was reduced following LCL161 exposure. Using the T-cell function assay, we found that perforin and granzyme B inside T-cell was significantly increased post LCL161 exposure. The presence of LCL161 significantly improved the T-cell killing activity towards rituximab-chemotherapy sensitive and more importantly resistant cell lines. XIAP knock out in target cells resulted in abrogation of the enhance cytotoxicity exhibited by T-cells exposed to LCL161. Conclusion: Our data suggest that besides its previously documented direct anti-tumor activity, LCL161 has dual functions modulating immune cells function. LCL161 improve T-cell function by increasing the number of CD8+ T-cells and intracellular levels of granzyme B and perforin leading to an increase in T-cell cytotoxicity. In addition, LCL161 decreases the number of T-reg cells further enhancing immune activation. Taking together, targeting IAPs would be an attractive approach that potentiate current CART-19 immunotherapy in the clinical setting. Disclosures Hernandez-Ilizaliturri: Astra Zeneca: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Seattle Genetics: Consultancy; Epyzome: Consultancy; Pharmacyclics: Consultancy; Amgen: Consultancy; Gilead: Consultancy.

Omipalisib (GSK458), a Dual an-PI3K/mTOR Inhibitor, Exhibits in Vitro and In Vivo activity in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Background: Reports of recurrent genomic alterations in Burkitt lymphoma (BL) have identified multiple recurrent alterations that result in activation of PI3K highlighting the importance of the PI3K/AKT/mTOR pathway in Burkitt lymphomagenesis. In a cell line model of resistant BL, we have previously identified an increase of PI3K/AKT/mTOR pathway activation suggesting a role in therapy resistance. While inhibition of PI3K-delta using the isoform specific inhibitor idelalisib has demonstrated clinical activity in indolent lymphomas, limited single agent activity has been observed in more aggressive variants. Pre-clinical investigation of idelalisib in BL indicated similar somewhat limited in vitro activity with synergistic activity in combination with chemotherapy. Broader inhibition of both upstream PI3K and downstream mTOR may exhibit more significant anti-lymphoma activity.Objectives: Investigate the in vitro and in vivo activity of the dual pan-PI3K/mTOR inhibitor omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models.Methods: Experiments were conducted in Raji, Raji 4RH (chemotherapy-rituximab resistant), Ramos, and Daudi BL cells. Cell viability following exposure to omipalisib +/- chemotherapy was analyzed using Cell-Titer Glo and Alamar blu assays. Induction of apoptosis was assessed by flow cytometry for Annexin V (AV)-propidium iodide (PI) staining. Downstream effects of omipalisib on PI3K/Akt/mTOR signaling were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using PI staining. Synergy of combination exposures was determined by calculation of the combination index (CI) using CalcuSyn software. In vivo activity was evaluated using disseminated Raji and subcutaneous Ramos SCID mouse xenograft models. The survival end point was hind limb paralysis in the disseminated model and tumor diameter >2cm in the subcutaneous model. Mice were treated with vehicle or omipalisib daily by oral gavage. Median survival was compared by Kaplan-Meier analysis.Results: Exposure of BL cells to omipalisib for 24-72 hours resulted in a dose- and time-dependent decrease in viable cells at nM concentrations (48h IC50 values: Raji=1.2uM, Raji 4RH=0.02uM, Ramos=0.01uM, Daudi=0.01uM) (Figure 1A). Marked induction of apoptosis occurred following 72h exposure to omipalisib primarily in chemosensitive cells with half-maximal effect noted at approximately 200nM, but requiring significantly higher concentrations to induce apoptosis in therapy resistant Raji 4RH cells (%AV positive at 200nM: Raji=40.7%, Raji 4RH=4.4%, Ramos=59.4% and Daudi=46.9%). Downstream of PI3K/Akt/mTOR, S6 and GSK3β showed reduced phosphorylation after 30 minute omipalisib exposure. G1 cell cycle arrest occurred in all cell lines following exposure to omipalisib for 72 hours; however, chemotherapy-resistant Raji 4RH cells arrested in G2/M at higher concentrations. BL cells exposed to omipalisib in combination with either doxorubicin or dexamethasone, exhibited synergistic anti-tumor activity (CI<0.9) with synergistic induction of apoptosis in therapy sensitive cells exposed to omipalisib and chemotherapy. NOD-SCID mice injected via tail vein with Raji-luc (provided by Dr. Mitchell Cairo) and treated with omipalisib demonstrated decreased luciferase signal compared to controls (Figure 1C) while mice with established subcutaneous Ramos xenografted tumors treated with omipalisib exhibited slower tumor progression compared to controls (Figure 1B), though with only modest prolongation of survival (median 28 vs 34 days, n=15/group, p<0.05).Conclusion: Dual PI3K-mTOR inhibitor omipalisib suppresses the PI3K/Akt/mTOR pathway leading to induction of apoptosis, impaired BL cell proliferation in vitro and in vivo and exhibits synergistic in vitro activity when combined with cytotoxic chemotherapy highlighting the relevance of PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High Mir-17-92 Expression Is Associated with in Vitro chemoresistance in Burkitt Lymphoma

Background: Copy number gain and/or MYC induction leads to increased expression of miRs in the miR-17~92 cluster. Development of Burkitt lymphoma (BL) is associated with translocation of MYC resulting in MYC expression and frequent recurrent copy number gains of chromosome 13q containing the miR-17~92 cluster of miRs. MiRs in the miR-17-92 cluster, including miR19 and miR17, have been associated with B-cell lymphomagenesis. Copy number gain of 13q and high expression of miR-17 have also been associated with possible inferior outcome in children with BL. MiR-17~92 miRs can decrease expression of the PI3K inhibiting protein PTEN leading to activation of the PI3K/AKT pathway, a pathway implicated in BL lymphomagenesis. These miRs also target the pro-apoptotic protein BIM, suppression of which has also been associated with chemoresistance in BL.Objectives: Investigate the in vitro effect of altered miR-17-92 expression on chemoresponsiveness in BL cells.Methods: MiR expression was determined by qPCR. The miR-17-92 locus was deleted in Raji cells using custom CRISPR-Cas9 lentiviral vectors. Single cell-derived clones, R5 and R24, were established and locus deletion determined by genomic DNA PCR. Protein expression and Caspase cleavage was assessed by western blotting. Apoptosis induction was determined by flow cytometry for Annexin V-propidium iodide staining. Cell viability following exposure to chemotherapy was determined by AlamarBlue assay. SCID mice were inoculated with Raji or R24 cells by tail vein injection and survival determined by Kaplan-Meier analysis. Hind limb paralysis was used as an end-point for survival. Antagomir targeting miR17 was transduced into Raji cells by electroporation.Results: Chemotherapy resistant Raji 4RH cells exhibit increased miR17 and miR19 expression and increased PI3K/AKT pathway activation compared to parental Raji. R5 and R24 Raji KO lines exhibit decreased miR17 and miR19 expression and increased expression of miR targets PTEN and BIM. When xenografted into SCID mice, R24 cells demonstrated a significant prolongation in survival compared to Raji (Raji vs R24: median 29 days vs not reached at 150 days, P<0.05) (Figure 1B). A significant decrease in [IC50] in R24 cells compared to Raji was observed following 48h exposure to doxorubicin (Raji vs. R24: 65±23nM vs 12±2nM), dexamethasone (1579±10e5nM vs 49±57nM), etoposide (571±106nM vs 106±22nM) or cisplatin (2.5±0.7uM vs 0.96±0.3uM) (Figure 1A). Raji R24 cells also demonstrate an increase in apoptosis induction following exposure to doxorubicin or etoposide. To investigate the effect of direct miR targeting, Raji cells were transfected with an antagomir targeting miR17 resulting in decreased viability following exposure to doxorubicin or cisplatin compared to Raji cells transfected with a control oligomer (Figure 1C). Antagomir transfected Raji cells express higher levels of PTEN and BIM compared to control Raji cells.Conclusion: High expression of miR17-92 cluster miRs is associated with in vitro chemotherapy resistance. Knockout of the MIR17HG gene in BL cells results in impaired proliferation, impaired in vivo engraftement with prolonged survival in SCID mice and increased in vitro chemoresponsiveness associated with an increase in expression of PTEN and BIM. Targeting of miR17 using an antagomir approach resulted in increased PTEN and BIM and increased chemoresponsiveness. These findings highlight the role of MYC-associated miRs in in vitro chemoresistance in BL cells warranting continued investigation as a possible therapeutic target for relapsed/refractory BL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Regulation of Cytokines/Chemokines Release and Anti-Tumor Effect of Expanded Natural Killer (NK) Cells By a Novel Fusion of N-820 (2B8T2M), an IL-15 Superagonist with 4 Single-Chain Anti-CD20 Antibody Domains, Against Rituximab Resistant Burkitt Lymphoma (BL)

Background: The prognosis of children with relapsed/refractory B-cell (CD20+) non-Hodgkin lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemo-radiotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, BJH, 2018). Rituximab has been widely used in frontline treatment of B-NHL, however, some patients retreated with rituximab will relapse which limits patient treatment options (Goldman/Cairo, Leukemia, 2013). Several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering of NK cells with chimeric antigen receptors (Chu/Cairo et al, Can Imm Res 2015), as well as second-generation engineered anti-CD20 antibodies (Tiwari/Cairo et al BJH 2015). To enhance NK based therapy, our group has successfully expanded the functionally activated peripheral blood NK cells (exPBNK) with irradiated feeder cells (Chu/Cairo, et al, Can Imm Res 2015). To enhance NK cell-based targeted therapy, we had successfully engineered expanded NK cells with chimeric antigen receptors (Chu/Cairo, et al, Can Imm Res 2015) and combined expanded NK with an anti-CD20 targeted IL-15 fusion protein N-820 (2B8T2M) targeting rituximab-sensitive and -resistant BL (Chu/Cairo, et al, ASH 2017). N-820 was generated by fusing ALT-803 to four single-chain antibody domains of rituximab (Liu/Wong, et al, JBC, 2016). ALT-803 is a superagonist of an IL-15 variant bound to an IL-15RαSu-Fc fusion with enhanced IL-15 biological activity (Zhu et al. 2009 J Immunol), longer half-life and increased potency (Han, et al. Cytokine. 2011). N-820 displayed tri-specific binding activity through its recognition of CD20 on tumor cells, activated NK cells to enhance antibody-dependent cellular cytotoxicity (ADCC), and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016).Objective: To investigate how N-820 modulates the crosstalk between BL tumor cells and expanded NK cells by monitoring cytokines, chemokines and growth factors released and the anti-tumor effects of N-820 on NK cells in BL xenografts.Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells and isolated as we have previously described with K562-mbIL15-41BBL (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 and N-820 were generously provided by Altor Bioscience. Equal doses of N-820, Rituximab (R), ALT-803, R+ALT-803, obinutuzumab (obinu, generously provided by Christian Klein, PhDfrom Roche) were used for comparison. IgG was used as control. Rituximab-sensitive Raji and -resistant BL cells Raji-2R and Raji-4RH were used as target cells. NK cells were sorted by Beckman Coulter MoFlo XDP high-speed sorter and NK purity was confirmed by flow cytometry. Cytokines, chemokines and growth factors mRNA levels in NK cells were monitored by real-time PCR. Secreted cytokines, chemokines and growth factors were examined by standard ELISAs. Raji-2R-Luc cells were injected into NSG mice. ExPBNK+N-820 and controls were injected to the xenografted NSG mice. The tumor burden was measured with the IVIS-200 system.Results: We found that ALT-803 and N-820 at equal doses significantly enhanced the expression of NK activating receptors such as NKG2D, NKp30, NKp44, and CD16 on exPBNK compared to IgG controls. Additionally, N-820 significantly enhanced exPBNK cytotoxicity against Raji, Raji-2R and Raji-4RH cells compared to the controls IgG, R, ALT-803, R+ALT-803, or obinu (p<0.001). We further examined 84 genes of cytokines, chemokines and growth factors by real time PCR. Among these 84 genes, N-820 up-regulated 26 genes and down-regulated 22 genes in exPBNK when co-cultured with Raji-2R compared to exPBNK alone. Using ELISAs, we confirmed that N-820 significantly enhanced IFN-g, granzyme B, and GM-CSF (Fig.1A) release from exPBNK against Raji-2R and Raji-4RH compared to IgG and R+ALT-803 (p<0.001). We also found that N-820 significantly reduced MDC (CCL22) (Fig.1B) release from Raji-2R cells compared to IgG (p<0.01) and R+ALT-803 (p<0.05). In NSG mice bearing Raji-2R tumor xenografts, we found that N-820 added to exPBNK significantly reduced tumor burden (p<0.05) and expanded mice survival (Fig.1C) (p<0.05) compared to control groups.Conclusions: N-820 significantly enhanced exPBNK cytotoxicity, IFN-g, granzyme B, GM-CSF release from exPBNK in-vitro, inhibited MDC release from BL and increased anti-tumor activity against BL xenografts in NSG mice. [Display omitted] DisclosuresJeng:Altor: Employment. Alter:NANTCell / Altor Bioscience: Employment. Rhode:NANTCell / Altor Bioscience: Employment, Patents & Royalties: Altor Bioscience. Lee:NantKwest: Employment. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wong:Altor: Employment, Equity Ownership, Patents & Royalties: Altor Bioscience. Cairo:Janssen: Research Funding.

Abstract 2565: EBV peptide-derived vaccine significantly enhanced in vitro cytotoxicity against EBV-positive B-cell lymphoma (EBV-BL) treatment using TMV-based delivery system

Abstract Background: EBV latent proteins (LMP-1/2) increase the chemoresistance in Burkitt Lymphoma (BL) cells by constitutively activating the CD40 receptor. It has also been demonstrated that B- cell lymphoma resistant to rituximab is a result of LMP-1 induced miR-155 expression and survival mediated by AKT phosphorylation (Hong et al.Gene Ther, 2012). EBV peptides delivered by a virus carrier (Tobacco mosaic virus;TMV) has the potential to boost immunity and stimulate potent T cell responses against EBV-associated antigens and induce a virus specific T cell response against EBV infected malignant cells (Miles/Cairo et al. BJH,2012, Kemnade/McCormick et al. Vaccine, 2014).We previously observed that obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al. BJH, 2015) in xenografted NSG mice. However, synergistic effects of obinutuzumab in-combination with LMP1/2-peptide vaccine against mature-B-NHL are unknown. Objective: To determine the efficacy of peptide specific cytotoxic T-lymphocytes (CTL) or the EBV vaccine alone/ in-combination with obinutuzumab against mature-B-NHL Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji cells (ATCC, USA) were cultured in 10% RPMI. Tumor cells were incubated with TMV-RIED IgG (LMP2) with obinutuzumab (100ug/ml) for 4 hrs with NK cells. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E:T ratio. Further, to generate LMP1/LMP2 peptide specific cytotoxic T cells (CTL), mature dendritic cells were pulsated with TMV-conjugated LMP1/LMP2 peptides for 24 hrs. PBMC isolated T cells were mixed with APC (LMPs-mature -DCs) at 1:20 ratio for activation and maturation. LMP-peptide specific CTLs were investigated in-vitro for cytotoxicity efficacy against BL cell lines. Results: LMP1 and LMP2 peptide were successfully conjugated with TMV. TMV conjugated and purified serum vaccine was tested against BL cell lines. Conjugated serum vaccine (LMP2-(RIED-TMV) +obinutuzumab+NK, compared to obinutuzumab+NK or vaccine+NK alone significantly enhanced in-vitro cytotoxicity 62.67±7.82% vs. 53.66±6.43% vs. 50.8±5.2% (p=0.04 and 0.02), respectively against BL (Raji). Furthermore, cytotoxicity of CTL (LMP1-MSD-TMV) or (LMP2-TYG-TMV) cells were significantly enhanced compare to IL2 treated T cells only, 58.5±8.6% or 48.56±15.18% vs. 21.42±13.3% in Raji (P=0.01, 0.02), and 57.8±7.9% or 45.52±19.4% vs. 14.48±4.2% in Raji4RH (P=0.003, 0.002), respectively. Conclusion: Our preliminary data indicates that LMP1 (MSD-TMV) and LMP2 (TYG-TMV) successfully conjugated with TMV and TMV-conjugated peptide specific CTL cells significantly enhanced cytotoxicity against BL cell lines compared to T cells only. Citation Format: Aradhana Awasthi Tiwari, Alison McCormick, Dina Edani, Aaron Newman, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. EBV peptide-derived vaccine significantly enhanced in vitro cytotoxicity against EBV-positive B-cell lymphoma (EBV-BL) treatment using TMV-based delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2565.

Therapeutic effects of 2B8T2M, a novel fusion of ALT-803, an IL-15 superagonist, with 4 single-chains of anti-CD20 antibody in combination with expanded natural killer cells against rituximab sensitive and resistant Burkitt lymphoma (BL).

32 Background: Patients retreated with rituximab often relapse which limit patient treatment options (Goldman/Cairo, Leukemia, 2013). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) to target BL (Chu/Cairo, et al, Can Imm Res, 2015). 2B8T2M was generated by fusing ALT-803, an IL-15 superagonist, to four single-chains of rituximab (Liu/Wong, et al, JBC, 2016). 2B8T2M displayed tri-specific CD20 binding activity, activated NK cells to enhance antibody-dependent cellular cytotoxicity, and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016). Methods: ALT-803 and 2B8T2M were generously provided by Altor BioScience Corporation. NK expansion, NK receptors expression and cytotoxicity were examined as we previous described (Chu/Cairo, et al, Can Imm Res 2015). IFNg and granzyme B levels were examined by ELISA assays. Equal doses of IgG, Rituximab, ALT-803, Rituximab+ALT-803, obinutuzumab (obinu, generously provided by Christian Klein, PhD from Roche) were used for comparison. Results: 2B8T2M significantly enhanced exPBNK cytotoxicity against rituximab-sensitive Raji cells compared to the controls IgG, Rituximab, ALT-803, Rituximab+ALT-803, obinu (p &lt; 0.001, E:T = 1:1). 2B8T2M also significantly enhanced exPBNK cytotoxicity against rituximab-resistant Raji-2R cells (p &lt; 0.001, E:T = 1:1) and resistant Raji-4RH cells (p &lt; 0.001, E:T = 1:1). Furthermore, 2B8T2M significantly enhanced IFN-g and granzyme B production from exPBNK against Raji, Raji-2R and Raji-4RH compared to IgG (p &lt; 0.001), rituximab (p &lt; 0.001), ALT-803 (p &lt; 0.001), Rituximab+ALT-803 (p &lt; 0.001), and obinutuzumab (p &lt; 0.001). Conclusions: 2B8T2M compared to rituximab, ALT-803 or obinutuzumab significantly enhanced exPBNK in vitro cytotoxicity against rituximab-sensitive and –resistant BL cells. The in vivo functions of 2B8T2M with exPBNK using humanized NSG models are under investigation.

Polatuzumab Vedotin; Antibody Drug Conjugates (ADC, anti-CD79b-vc-MMAE, PV) Significantly Enhances in-Vitro Cell Death Against CD79b+ Burkitt Lymphoma (BL) /Primary Mediastinal Large B Cell Lymphoma (PMBL) and Improved Overall Survival Against Rituximab Sensitive/Resistant-BL NSG Xenograft Mice

Background: Pediatric Burkitt Lymphoma (PBL) represents the most common malignancy in childhood and adolescent non-Hodgkin Lymphoma (NHL) but occurs in less than 5% of adult NHL (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012). Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood. 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). The prognosis of PBL has significantly improved over 30 years through the use of short and intense multi-agent chemotherapy, however, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease and a dismal prognosis (≤ 20% 5 years, EFS,Cairo et al.Blood. 2007; Cairo et al. JCO.2012, Miles/Cairo et al. BJH. 2012). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease.The anti-CD79b-vc-MMAE antibody drug conjugates (ADC, Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res. 2009; Dornan et al. Blood. 2009). More recently PV has been safe and well tolerated in adults with CD79b refractory NHL (Palanca-Wessels et al. Lancet Oncol.2015), However, PV preclinical activity against mature BL/PMBL is unknown.Objective: To determine the efficacy of the ADC (anti-CD79b-vc-MMAE) against CD79b+ PMBL in-vitro and rituximab (RTX) sensitive/resistant BL tumor cell lines in-vitro/ in-vivo.Methods: Raji/Raji4RH (provided by M. Barth, Roswell Park Cancer Institute) and PMBL: Karpas1106P and MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with hu anti- CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and cell proliferation was analyzed by alamar blue by flow-cytometry, n=3. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS only (control), isotype control (IgG), PV (5mg/kg), anti-CD79B mAb (5 mg/kg) and MMAE (5 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji and Raji4RH cells at 5x106 tumor cells/mouse as we have previously demonstrated (Awasthi/Cairo et al, BJH, 2015). Mice were treated twice a week for 6 weeks. Tumor burden was monitored by IVIS spectrum system.Results: Hu- anti -CD79b-vc-MMAE compared to hu anti-CD79b Ab or control hu IgG1 Ab alone (10µg/ml, 24hrs), significantly enhanced cell death ( apoptosis) in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p=0.0008 and p=0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p=NS and p=0.03), Karpas1106P, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p=0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p=0.002 and 0.0001), respectively. Hu anti- CD79b-vc-MMAE also significantly reduced cell proliferation compared to hu anti- CD79b Ab or control hu anti -IgG1 Ab alone (20µg/ml, 24hrs). Raji, 56.1±5.1% vs 38.1±0.7% vs. 14.8±0.4% (p=0.001 and p=0.0008), Raji4RH, 53.4±5.4% vs 23.4±5.51% vs. 11.3±0.8% (p=0.004 and 0.006), Karpas1106P, 46.4±0.3 %vs 29.0±1.5% vs. 15.9±0.6% (p=0.005 and 0.00007) and MedB-1, 47.2±7.5% vs 27.7±8.5% vs. 12.3±0.5% (p=0.01 and p=0.0005), respectively. Further, median survival time in mice receiving 5 mg/kg of PV was significantly increased when compared to mice receiving 5 mg/kg of anti-CD79b antibody or IgG isotype control in Raji and Raji4RH ( 35.5 vs.17 vs. 19.5 days, p=0.0001, 0.0003 and 50 vs. 18 vs. 18.5 days, p=0.0001, 0.0001, respectively, Figure. A and B)Conclusion: Our preliminary data indicates thathu anti- CD79b-vc-MMAE significantly enhances cell death and reduced cell proliferation in RTX sensitive/ and resistant CD79b+ BL and PMBL compared to CD79b Ab or isotype control IgG1 Ab alone. Furthermore, PV significantly increased survival in BL (RTX sensitive/ resistant) NSG xenografts compared to an equal dose of anti-CD79b alone. [Display omitted] DisclosuresCairo:Celgene: Research Funding.

Omipalisib (GSK458), a Novel Pan-PI3K/mTOR Inhibitor, Exhibits In Vitro Anti-Lymphoma Activity in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Background: Despite significant gains achieved in the treatment of Burkitt lymphoma (BL), current multi-agent immunochemotherapeutic regimens lead to high rates of acute toxicity, and relapsed/refractory disease still represents a significant hurdle with survival expected in only about 20-30% of such patients. Novel targeted therapeutic approaches are necessary to reduce treatment related toxicity in the up-front setting and improve survival in the relapsed/refractory setting. Analyses of genomic abnormalities in BL have identified increased activation of the PI3K/Akt/mTOR pathway in BL, induced by tonic B-cell receptor signaling and increased expression of Myc induced microRNAs (miRs), as having a significant role in Burkitt lymphomagensis. Additionally, recent reports have implicated higher expression of PI3K activating, Myc induced miRs in pediatric patients with a higher risk of relapse. While focused targeting of PI3K with the PI3K-delta isoform specific inhibitor idelalisib has led to significant activity in indolent B-cell lymphomas, limited activity has been noted in the setting of more aggressive forms. A broader inhibition of both upstream and downstream components of the pathway may exhibit more significant anti-lymphoma activity. To this end, we investigated the in vitro effects of PI3K/Akt/mTOR pathway inhibition with the dual pan-PI3K/mTOR inhibitor Omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models.Methods: The in vitro effect of omipalisib was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell viability following exposure to omipalisib alone and in combination with cytotoxic chemotherapeutic agents was analyzed using Cell-Titer Glo and Alamar Blue assays. Apoptosis was analyzed using western blotting for PARP and by flow cytometry with Annexin V-propidium iodide staining. Downstream targets in the PI3K/Akt/mTOR pathway were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using propidium iodide staining. Synergistic activity of combination exposures was determined by calculation of a combination index using CalcuSyn software.Results: In vitro exposure of BL cell lines to omipalisib in concentrations ranging from 0.05μM to 50μM for 24, 48 or 72 hours resulted in a dose and time dependent decrease in viable cells with significant activity noted at even low nM concentrations (48 hour IC50 values: Raji=1.2μM, Raji 4RH=0.02μM, Raji 8RH=1.9μM, Ramos=0.01μM, Daudi=0.01μM). Flow cytometry for Annexin V and propidium iodide, after 72 hours of single agent exposure to omipalisib, showed a marked induction of apoptosis in all cell lines. For example, at an omipalisib concentration of 200nM, the percentage of Annexin V positive cells were Raji=40.7%, Raji 4RH=4.4%, Raji 8RH=41.5%, Ramos=59.4% and Daudi=46.9%. Approximately ten-fold higher omipalisib concentrations were required to induce similar degrees of apoptosis in the chemotherapy resistant Raji 4RH cell line compared to chemotherapy sensitive cell lines. Western blotting for downstream targets of the PI3K/Akt/mTOR pathway, including S6 and GSK3Β, showed a reduction in phosphorylation after 30 minutes of exposure to omipalisib in all cell lines. Determination of cell cycle progression following exposure to omipalisib for 72 hours at concentrations ranging from 0.006μM to 25μM showed dose-dependent cell cycle arrest in G1 phase in all cell lines; however the chemotherapy resistant Raji 4RH cells arrested in G2/M at higher concentrations. When BL cells were exposed to omipalisib in combination with either doxorubicin or dexamethasone, synergistic anti-tumor activity was observed in all cell lines tested.Conclusion: Inhibition of PI3K and mTOR by the dual inhibitor omipalisib suppresses activation of the PI3K/Akt/mTOR pathway leading to impaired BL cell proliferation with G1 cell cycle arrest and induction of apoptosis in chemotherapy-sensitive and -resistant cell line models of BL. Inhibition of the PI3K/Akt/mTOR pathway with omipalisib also increases the in vitro response to cytotoxic chemotherapeutic agents. Our findings note the pre-clinical activity of PI3K/Akt/mTOR pathway inhibition in BL and highlight the relevance of pursuing PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL. DisclosuresNo relevant conflicts of interest to declare.

Targeting BET Bromodomains in Pre-Clinical Models of Burkitt Lymphoma

Background: Burkitt lymphoma (BL) is the most common NHL type in children. Although treatment for pediatric BL has improved significantly, there is an urgent need for novel therapies that reduce the toxicity of modern treatment regimens and improve on the dismal survival observed in the relapsed/refractory setting where only about 20-30% of patients survive their disease. Recent reports have implicated co-activation of c-Myc and PI3K in Burkitt lymphomagenesis. Genomic analysis of recurrent oncogenic mutations in BL have identified tonic B-cell receptor signaling and the over-expression of Myc induced microRNAs from the MIR17-92 family, e.g. mir17 and mir19, as possible mechanisms of PI3K activation in BL. Mir17 and mir19, have been implicated in Burkitt lymphomagenesis and their overexpression may be associated with a higher risk of relapse. The protein phosphatase and tensin homolog (PTEN) is a major regulator of PI3K pathway activation. MIR17-92 cluster members have been shown to target PTEN leading to increased PI3K activation. We have previously identified increased expression of mir17 and mir19 along with increased activation of AKT in cell line models of chemotherapy resistant BL suggesting a potential mechanism for increased resistance. BET bromodomains interact with chromatin and enhance transcriptional activation of numerous genes including c-Myc. Thus, BET bromodomains represent a promising target in BL. To investigate the effects of inhibition of c-Myc driven activation of the PI3K/AKT/mTOR pathway, we characterized the activity of the highly potent small molecule bromodomain inhibitor JQ1 in chemotherapy sensitive and resistant BL cell lines. Additionally, we analyzed the ability to enhance anti-lymphoma activity of PI3K/Akt/mTOR pathway inhibition in BL by the combination of BET bromodomain inhibition and targeted inhibition of the PI3K/AKT/mTOR pathway.Methods: The in vitro activity of JQ1 was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell Viability following exposure to JQ1 alone and in combination with the PI3K/mTOR inhibitor omipalisib (GSK458)) was analyzed using Cell-Titer Glo or Alamar Blue assays following 24, 48, and 72 hour exposure over a range of inhibitor concentrations. Induction of apoptosis was analyzed using western blotting for cleaved PARP. C-Myc expression following JQ1 exposure was determined by western blot following 48 hour JQ1 exposure. The effect of JQ1 exposure on the expression of c-Myc induced microRNA expression was determined by qRT-PCR in cells exposed to JQ1 for 48hours. Synergy of combination exposures was determined using CalcuSyn to generate combination index (CI) values.Results: In vitro exposure of BL cell lines to JQ1 for 24, 48, and 72 hours resulted in a significant dose and time dependent decrease in viable cells (72 hour IC50 values: Raji 0.12µM, Raji 4RH 1.7µM, Raji 8RH 0.7µM, Ramos 0.22µM and Daudi 4µM). There was an increase in cleaved PARP after 72 hour exposure indicating induction of apoptosis. While single agent effect was seen in the resistant Raji 4RH cell line, activity was noted to primarily occur at the higher end of the dosing. Western blot analysis demonstrated a reduction in c-Myc expression following exposure to JQ1 1µM for 24 hours (relative band intensity normalized to control: Raji=0.12, Raji 4RH=0.18, Raji 8RH=0.11). qPCR showed a reduction in Mir17 relative transcription levels after 48 hours of exposure to JQ1 2.5mM (relative expression compared to control: Raji=0.72, Raji 4RH=0.98, Raji 8RH=0.83, Ramos=0.57, Daudi=0.46). When combined with omipalisib, an increased effect on cell viability was noted. The combination effect was noted to be synergistic (CI<0.9) at multiple dose combinations while other combinations exhibited primarily additive effects.Conclusion: In vitro inhibition of BET bromodomains with JQ1 results in a decrease in c-Myc expression and a decrease in c-Myc dependent miR expression with impaired proliferation and induction of apoptosis in chemotherapy-sensitive and -resistant BL cell lines. Augmented, and in some cases synergistic, activity is also noted with dual inhibition of BET bromodomains and the PI3K/Akt/mTOR pathway in BL cell lines. DisclosuresNo relevant conflicts of interest to declare.

The XIAP Inhibitor BMT-062789 Displays Anti-Tumor Activity in Cell Line Models of De Novo and Rituximab Relapse/Refractory Lymphoma

The addition of rituximab to front line therapy for aggressive lymphomas has improved clinical outcomes, but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for rituximab associated chemotherapy cross-resistance our group developed several rituximab resistance cell lines (Raji 4RH and RL 4RH), which also display significant resistance to a wide range of chemotherapy agents. These rituximab resistant cell lines (RRCL)s exhibit multiple deregulations in the BCL-2 and inhibitor of apoptosis (IAP) protein families, including loss of the pro-apoptotic proteins Bax and Bak. We previously demonstrated that the X linked inhibitor of apoptosis protein (XIAP) is critically required for chemotherapy resistance in the RRCLs, and that an shRNA knockdown of XIAP increased chemotherapy response in both in vitro and in vivo models of rituximab resistant lymphoma. BMT-062789 is a heterodimeric mimetic of the second mitochondrial activator of caspases (SMAC) developed by Bristol-Myers Squibb, which can inhibit both the caspase 9 and caspase 3/7 binding domains of XIAP. BMT-062789 demonstrated dose and time dependent single agent anti-tumor effect (as measured by the Cell TiterGlo luminescent viability assay) in a panel of lymphoma cell lines, with IC50 values of less than 5uM for all cell lines tested except the rituximab resistant cell line Raji 4RH. The Burkitt's lymphoma cell line Daudi and diffuse large B-cell lymphoma cell line U2932 were particularly sensitive to BMT-062789 with IC50 values of 0.91uM and 0.76uM respectively. To investigate if the observed anti-tumor effect of BMT-062789 was due to increased apoptosis we exposed rituximab sensitive (Raji, RL) and rituximab resistant (Raji 4RH, RL 4RH) cells to escalating doses of BMT-062789 with or without the addition of 20uM etoposide for 48 hours. The induction of apoptosis was measured by flow cytometry with an Annexin-V:PE-Cy7 conjugate and Sytox blue (a DNA stain). 2uM BMT-062789 alone triggered apoptosis in 75% of Raji cells and 65% of RL cells. It is also worth adding that 2uM BMT-062789 induced higher rates of apoptosis than 20uM etoposide alone in both cell lines. The combination of 2uM BMT-062789 and 20uM etoposide together triggered apoptosis in 80% of Raji cells and 85% of RL cells, indicating that BMT-062789 may be able to augment the anti-tumor activity of conventional chemotherapy. More importantly, the combination of BMT-062789 and etoposide was also able to induce apoptosis in the rituximab resistant cell line models Raji 4RH and RL 4RH. 3uM BMT-062789 in combination with 20uM etoposide triggered apoptosis in 90% of Raji 4RH cells and 55% of the RL 4RH cells, which is a substantial improvement compared to 15% apoptosis with 20uM etoposide alone in each cell line. Additional studies are in progress to evaluate the anti-tumor effect of BMT-062789 in ex vivo samples from lymphoma patients with de novo and relapse/refractory disease. In summary, the novel heterodimeric XIAP inhibitor BMT-062789 has anti-tumor effect at low micromolar concentrations in lymphoma cell line models, including models of rituximab resistant disease. These results support earlier studies by our group indicating that XIAP is critical for survival in models of rituximab resistant lymphoma, and establish that XIAP inhibitors may have potential clinical value for the treatment of both de novo, and rituximab relapse/refractory lymphomas. DisclosuresNo relevant conflicts of interest to declare.

JQ1, a c-MYC Inhibitor Targets Hexokinase II and Enhances the Cytotoxic Effects of Chemotherapeutic Agents in Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Results from thecollaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated a poor clinical among patients treated with second-line chemo-immunotherapy in the post-rituximab era, stressing the need to identify and/or optimize novel targeted agents. C-myc expression was found correlate with a poor clinical outcome in patients with newly diagnosed or relapsed/refractory DLBCL. Previously, we found that JQ1 overcomes rituximab-chemotherapy resistance in lymphoma pre-clinical models. JQ1 exhibited synergistic activity when combined with chemotherapy agents. We also demonstrated that hexokinase II was highly expressed in rituximab resistant cell lines (RRCL), and its expression was associated with a deregulation in the glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. In our current work, we evaluated molecular studies dissecting the cellular pathways affected by JQ1.Methods: A panel of rituximab-sensitive (RSCL) and RRCL cell lines were exposed to escalating doses of JQ1 (0-8μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism 6 software. Loss of mitochondrial membrane potential (Δψm) following JQ1 exposure was assessed by DiOC6 staining. Changes in c-MYC, HKII, HKI, Voltage dependent anion channel protein (VDAC) and p21 expression levels were determined by western blotting. HKII was silencing using siRNA interference in RRCL (Raji-4RH) and changes in HKII, HKI, VDAC, c-MYC and p21 protein expression was determined. HKII knockdown or scramble Raji-4RH cells were exposed to different chemotherapy agents and JQ1; cell viability and mitochondrial membrane potential was accessed.Result:JQ1 induced a dose-and time- dependent cell death in cell lines. In vitro exposure cells to JQ1 resulted in a loss of mitochondrial potential and induced G1 cell cycle arrest. In vitro exposure to JQ1 decreased c-MYC and HKII expression levels and induced of p21 expression. Complete silencing of HKII re-sensitized cell to chemotherapy (doxorubicin, vincristine and bortezomib). On the other hand, HKII knockout abrogated JQ1 anti-tumor activity.Conclusion: Our data suggests that JQ1 had anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The inhibition of HKII following JQ1 exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic effect. Our finding highlights a better understanding in the molecular events triggered by JQ1 as a potential role in the treatment of B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2211: Enhanced in vitro and in vivo targeting of rituximab-sensitive and -resistant Burkitt lymphoma (BL) by anti-CD20 chimeric antigen receptor (CAR)-modified expanded natural killer cells in combination with a histone deacetylase inhibitor, romidepsin

Abstract Background: For BL patients who relapse, the prognosis is dismal due to chemo-immunotherapy resistance (Cairo et al, JCO, 2012, Goldman/Cairo et al, Leukemia, 2013). Our group has successfully modified expanded peripheral blood Natural killer cells (exPBNK) with an anti-CD20 CAR to target rituximab sensitive/resistant CD20+ BL cells in vitro and in NSG mice (Chu/Cairo, et al, Can Imm Res 2015). Romidepsin is a histone deacetylase (HDAC) inhibitor that increases the expression of NKG2D ligands in BL (Chu/Cairo, et al, Cytotherapy, 2014). Objective: We investigated the effect of romidepsin alone and in combination with anti-CD20 CAR modified exPBNK cells against CD20+ BL cells in vitro and in humanized BL NSG mice. Methods: Anti-CD20 CAR modified exPBNK cells were produced as we have described (Chu/Cairo, et al, Can Imm Res 2015). HDACs levels in BL cells were examined by western blot. Raji, Raji-2R and Raji-4RH cells were treated with 10ng/ml romidepsin, generously provided by Celgene. Raji-Luc engrafted mice were injected i.p. with romidepsin (2.2mg/kg) or PBS once a week for 3 consecutive weeks followed by anti-CD20 CAR exPBNK cell or mock exPBNK cell injections 24hrs later. Tumor regression and/or progression were monitored weekly by tumor volume measurement and by in vivo bioluminescent imaging (Chu/Cairo, et al, Can Imm Res 2015). Results: HDAC 1, 3, 6 levels were significantly increased in Raji, Raji-2R, Raji-4RH, Ramos, and Daudi compared to white blood cells. Total H3K9 acetylation was enhanced in Raji, Raji-2R, and Raji-4RH following romidepsin. More importantly, romidepsin significantly inhibited BL cell proliferation (p&amp;lt;0.001), induced apoptosis only in rituximab sensitive BL cells with increased active caspase 3, and induced cell cycle arrest in resistant BL cells (p&amp;lt;0.001). Romidepsin significantly inhibited in vivo Raji and Raji-2R cells growth in xenografted NSG mice with reduced tumor size (p&amp;lt;0.05) and bioluminescence (p&amp;lt;0.05). In vitro cytotoxicity of anti-CD20 CAR exPBNK cells was significantly enhanced against romidepsin treated BL cells compared to the untreated at E:T = 3:1 (P&amp;lt;0.001; n = 4), or compared to the mock exPBNK (P&amp;lt;0.05; n = 4). In humanized Raji xenograft NSG mice, survival time in romidepsin+CAR exPBNK treated mice was significantly extended compared to the untreated mice (median 28 days, P&amp;lt;0.001), the mock exPBNK treated mice (median 29 days, P&amp;lt;0.001), the CAR exPBNK treated mice (median 42.5 days, P&amp;lt;0.05), the romidepsin treated mice (median 30 days, P&amp;lt;0.001), and the romidepsin+mock exPBNK treated mice (median 34.5 days, P&amp;lt;0.05). Conclusion: These results suggest the therapeutic potential of the combination of anti-CD20 CAR modified exPBNK cells and romidepsin against chemo-immunotherapy resistant BL. Citation Format: Yaya Chu, Ashlin Yahr, Bokun Cheng, Bian Hang, Janet Ayello, Matthew Bath, Mitchell S. Cairo. Enhanced in vitro and in vivo targeting of rituximab-sensitive and -resistant Burkitt lymphoma (BL) by anti-CD20 chimeric antigen receptor (CAR)-modified expanded natural killer cells in combination with a histone deacetylase inhibitor, romidepsin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2211.

Abstract 3893: Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways

Abstract Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007) and further improved with the addition of rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012, Barth et al. BJH, 2013). Obinutuzumab, a novel glycoengineered type II CD20 Ab, mediates enhanced cell death &amp; ADCC against B-cell lymphoma vs. RTX (Awasthi/Cairo et al. BJH, 2015), and was recently FDA and EMA approved for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathway altered differentially following obinutuzumab vs RTX against RTX-sensitive/resistant BL Methods: Raji (CD20+) and Raji-4RH) cells were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab, and/or RTX for 24 hrs. For phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji /Raji4RH in the presence/absence of obinutuzumab or rituximab or isotype control. Silencing of PLCG2 (Dharmacon, USA) and MAPK1 (Sigma Aldrich, USA) in Raji/Raji4RH cell lines was carried out according to the manufacturer's instructions Results: In all 978 unique phosphorylated proteins were identified. Out of these 661 phosphoproteins were identified after obinutuzumab vs. 615 in RTX treatment, respectively. For the Raji4RH, 534 phosphoproteins were identified after obinutuzumab and 534 in RTX treatment, respectively (Fig.1). Functional annotation of proteins differentially phosphorylated in response to obinutuzumab vs. RTX (&amp;gt;1.5-fold) reveals the involvement of the BCR (PLCG2, BTK &amp; GSK3B), FC gamma phagocytosis (FCRG2B, MAPK1 &amp; RAF1), and Natural killer cell-mediated cytotoxicity (MAPK1, RAF1&amp; PLCG2) signaling pathways (Fig. 2). Differential phosphorylations of proteins involved in BCR or cytotoxicity pathways were validated by western blot after incubation with obinutuzumab vs. RTX in Raji/ Raji4RH cell lines, revealed up regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab vs. RTX treatment in Raji. Silencing, of PLCG2 and MAPK1 pathway, significantly increased cell proliferation and decreased cytotoxicity after obinutuzumab treatment in Raji (P = 0.0002 &amp; 0.000002) but no change in Raji4RH. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, and cytotoxicity signaling pathways. Obinutuzumab function differentially in RTX resistant and sensitive BL cell lines which may provide insights into alternate therapeutic strategy in RTX resistant BL. Citation Format: Aradhana Awasthi Tiwari, Delphine C.m. Rolland, Mona Elmacken, Janet Ayello, Lisa Kurien, Carmella van de Ven, Venkatesha Basrur, Kevin Conlon, Damine Fermin, Matthew J. Barth, Christian Klein, Kojo S.j. Elenitoba-Johnson, Megan Lim, Mitchell S. Cairo. Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3893.

PI3K/Akt/mTOR Pathway Inhibition in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Introduction: Burkitt lymphoma (BL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) in children. Despite significant improvements in survival with de novo disease, treatment of relapsed or refractory BL remains a significant hurdle with survival in only about 20% of patients. Novel therapeutic approaches are necessary to improve outcomes in this group of childhood B-NHL patients with the worst prognosis. Recent literature has identified a high rate of recurrent mutations that result in activation of the PI3K/Akt pathway in BL and have implicated activation of PI3K/Akt in coordination with Myc in BL lymphomagenesis. Our laboratory has developed rituximab and chemotherapy resistant cell line models and subsequently found that these cell lines exhibit increased activation of Akt. We hypothesized that increased activation of Akt may be contributing to chemoresistance and that targeting the PI3K/Akt/mTOR pathway may increase chemoresponsiveness. To that end, we have investigated the effect of inhibiting the PI3K/Akt/mTOR pathway with either the PI3K-delta inhibitor idelalisib or the pan-PI3K/mTOR inhibitor BEZ-235 in cell line models of BL.Methods: The in vitro effect of idelalisib or BEZ-235 was investigated in BL cell lines including Raji, Raji 2R and Raji 4RH (rituximab-chemotherapy resistant), Raji 7R and Raji 8RH (rituximab resistant), Ramos and Daudi. Cell viability following inhibitor exposure was assessed by Alamar blu and cell-titer glo assays. The effect of inhibitor exposure on cell cycle progression was determined by flow cytometry using propidium iodide staining. Inhibition of Akt activation following inhibitor exposure was determined using phospho-flow cytometry. The activity of cytotoxic chemotherapeutic agents following inhibition by idelalisib or BEZ-235 was assessed using Alamar blu and cell titer glo assays.Results: In vitro exposure of BL cell lines to idelalisib in concentrations from 0.1-100µM for 24, 48 or 72 hours resulted in a dose and time-dependent decrease in viable cells in all cell lines tested with IC50 concentrations of 60-300uM. Pre-treatment with the pan-caspase inhibitor QVD resulted in a small reversal in the decrease in cell viability suggesting only a minimal portion of the activity was caspase dependent. When induction of apoptosis was measured using annexin V-propidium iodide staining, little induction of apoptosis was observed with single agent idelalisib at concentrations up to 100uM. Determination of cell cycle progression following exposure to idelalisib at 1, 10, 50 or 100 uM for 24, 48 or 72 hours indicated a time and dose dependent cell cycle arrest in all cell lines. In chemotherapy-sensitive cell lines the arrest was primarily noted in G1, while the chemotherapy-resistant Raji 2R and Raji 4RH cell lines exhibited arrest primarily in G2/M. A significant reduction in cell viability following chemotherapy exposure for 48 hours was noted in chemotherapy resistant Raji 2R cells following pre-treatment for 48 hours with idelalisib 10uM compared to non-idelalisib exposed cells (doxorubicin 10uM 55% vs 77%, p<0.001; vincristine 0.05uM, 48% vs 61%, P<0.001). At higher idelalisib pre-treatment concentrations (50uM) additional synergistic activity was observed in Raji 2R cells (cisplatin 48% vs 61%, p<0.001; dexamethasone 67% vs 87%, p<0.01). To further assess the effect of dual inhibition of PI3K and mTOR, cell lines were exposed to the dual inhibitor BEZ-235. BEZ-235 exhibited a more potent decrease in cell viability compared to idelalisib with activity at nM concentrations. Unlike idelalisib, exposure to BEZ-235 resulted in significant induction of apoptosis by Annexin V-propidium iodide staining. BEZ-235 also exhibited synergistic activity in combination with chemotherapy in all cell lines. At equivalent dosing, BEZ-235 exposure resulted in a more significant decrease in Akt phosphorylation compared to idelalisib as determined by flow cytometry for p-Akt at Ser and Thr phosphorylation sites.Conclusions: Chemotherapy sensitive and resistant BL cell line models are susceptible to inhibition of the PI3K/Akt/mTOR pathway. Targeted inhibition of this pathway leads to a decrease in AKT activation, decrease in cell viability, cell cycle arrest and an increase in sensitivity to cytotoxic chemotherapeutic agents. Broader inhibition of both PI3K and mTOR is more effective than more targeted inhibition of PI3K-delta alone. DisclosuresNo relevant conflicts of interest to declare.