ALBERT

Description: Cancer cells have altered energy demand due to their increased proliferative capacity. In Waldenstrom Macroglobulinemia (WM), an indolent yet incurable B-cell lymphoma, the homeostasis of the bone marrow (BM) environment is disturbed due to the infiltration of the lymphoplamacytic cells that continuously produce monoclonal IgM. An alteration in energy demand could skew the balance of key proteins and metabolites towards a permissive niche for WM tumor cells growth, and unfavorably effect on the immune response against tumor cells. Therefore, the aim of our study was to identify how changes in certain metabolites and/or proteins could contribute to the pathobiology of WM and whether the cytokine and chemokine composition of the BM microenvironment play a role in such changes. WM patient's samples including BM plasma, peripheral blood serum and BM cells (n=101) as well as equivalent normal counterparts (n=86) were collected and used for metabolomics analysis. Comprehensive targeted metabolomics analysis was performed using Capillary Electrophoresis Time-of-Flight Mass spectrometry (CE-TOFMS), CE-triple quadrupole mass spectrometry (CE-qQqMS) and Liquid Chromatography (LC-TOFMS). Normal and WM peripheral blood serums samples were also used for untargeted proteomics analysis using a fully automated proteomic technology platform that includes an Agilent 1200 Series Quaternary HPLC system connected to a Q Exactive Plus mass spectrometer. Real-time PCR analysis was performed to detect the gene expression of the relevant metabolite transporters located on the cell membrane. BM cells from control and WM patients' samples were used for flow cytometry analysis. IHC was used to detect the proteins on the BM tissues. Principal Component Analysis (PCA) and Hierarchal Clustering Analysis (HCA) on both metabolomics and proteomics data identified two distinct clusters for disease and normal samples, indicating that there are differentially expressed proteins and metabolites in WM versus normal samples. Furthermore, pathway analysis showed that the majority of the altered metabolites were the members of the glutathione (GSH) metabolism pathway. This finding was further validated not only by data obtained from metabolomics analysis of BM cells and BM plasma, but also by proteomic data WM patients serum, implying that GSH metabolism is key to the biology of WM. Moreover, stimulation of WM cell lines by IL-6 and IL-21, cytokines involved in inducing WM cell proliferation and IgM secretion, resulted in increased gene expression of the transporters mediating uptake of metabolites required for GSH synthesis, including SLC7A11, 4F2HC and LAT1, indicating that cytokines in the WM BM could modulate GSH metabolism. In addition, IHC staining of the BM tissues as well as flow cytometry analysis of patients' lymphoplasmacytic cells identified glutathione peroxidase as one of the major proteins modulating GSH metabolism in WM. In summary, our data highlight a central role for GSH metabolism in WM disease biology and indicate that intervening with the metabolic processes could be a potential therapeutic strategy for patients with WM. Disclosures Ansell: AI Therapeutics: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Bristol Myers Squibb: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials.

Description: Background. Recent clinical trials based on immunotherapies targeting the PD-1/PD-L1 pathway have shown striking durable responses in a subset of patients with solid cancers. The Programmed death 1 (PD-1) protein is a key immune checkpoint inhibitor expressed by activated T cells. Its ligand, PD-L1, was reported highly expressed by tumor cells in some diffuse large B-cell lymphomas (DLBCL) of non-Germinal Center phenotype. We recently reported on the French multicenter GOELAMS075 trial that pre-treatment soluble PD-L1 (sPD-L1) in plasma was elevated in DLBCL patients compared to controls, and that elevated sPD-L1 was associated with inferior overall survival (OS), independent of the International Prognostic Index (IPI) and other clinical factors (Rossille et al., Leukemia 2014). Here, we replicate and extend these findings in two independent studies from Australia and the US. Methods. The protein expression of sPD-L1 was evaluated using a commercial ELISA kit. The French discovery cohort consisted of 288 adults with newly diagnosed aggressive DLBCL, age 18 to 60 years, and treated with R-CHOP or high dose chemotherapy plus rituximab followed by autologous stem cell support (clinicaltrials.gov: NCT00561379); there were also 60 controls. The Australian study consisted of 51 DLBCL patients age 18 to 71 years, all stages, treated with R-CHOP14, along with 57 controls. The US study was an observational cohort from the Iowa/Mayo Lymphoma SPORE and consisted of 225 DLBCLs, age 19 to 92 years, all stages, treated with immunochemotherapy, along with 98 controls. Plasma samples were collected pre-treatment using EDTA tubes for the Australian and the US cohorts, BDª P100 tubes for the French cohort. sPD-L1 expression was measured in Rennes, France (French & US samples) and in Brisbane, Australia (Australian samples).The Kaplan-Meier method and Cox regression were used to model the association of sPD-L1 with OS. The 95th percentile of the sPD-L1 levels in each matched control group was used as the cutoff point to define elevated sPD-L1 levels. Results. Replicating the French findings, sPD-L1 levels were significantly higher for DLBCL patients compared to controls in both the US (P

Description: Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p 〈 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line 'Karpas299'. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response. Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. Disclosures Parikh: Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Description: Key Points Alternatively polarized macrophages are abundant constituents of the tumor microenvironment in T-cell lymphoproliferative disorders. GATA-3 expression identifies a subset of PTCL, NOS with a distinct cytokine profile and inferior survival.

Description: Introduction: As previously reported, the combination of brentuximab vedotin with doxorubicin, vinblastine and dacarbazine (A+AVD) demonstrated a statistically significant improvement in modified progression free survival (modified PFS) compared with doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) in patients with newly diagnosed Stage III or IV classical HL in the phase 3 ECHELON-1 trial (NCT01712490). The benefit of A+AVD in the ITT population observed in the primary analysis was maintained at 3-years median follow-up [3-year PFS: A+AVD: 83.1% (79.9-85.9), ABVD: 76% (72.4-79.2)] and appears independent of interim PET status, disease stage, and prognostic risk factors. Here we present the efficacy and safety results of longer follow-up at a median 43.3 months. Methods: Newly diagnosed patients with Stage III or IV cHL were randomized 1:1 to receive A+AVD (n=664) or ABVD (n=670) intravenously on days 1 and 15 of each 28-day cycle for up to 6 cycles. The primary endpoint of the study was modified PFS per independent central review. The present follow-up PFS analysis is exploratory and per investigator assessment, with a cutoff date of June 17th, 2019. Patients with ongoing peripheral neuropathy (PN) at end of treatment were followed for resolution or improvement (defined as improved by ≥1 grade from worst grade as of the latest assessment) during the post-treatment follow-up period. Results: With a median follow-up of 43.3 months, the 42-month PFS per investigator for all patients was 82.4% (95% CI, 79.1-85.2) on the A+AVD arm and 76.2% (95% CI, 72.6-79.4) on the ABVD arm [overall HR 0.697 (95% CI, 0.547-0.890)]. Exploratory subgroup analyses of PET2(+) and PET2(-) patients showed a treatment effect in favor of A+AVD. The 42-month PFS in PET2(-) patients was 85.0% (95% CI, 81.6-87.7) for A+AVD and 79.6% (95% CI, 75.9-82.8) for ABVD [overall HR 0.695 (95% CI, 0.526-0.919)]; in PET2(+) patients 42-month PFS was 68.3% (95% CI, 54.5-78.7) for A+AVD and 51.5% (95% CI, 38.2-63.4) for ABVD [overall HR 0.552 (95% CI, 0.308-0.989)]. Upon continued follow-up, 81% (356/442) of patients with PN in the A+AVD arm had either complete resolution (64%, 283/442) or improvement (17%, 73/442) of their PN events compared with 83% (236/286) with either complete resolution (74%, 212/286) or improvement (8%, 24/286) in the ABVD arm. Among patients with ongoing PN after continued follow-up, the majority were Grade 1/2 events, with 89% (141/159; 59% Grade 1) and 95% (70/74; 65% Grade 1) on the A+AVD and ABVD arms, respectively. Overall survival data are not yet mature; per protocol, the final analysis will be performed after 112 deaths have occurred. Additional follow-up at an estimated median of ~4 years, including data from prespecified subgroups, will be presented. Conclusions: With a median follow-up of 43.3 months, A+AVD continues to provide a robust, durable benefit for patients with previously untreated Stage III or IV cHL compared with ABVD; the benefit is evident regardless of patient status at interim PET [PET2(+) or PET2(-)] and without the need for treatment intensification. PN continued to completely resolve or improve in patients on the A+AVD and ABVD arms. Together, these data further support the clinical advantages of A+AVD versus ABVD as treatment for patients with previously untreated Stage III or IV cHL. Disclosures Bartlett: Affimed Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Dynavax: Research Funding; Forty-Seven: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Immune Design: Research Funding; Janssen: Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Medimmune: Research Funding; Merck: Research Funding; Millennium: Research Funding; Novartis: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Seattle Genetics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Straus:Elsevier (PracticeUpdate): Consultancy, Honoraria; Hope Funds for Cancer Research: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Honoraria. Dlugosz-Danecka:Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Macrogenomics: Research Funding; Roche: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Illes:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Feldman:Takeda: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Cell Medica: Research Funding; Amgen: Research Funding; Viracta: Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Honoraria, Other: Travel expenses, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Pharmacyclics: Honoraria, Other: Travel expenses, Speakers Bureau; Pfizer: Research Funding; Portola Pharma: Research Funding; Roche: Research Funding; Eisai: Research Funding; Corvus: Research Funding; Roche: Research Funding; AbbVie: Honoraria, Other: Travel expenses, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Other: Travel expenses, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Trillium: Research Funding. Smolewski:Roche: Other: Travel Expenses. Savage:Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Walewski:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding; Takeda: Honoraria, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria; Gilead: Other: Travel Expenses; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Zinzani:Portola: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy; Eusapharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics: Honoraria, Speakers Bureau. Hutchings:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding; Genmab: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding; Novartis: Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding. Radford:AstraZeneca: Equity Ownership, Research Funding; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; GSK: Equity Ownership. Munoz:AstraZeneca: Speakers Bureau; Kite Pharma: Consultancy, Research Funding, Speakers Bureau; Pharmacyclics LLC an AbbVie Company: Consultancy, Research Funding, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Fosunkite: Speakers Bureau; Kyowa: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Portola: Research Funding; Incyte: Research Funding. Kim:Roche: Research Funding; Kyowa-Kirin: Research Funding; Novartis: Research Funding; J&J: Research Funding; Mundipharma: Research Funding; Celltrion: Research Funding; Donga: Research Funding. Advani:Cell Medica, Ltd: Consultancy; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Kura: Research Funding; Infinity Pharma: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celmed: Consultancy, Membership on an entity's Board of Directors or advisory committees; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agensys: Research Funding; Stanford University: Employment, Equity Ownership; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Research Funding; Kyowa Kirin Pharmaceutical Developments, Inc.: Consultancy; Regeneron: Research Funding; Millennium: Research Funding; Janssen: Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc./Kite Pharma, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forty-Seven: Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ansell:Mayo Clinic Rochester: Employment; Affimed: Research Funding; Mayo Clinic Rochester: Employment; Trillium: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding; LAM Therapeutics: Research Funding; Regeneron: Research Funding; Affimed: Research Funding; Trillium: Research Funding; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding; Mayo Clinic Rochester: Employment; LAM Therapeutics: Research Funding; LAM Therapeutics: Research Funding; Seattle Genetics: Research Funding; Mayo Clinic Rochester: Employment; Mayo Clinic Rochester: Employment; Trillium: Research Funding; LAM Therapeutics: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; LAM Therapeutics: Research Funding; Regeneron: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Affimed: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Trillium: Research Funding; Mayo Clinic Rochester: Employment. Younes:Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Curis: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Abbvie: Honoraria; Takeda: Honoraria; Pharmacyclics: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Biopath: Consultancy; Xynomics: Consultancy; Epizyme: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; HCM: Consultancy; BMS: Research Funding; Syndax: Research Funding. Gallamini:Takeda: Consultancy; Roche: Consultancy. Miao:Millennium Pharmaceuticals, Inc., Cambridge, MA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment, Equity Ownership. Liu:Takeda: Employment. Fenton:Seattle Genetics, Inc.: Employment, Equity Ownership. Forero-Torres:Seattle Genetics: Employment, Equity Ownership, Honoraria, Research Funding.

Description: Background: While B-cell receptor (BCR) signaling is essential for the development, of normal B cells, its aberrant hyper-activation results in neoplastic transformation of B-lymphocytes. Recent investigations using small molecule inhibitors validate the BCR pathway as a valuable target. Bruton’s tyrosine kinase (BTK) is one of the components of a signaling hub that transduces signals from the BCR into the cell for its activation and has been shown to be a therapeutic target. Ibrutinib (PCI-32765), an irreversible BTK inhibitor has shown clinical efficacy in CLL, mantle cell lymphoma (MCL) and Waldenströms macroglobulinemia (WM). Ibrutinib binds to cysteine-481 of the BTK protein and blocks its phosphorylation, resulting in termination of BCR-mediated activation of cells with a concomitant induction of death. Despite the clinical success of ibrutinib, a high percentage of patients achieve only partial response and eventually acquire resistance to the drug, resulting in aggressive relapse of the disease. A mutation of Cys481-Ser in BTK (ibrutinib-BTK binding site) has been reported to be one of the reasons for the development of ibrutinib resistance (IR). To understand the mechanisms resulting in acquisition of IR, we developed preclinical models of IR in WM and MCL. Materials: Ibrutinib was obtained from Pharmacyclics, CA. Validated human WM models (BCWM.1, RPCI-WM1 and MWCL.1 cell lines) and human MCL models (Jeko-1 and Maver cell lines) were used for the study. Results: BTK was constitutively phosphorylated at Y223 and Y551 in all the cell lines tested and this was inhibited by ibrutinib in a dose dependent manner. Phosphorylation of other kinases in the cascade such as SYK (Y323 and Y525/526) and PLCg2 (Y759 and Y1217) were also inhibited while AKT phosphorylation at both Ser473 and Thr308 was consistently increased in presence of ibrutinib. Treatment with ibrutinib induced cell cycle arrest in the G1 phase by 24h followed by apoptosis. Cell growth assays (MTS assay) showed that BCWM.1 was the most sensitive cell line followed by MWCL-1, RPCI-WM1, Maver and Jeko-1. Exposure of WM and MCL cells for prolonged periods of time with progressively increasing concentrations of ibrutinib resulted in outgrowth of clones (IR WM and MCL cell lines) that were resistant to apoptosis with a slow growth rate as compared to wild type parental cells. IR cells attained 2 – 20 fold resistance to ibrutinib as compared to the respective parental lines as determined by MTS assay. Sequence analysis of the BTK gene in all the cell lines revealed no mutation in IR cells at Cys481 suggesting that in an acquired IR state, resistance to ibrutinib can be developed independent of BTK Cys481 mutation. Interestingly, we found p-BTK levels to be markedly reduced in IR cells. Ibrutinib reversal experiments suggested that while a continuous presence of ibrutinib is needed for inhibition of BTK phosphorylation, a stable IR state could be maintained (for 〉1 month) in the absence of ibrutinib. This suggested the cells reliance on a parallel survival pathway, independent of BTK phosphorylation. Focused mRNA (Nanostring nCounter assay) and immunoblot analysis revealed significant changes in the expression profiles of several cellular elements. These included transcription factors such as PU.1, IRF4, BLIMP1, BCL-6 b-catenin as well as the phosphorylated ERK1/2, STAT1 and 3 suggesting a reprogramming of critical cellular networks, which IR tumor cells might be utilizing to overcome ibrutinib-induced cytotoxicity. Importantly, we observed that IR cells retained high levels of p-AKT and showed an increase in expression of BCL2 family members, as well as BCL-2 itself. Treatment of IR cells with ibrutinib +/- MK2206 (AKT inhibitor), or ABT-199 (BCL-2 inhibitor), synergistically induced cytotoxicity in IR cells, suggesting the importance of these parallel survival pathways (AKT/BCL2) in maintaining an IR state. Conclusion: Here we demonstrate that in the absence of BTK Cys481 mutation, an IR state is associated with reprogramming of transcriptional networks countering ibrutinib-induced toxicity by activation of AKT and BCL-2. Our current data exposes multiple vulnerabilities within IR cells, which can be therapeutically exploited to potentially delay onset of IR, by targeting alternative oncogenic mechanisms that are activated in presence of sustained BTK inhibition. Disclosures No relevant conflicts of interest to declare.