Natural killer (NK) cells are innate lymphocytes that play essential roles in mediating antitumor immunity. NK cells respond to various inflammatory stimuli including cytokines and stress-induced cellular ligands which activate germline-encoded activation receptors (NKRs), such as NKG2D. The signaling molecules activated downstream of NKRs are well defined; however, the mechanisms that regulate these pathways are not fully understood. IQ domain-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein. It regulates diverse cellular signaling programs in various physiological contexts, including immune cell activation and function. Therefore, we sought to investigate the role of IQGAP1 in NK cells. Development and maturation of NK cells from mice lacking IQGAP1 (Iqgap1−/−) were mostly intact; however, the absolute number of splenic NK cells was significantly reduced. Phenotypic and functional characterization revealed a significant reduction in the egression of NK cells from the bone marrow of Iqagp1−/− mice altering their peripheral homeostasis. Lack of IQGAP1 resulted in reduced NK cell motility and their ability to mediate antitumor immunity in vivo. Activation of Iqgap1−/− NK cells via NKRs, including NKG2D, resulted in significantly reduced levels of inflammatory cytokines compared with wild-type (WT). This reduction in Iqgap1−/− NK cells is neither due to an impaired membrane proximal signaling nor a defect in gene transcription. The levels of Ifng transcripts were comparable between WT and Iqgap1−/−, suggesting that IQGAP1-dependent regulation of cytokine production is regulated by a post-transcriptional mechanism. To this end, Iqgap1−/− NK cells failed to fully induce S6 phosphorylation and showed significantly reduced protein translation following NKG2D-mediated activation, revealing a previously undefined regulatory function of IQGAP1 via the mechanistic target of rapamycin complex 1. Together, these results implicate IQGAP1 as an essential scaffold for NK cell homeostasis and function and provide novel mechanistic insights to the post-transcriptional regulation of inflammatory cytokine production.

Background: Patients who relapse with CD20+ B-NHL and B cell Acute lymphoblastic leukemia (B-LL) have a dismal prognosis, which is often associated with chemotherapy resistance and may require alternative therapeutic strategies (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013). Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe and well-tolerated and is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Goldman/Cairo et al. Leukemia, 2013). Resistance to RTX, however, may predispose patients with CD20+ B-LL to an increase risk of relapse and/or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab, a novel type II glycoengineered CD20 antibody, mediates enhanced cell death and ADCC vs. RTX (Bologna L et al. JI, 2012), and was recently approved by FDA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate anti-tumor activity of obinutuzumab vs RTX against RTX resistant and sensitive BL and pre-B-ALL in xenografted NSG mice. Methods: Raji (CD20+) and Loucy (T-ALL, CD20-), (ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. The lentiviral construct, pSico PolII-eGFP-Luc2, was transfected into Raji, Raji 4RH (RTX resistant), U698M and Loucy. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), mice were bred in house under pathogen free conditions, NSG mice were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab 10 mg/kg (generously supplied by Christian Klein, PhD (Roche)), obinutuzumab (30 mg/kg), and rituximab (30 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji, Raji4RH, U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were then injected every 7 days with the respective therapy for 8 weeks. Mice were closely monitored for tumor burden and survival for up to 12 weeks ( approx. 80 days) via bioluminescent imaging (BLI) using the IVIS Spectrum system. Results: We demonstrated that obinutuzumab was significantly more effective than RTX when administered at the same doses in BL (RTX resistant/sensitive) and pre-B-ALL xenografts. Overall survival in mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in BL; Raji (p=0.0002), Raji4RH (p=0.01) and U698-M (p=0.001), respectively. Conclusion: These preliminary studies demonstrate that RTX sensitive/resistant BL and pre-B-ALL xenografted mice display significantly increased survival when given 30 mg/kg of obinutuzumab and decreased tumor burden in BL and Pre-B-ALL xenografts compared to equal dose of RTX. Citation Format: Aradhana A. Tiwari, Janet Ayello, Carmella van de Ven, Matthew J. Barth, Mitchell S. Cairo. Obinutuzumab (GA101) significantly increases overall survival against CD20+ rituximab-sensitive and -resistant Burkitt (BL) and acute lymphoblastic leukemia (B-ALL): potential targeted therapy in patients with high risk BL and pre-B-ALL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2014-2902

Multi-agent chemotherapy regimens have significantly improved long-term survival of pediatric Burkitt Lymphoma (BL) to over 80%. However, patients with refractory or relapsed disease have a dismal prognosis, stressing the need to identify mechanisms of therapeutic resistance and develop novel therapeutic approaches. Recent data demonstrated tonic B-cell receptor (BCR) activation of PI3K in BL (Srinivisan et al, Cell 2009); and implicated PI3K, in coordination with MYC, in Burkitt lymphomagenesis (Sander et al, Cancer Cell 2012). In order to investigate mechanisms of resistance, our laboratory generated several resistant BL cell lines. We exposed Raji cells to escalating doses of rituximab +/- human serum and generated several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or therapy (rituximab-chemotherapy) resistant (Raji 2R and Raji 4RH) (TRCL) cell lines. We then screened for aberrant activation of signal transduction pathways using western blot, phospho-flow cytometry and phosphoproteomics to define pathways associated with the development of resistance. While total Akt expression was similar between all cell lines tested, Raji 2R and Raji 4RH cells had increased phosphorylation of Akt at Ser473 and Thr308, indicating Akt activation, when compared to Raji, Raji 7R and Raji 8RH by western blot and phospho-flow cytometry. Phosphoproteomic analysis comparing Raji and Raji 4RH identified an increase of at least 2 fold in phosphorylation of 315 proteins in Raji 4RH cells, including multiple direct targets of AKT (e.g. GSK3B, WEE1, FOXO1 and PRAS40). Altered phosphorylation of proteins downstream of Akt (e.g. BAD, 4EBP1, GSK3B and ERK) was also detected by western blot in Raji 2R and Raji 4RH compared to Raji, Raji 7R and Raji 8RH. These findings suggest that activation of PI3K/Akt may play a role in the development of chemoresistance noted in our cell line model. KEGG analysis of phosphoproteome and gene expression comparison of Raji to Raji 4RH cells identified the BCR pathway as one of the top pathways altered. Additionally, the microRNA (miR) 17-92 has been noted to increase Akt activation through its inhibitory effects on PTEN, a key regulator of Akt activation. A recent copy number analysis indicated that a recurrent gain in 13q, containing the MIR17HG locus, was also associated with higher mir17 RNA levels and early relapse in pediatric BL patients (Schiffman et al, Br J Haematol 2011). An analysis of miR expression in Raji 2R and Raji 4RH compared to Raji noted a 1.93 and 3.77 fold increase in miR17 respectively. These findings suggest possible mechanisms for increased PI3K/Akt activation in our chemoresistant cell lines. In vitro exposure to MK-2206, a pan-Akt inhibitor, resulted in dose- and time-dependent decreases in viability of TRCLs (IC50 at 72 hours: Raji 2R = 2.6µM; Raji 4RH = 3.2µM) and to a slightly lesser degree Raji and RRCL (Raji= 4.4µM; Raji 7R = 4.0µM; Raji 8RH = 5.2µM) (TRCL vs. Raji/RRCL, p<0.05), by alamarBlu assay. Cell cycle analysis following exposure to MK2206 identified G1 cell cycle arrest in Raji/RRCL, but G2/M cell cycle arrest was observed in TRCL, suggesting differing mechanisms of anti-proliferative effect between sensitive and resistant cells. The combination of MK2206 and either doxorubicin or dexamethasone resulted in synergistic decreases in cell viability at a variety of dose combinations in both sensitive and resistant cells, as well as in BL Ramos and Daudi cells, using the Chou-Talalay method to measure synergy. In Raji and Ramos, the combination of MK2206 and doxorubicin also resulted in a higher degree of PARP cleavage compared to either agent alone, though no PARP cleavage was noted in Raji 2R and Raji 4RH cells likely related to impaired apoptotic potential previously noted in these cell lines. An siRNA knockdown of Akt in Raji cells resulted in a decrease in viable cells compared to controls following exposure to doxorubicin. Similar effects on cell proliferation, cell cycle and synergy were seen with upstream inhibition of PI3K by the PI3Kδ inhibitor idelalisib. Our data suggests that constitutive activation of the PI3K/Akt pathway, through activation of the BCR pathway or over-expression of miR17, is associated with the development of resistance in BL cell lines, and inhibition of PI3K/Akt can sensitize BL cells to the effects of some chemotherapeutic agents. Targeting the PI3K/Akt signaling pathway may be a clinically relevant approach in pediatric BL. Disclosures Cairo: Celgene: Research Funding.

Introduction Mature B-cell lymphoma (MBL) accounts for approximately 60% of all non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007). MBL in children and adolescents is comprised of approximately 75-80% Burkitt lymphoma (BL), 15-20% diffuse large B cell lymphoma (DLBCL) and 1-2% primary mediastinal B-cell lymphoma (PMBL). However, despite the differences in histology they are treated uniformly in children and adolescents (Cairo, BJH, 2016). The use of short and intense multiagent chemotherapy alone and in combination with rituximab (R) results in ≥90% event-free survival (EFS) in children and adolescents with BL and DLBCL (Cairo et al, JCO, 2012; Goldman/Cairo Leukemia, 2013; BJH, 2014). However, similar chemotherapy approaches in adolescents and young adults with PMBL have only resulted in a 60-70% EFS (Gerrard/Cairo et al, Blood, 2013). Furthermore, patients with induction failure, recurrent or refractory MBL have a dismal prognosis with ≤30% overall survival (OS) with current re-induction platforms and autologous stem cell transplantation (Cairo et al, BJH, 2018). Newer therapeutic strategies are urgently needed for recurrent/refractory patients with MBL, especially those with the BL subtype and newly diagnosed and refractory patients with PMBL (Cairo, BJH, 2019). We previously demonstrated that Obinutuzumab (O), a new type II anti-CD20 monoclonal antibody, is an active agent against both BL and PMBL (Awasthi/Cairo et al, BJH, 2015). Additionally, we have previously demonstrated high expression of CD79b in children and adolescents with BL and PMBL (Miles/Cairo et al, BJH 2007). Polatuzumab vedotin (PV), an anti-CD79b antibody-drug conjugate, recently approved by the FDA in adults with recurrent/refractory DLBCL has been demonstrated to be an active agent alone and in combination with chemotherapy in all molecular forms of adult DLBCL (Pfeifer et al, Leukemia, 2015; Corrinna et al, Lancet Oncology, 2015; Morschauser et al, Lancet Onc, 2019; Tilly et al, Lancet Onc, 2019). However, the efficacy of PV alone or in combination with R or O against other subtypes of B-NHL, including BL and PMBL, is relatively unknown. Objective To determine the in-vitro cytotoxicity and changes in in-vivo survival in a BL NSG xenograft model of PV alone or in combination with O vs R against R-sensitive and R-resistant BL and PMBL. Methods We compared the cytotoxicity of PV±R (10 ug/ml) vs PV±O (10 ug/ml) against R-sensitive BL (Raji) and R-resistant BL (Raji 4RH) and PBML (Karpas-1106p) alone or with ex-vivo expanded NK cells (Chu/Cairo et al, Can Imm Res, 2015) (10:1 E:T) by the DELFIA cytotoxicity assay. PV and O were generously supplied by Genentech and Roche, respectively. Furthermore, NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) were xenografted with luciferase positive BL (Raji, Raji 4RH) and PMBL cells (Karpas-1106p) and treated with PV (5 mg/kg) + O (20 mg/kg) vs PV + R (20 mg/kg) vs PV vs IgG isotype control vs PV + O + NK vs PV + R + NK vs PV + NK vs O + NK vs R + NK vs PBS (without tumor) and tumor burden was compared between treatment groups using the IVIS bioluminescence system and probability of OS by KM method as we have previously described (Chu/Cairo et al, Can Imm Res, 2015). Results PV + O + NK induced significantly more in-vitro cytotoxicity against BL Raji compared to PV + R + NK (p<0.007) vs PV, R or O + NK (p<0.0001), BL (Raji 4RH [R-resistant BL]) PV + O vs PV + R (p<0.004) and O, R or PV (p<0.002) and against PMBL (Karpas-1106p) PV + O + NK vs PV + R + NK (p<0.006) vs O, R, or PV + NK (p<0.001) (Fig. 1A, 1B, 1C, respectively). Most importantly, PV + O + NK vs PV + R + NK vs PV + NK + IgG isotype control significantly enhanced probability of survival in BL (Raji) and BL (Raji 4RH) NSG xenografts (p<0.03 and (p<0.05), (95.5 vs 50.4 days) (185 vs 47 days), respectively (Fig. 2A and 2B, respectively). Conclusions PV alone and in combination with O significantly induces cytotoxicity against BL, R-resistant BL and PMBL. Most importantly, PV + O + NK cells result in a significantly improved OS in BL and R-resistant BL NSG xenografts. These preclinical results suggest that PV should be considered for investigation in patients with both recurrent/refractory BL and PMBL and in patients with newly diagnosed disease with either high risk features or those with poor responses to induction therapy. Authors AA, MB & AA are all considered co-primary first authors Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Cairo:Jazz Pharmaceuticals: Other: Advisory Board, Research Funding, Speakers Bureau; Osuka: Research Funding; Miltenyi: Other: MTA.

Background: Childhood, Adolescent and Young Adult (CAYA) B-NHL represents the third most common malignancies in children under the age of 15yrs (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012).The prognosis of mature-B-NHL (which include BL/PMBL) has significantly improved over the last 40 years through the use of short and intense multi-agent chemo-immunotherapy, however; a subset of patients with relapsed/refractory disease has chemoimmunotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al.Blood. 2007; Cairo et al. JCO.2012, Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). It is therefore critical to investigate and develop targeted translational strategies in BL/PMBL to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. Blinatumomab, targeting CD19, has demonstrated encouraging clinical activity against pre-B-ALL (Topp et al, Leukemia, 2018) and highly expressed in BL/PMBL. Objectives: To determine in-vitro activity of blinatumomab against rituximab sensitive/resistant BL and PMBL cell lines. Methods: BL; Raji, Raji-4RH, and PMBL: Karpas1106p/MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with/without blinatumomab (generously supplied by Amgen) for 4 hr. with T-cells. CD3+ isolated and expanded T-cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA®cytotoxicity assay at 10:1 E: T ratio and cytokines secretion was measured by multiples ELISA kit. CD3+/CD107a+, granzyme b and perforin T-cell expression level were measured by flow-cytometry. Results: Blinatumomab+T-cells compared to T-cells only elicited a significant increased cytotoxicity against, Raji 58.18±7.6% vs 27.3.81±11.2% (p=0.007), Raji-4RH 67.4±8.0% vs 27.6±2.5%, (p=0.004), Karpas1106p, 75.7±3.06 % vs. 17.86± 4.82% (p=0.003) and MedB1, 65.17±13.3% vs18.1±2.03%, respectively (p=0.05). Blinatumomab treated T-cells also increased IFN-γ secretion compared to IL2-T cells, Raji 1 (p=0.002) and Raji-4RH (p=0.02) respectively. Furthermore, CD107a, granzyme b and perforin expression were significantly enhanced with blinatumomab treated/activated T-cells against; Raji (p=0.03, p=0.003 & p=0.009), Raji-4RH (p=0.03, p=0.02 & p=0.03) and Karpas1106p (p=0.006, p=0.03 & p=0.02) respectively. Conclusion: Blinatumomab significantly enhances T-mediated in-vitro cytotoxicity and cytokine secretion against BL/PMBL. Further, blinatumomab treated and activated T-cells significantly enhanced CD107a, granzyme b and perforin expression. These preliminary studies demonstrate that BiTE (CD3/CD19) would be a novel agent to investigate as immunotherapy therapy in patients with relapse refectory BL/PMBL. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Janet Ayello, Mitchell S. Cairo. Blinatumomab enhanced anti-tumor activity against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2381.