ALBERT

Description: Tumor immunotherapy based on the use of Chimeric Receptor Modified T cells (CAR T cells) is a promising approach for the treatment of a refractory hematological cancer. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable. In order to enhance the effectiveness of CAR-based immunotherapy we tested the immunoadjuvant properities of lenalidomide in combination with CAR19 T cells in a mouse model of B cell lymphoma. CAR19 construct which was used is composed of anti-CD19scFv joined with signaling domain of 4-1BB and TCR zeta and was delivered into T cell via lentiviral transduction. Lenalidomide is an immunomodulatory drug used for the treatment of multiple myeloma and selected B-cell malignancies, e.g. mantle cell lymphoma (MCL) or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). However, the precise mechanism of action is not very well understood and it is believed that is mediated by a modulation of activity of E3 ubiquitin ligase cereblon which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors resulting in changes of expression of various receptors on the surface of tumor cells. To test our hypothesis, immunodeficient NSG mice (NOD-SCID-gamma chain null mice) were s.c. transplanted with various human B cells lymphoma cells (MCL or ABC-DLBCL) followed by i.v treatment with CAR19 T cells with or without daily i.p. lenalidomide. First, when we measured the growth of tumors following treatment with CAR19 T cells plus lenalidomide we found that this combination more effectively suppressed growth of s.c. B-NHL tumors than treatment with only CAR19 T cells or only lenalidomide (Figure 1, 1x10e7 Nemo tumor cell s.c., followed with 2 doses of 1x10(7) CAR19 T cells + Lenalidomide daily, tumor weight was measured 14 days after treatment). Additionally, in this experiment lenalidomide significantly enhanced infiltration of residual tumors by CD8+CAR19 T cells (not shown). Next, we tested the response of CAR19 T cells in vitro to B-NHL cells in the presence or, absence of lenalidomide to determine the costimulatory effect of lenalidomide on signaling via CAR, our data show that lenalidomide significantly enhanced functional response of CAR19 T cells following recognition of B cells in vitro which is demonstrated by enhanced production of IFN-gamma and by increased expression of CD69 by CAR19 T cells, interestingly, this effect was seen only if CAR19 T cells but not B-NHL cells were pre-treated with lenalidomide or, when we activated CAR19 T cell with antibody to CAR but not with antibody to CD3. Thus, our data indicate that lenalidomide might work through direct effects on T cells and specifically enhance signaling via CAR. The biochemical events underlying this costimulatory effect of lenalidomide on signaling by CAR are currently being investigated. In summary, our data support the use of lenalidomide for augmentation CAR-based immunotherapy in clinical settings. Figure 1 Figure 1. Disclosures Klener: Cellgene: Research Funding.

Description: Mature B-cell lymphomas represent a heteregenous group of malignancies, which are considered to arise along different steps of B-cell development, particularly from germinal or post-germinal center B-cells. Recent studies, however, indicate that hematopoietic stem and progenitor cells (HSPC) could be involved in the pathogenesis of these diseases. We analyzed HSPC populations from 131 patients with mature B-cell malignancies, including chronic lymphocytic leukemia (CLL, n=20), mantle cell lymphoma (MCL, n=26), diffuse large B-cell lymphoma (DLBCL, n=35), follicular lymphoma (FL, n=25), and multiple myeloma (MM, n=25). For comparison, HSPC populations obtained from 22 healthy donors were used. Hematopoietic stem cells (HSC, Lin-CD34+CD38-CD90+CD45RA-), multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-), multi-lymphoid progenitors (MLP, Lin-CD34+CD38-CD90-CD45RA+), and pro-B cells (CD34+CD38+CD10+CD19+) were analyzed by flow cytometry. Proportions of HSPC and pro-B cells were related to CD34+CD38- and CD34+CD38+ cells, respectively. HSC and pro-B cell populations were sorted directly into tubes containing lysis solution for subsequent gene expression analyses. Preamplified cDNA was used for quantitative PCR of selected gene targets (n=28 in HSC, and n=27 in pro-B). The expression of targets was normalized to GAPDH expression. Corresponding cell populations isolated from healthy donors were used as controls. The aforementioned flow cytometry analyses revealed significantly decreased MLP population in all 5 tested diagnoses: CLL (p

Description: Introduction: Role of angiogenesis in growth, spread and survival of malignant lymphomas remains largely unexplored. Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), has been approved for treatment of several types of solid tumors including colorectal carcinoma or breast carcinoma, in all cases in combination with chemotherapy. Mantle cell lymphoma (MCL) is a type of B-cell non-Hodgkin lymphoma characterized by frequent extranodal involvement. Two agents approved for the therapy of MCL including lenalidomide and temsirolimus exert their anti-lymphoma activity at least partially by inhibition of angiogenesis. Our aim was to investigate role of VEGF-dependent angiogenesis on growth and spread of MCL in vivo using different murine models of MCL including patient-derived xenografts (PDXs). We also analyzed a potential anti-lymphoma synergy of a multi-level inhibition of angiogenesis achieved by bevacizumab and temsirolimus. Methods: Experimental therapies were implemented using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Therapies were initiated three to five days after subcutaneous (SC) or intravenous (IV) injection of MCL cells (= day 1, D1). Therapies were terminated when SC tumor reached 2 cm, or when mice developed signs of terminal disease. Mice were xenografted either with MCL cell lines (JEKO-1, HBL2) or with PDX cells (VFN-M1, VFN-M2) derived in our laboratory from patients with relapsed MCL. Both PDXs were confirmed by NGS to keep majority of somatic mutations with the primary MCL cells. Growth curves of calculated tumor volumes and overall survivals were compared in case of SC and IV-xenografted animals, respectiverly, in the treated mice compared to untreated controls. VEGF levels were analyzed in different tissues (blood, tumor lysate, spleen lysate) by commercially available ELISA kit. TaqMan Angiogenesis Array was used to compare gene expression changes associated with bevacizumab failure. Results: Bevacizumab significantly inhibited growth of SC MCL tumors all tested models (Figure 1) (JEKO-1: 0.71g ±0.24g vs 3.22g ±0.65g, p=0.0061; HBL-2: 0.95g ±0.11g vs 4.2g ±0.28g, p

Description: Introduction Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2 and good sensitivity to a small molecule BCL2 inhibitor venetoclax. In the present study we analyzed molecular mechanisms of venetoclax resistance in MCL cells, and tested strategies to overcome it based on concurrent targeting of BCL2 a MCL1. Methods Cell death was determined by flow cytometry using Annexin-V/PI staining. Establishment of MCL cell clones with knock-down or transgenic overexpression of MCL1, BIM and NOXA, western blotting, immunohistochemistry of formalin-fixed paraffin-embedded tissue sections, and immunoprecipitation experiments were carried out as previously described (Klanova et al, Clin Cancer Res, 2016). All PDXs were derived in our laboratory from patients with relapsed MCL. All PDX were confirmed by NGS to keep majority of somatic mutations with the primary MCL cells from which they were derived. Samples were sequenced using SureSelectXT Human All Exon V6+UTR (Agilent Technologies, Santa Clara, CA) on the NextSeq 500 (Illumina, San Diego, CA) instrument according to manufacturer's protocols. Experimental therapies were implemented using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Therapy was initiated when all mice developed palpable subcutaneous tumors (= day 1, D1). Venetoclax (VTX) and S63845 were from MedchemExpress, carfilzomib (CFZ) was from Charles University General hospital pharmacy. Carfilzomib (4 mg / kg) was administered intravenously (IV) on days 1 and 6. Venetoclax (40 mg / kg) was given by oral gavage on days 1, 2, 3, 6 and 7. S63845 (25 mg / kg) was administered IV on days 1, 2, 3, 6 and 7. Tumor volumes were calculated using the following formula: π / 6 × tumor length × width × height. Results By transgenic overexpression or shRNA-mediated knock-down we confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. We demonstrated that both BIM and NOXA are differentially expressed between MCL cell lines on one side, and primary MCL cells and patient-derived xenograft (PDX) cells on the other side. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, biallelic deletions of BIM harbored by three commonly used MCL cell lines (JEKO-1, MINO and Z138), and previously reported to be present in approx. 30% of MCL patients, were not found in primary MCL cells. As a consequence, vast majority of the in vitro data was implemented on venetoclax-sensitive cell lines HBL2 and MAVER-1, whose patterns of expression of BCL2, MCL1, BIM and NOXA are similar to primary MCL cells. We demonstrated that MCL1, another key anti-apoptotic protein, plays an essential role in mediating resistance to venetoclax. First, MCL1 functions as a buffer for BIM released from BCL2 upon binding of venetoclax thereby preventing activation of BAX and induction of apoptosis. Second, marked upregulation of MCL1 protein was associated with acquired resistance to venetoclax in two most sensitive MCL cell lines HBL2 and MAVER-1. Based on the in vitro data we proposed two experimental treatment strategies that co-targeted MCL1 (along with inhibition of BCL2 with venetoclax): a direct blockage with a highly specific small molecule MCL1 inhibitor S63845, and an indirect blockage achieved by proteasome inhibitor carfilzomib that upregulates the proapoptotic protein NOXA that specifically binds and blocks MCL1. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo inducing the longest "remissions" of MCL bearing mice (i.e. temporary disappearance of subcutaneous MCL tumors) using a panel of four different PDXs derived from patients with relapsed / refractory MCL with complex karyotype changes (Figure 1). The combination of carfilzomib and venetoclax was far less effective, and at the same time more toxic suggesting functional blockage of MCL1 induced by overexpressed NOXA is either incomplete or insufficient. Conclusions Our data strongly support investigation of venetoclax in combination with S63845 as an innovative proapoptotic treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds. Figure 1 Figure 1. Disclosures Trněný: Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Gilead: Honoraria; Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Abbvie: Honoraria, Research Funding; F. Hoffman-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Sandoz: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board.

Description: Key Points PI3Kα/δ inhibition induces cytotoxicity in ABC DLBCLs through downregulation of NF-κB signaling. Inhibition of AKT induces cytotoxicity by downregulation of MYC in PTEN-deficient DLBCL models in vivo and in vitro.

Description: Key PointsPevonedistat (MLN4924), a NEDD8-activating enzyme inhibitor, is active in MCL preclinical models and potentiates rituximab activity. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL.

Description: Introduction: Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent type of B-cell non-Hodgkin lymphomas (B-NHL) in the Western hemisphere. While BCL2 gene deregulation was repeatedly associated with poor prognosis, the role of MCL1 in the biology of DLBCL remains largely unknown. ABT199 is a highly-selective inhibitor of BCL2 protein currently evaluated in clinical trials. Homoharringtonine (HHT) is a plant alkaloid and as a semisynthetic compound (omacetaxine) it was approved for the treatment of relapsed chronic myelogenous leukemia (CML). Anti-tumor activity of HHT includes downregulation of the anti-apoptotic protein MCL1. Aim: The aim of the project was to evaluate the preclinical anti-lymphoma efficacy of BCL2 and MCL1-targeting agents ABT199 and HHT in DLBCL. Methods: Immunophenotype of primary DLBCL samples was determined by immunohistochemistry (IHC) using the Hans algorithm. Sensitivity of DLBCL cell lines to ABT199 and HHT was determined by Annexin V-based apoptotic assay and WST8-based cell proliferation assay. DLBCL clones with downregulation of selected anti-apoptotic proteins were derived using pLKO1-based lentiviral particles containing shRNAs against BCL2, BCL-XL and MCL1. For upregulation, BCL2, BCL-XL and MCL1 were cloned in the lentiviral expression vector pCDHNeo and the prepared lentiviral particles were used for the transduction of DLBCL cell lines. Results: We analyzed molecular mechanisms of cytotoxic activity of HHT in 7 DLBCL cell lines, and confirmed decreased expression of MCL1 protein in all cases. By semi-quantitative protein expression analysis (western blot or IHC) we demonstrated that BCL-XL and MCL1 were detectable in all DLBCL cell lines (n=18) and primary samples (n=114, GCB=51, ABC=63), while BCL2 was not detectable in 6 out of 18 DLBCL cell lines and 32 out of 114 primary DLBCL samples. 8 out of 12 BCL2-positive DLBCL cell lines were sensitive to 1 microM ABT199 (i.e. did not survive 1 microM ABT199 by standard proliferation assay). In contrary, 6 out of 6 BCL2-negative DLBCL cell lines were resistant to 1 microM ABT199. 11 out of 12 BCL2-positive DLBCL cell lines were sensitive to 30 nM HHT (considered a steady-state plasma level in CML patients treated with HHT). 5 out of 6 BCL2-negative DLBCL cell lines were sensitive to 30 nM HHT. Significant drug synergism between ≤1 microM ABT199 and ≤ 30 nM HHT was observed in 8 out of 12 BCL2-positive, but only in 1 out of 6 BCL2-negative DLBCL cell lines. We demonstrated that high expression of BCL2 positively correlated with sensitivity to ABT-199, irrespective of expression levels of BCL-XL and MCL1. Expression levels of BCL2 and BCL-XL negatively correlated with sensitivity to HHT. Expression level of MCL1 did not correlate with sensitivity to HHT. Both targeted downregulation and transgenic overexpression of BCL-XL in selected DLBCL cell lines confirmed that the expression of BCL-XL negatively correlates with sensitivity to HHT (but not to ABT199). While increase in sensitivity to HHT was observed in 3 out of 3 DLBCL cell lines with targeted knock-down of BCL2, increase in sensitivity to ABT199 was observed only in 1 out of these 3 DLBCL cell lines. Targeted knockdown of MCL1 was associated with increased sensitivity to HHT in 1 out of 2 DLBCL cell lines, but with no change of sensitivity to ABT199. Conclusions: HHT is a promising anti-DLBCL agent in both BCL2-positive and BCL2-negative cases. ABT199, as a single-agent or in combination with HHT, effectively eliminates BCL2-positive DLBCL cells. Based on the observed data two biological categories of DLBCL might be assumed: BCL2-dependent (ABT199-sensitive, HHT-sensitive) and MCL1-dependent (ABT199-resistant, HHT-sensitive) DLBCL. Grant support: IGA-MZ: NT13201-4/2012, GACR14-19590S, UNCE 204021, SVV-2013-266509, PRVOUK P24/LF1/3, GA-UK 1270214 Disclosures No relevant conflicts of interest to declare.

Description: Despite the development of novel targeted drugs, the molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) still poses a major therapeutic challenge. DLBCL can be classified into at least two major subtypes, i.e. germinal center B-cell-like (GCB) and the aggressive activated B-cell-like (ABC) DLBCL, each characterized by specific gene expression profiles and mutation patterns. Here we demonstrate a broad anti-tumor effect of dimethyl fumarate (DMF) on both DLBCL subtypes, which is mediated by the induction of ferroptosis, a form of cell death driven by the peroxidation of phospholipids. Due to high expression of arachidonate 5-lipoxygenase in concert with low glutathione and glutathione peroxidase 4 levels, DMF induces lipid peroxidation and thus ferroptosis particularly in GCB DLBCL. In ABC DLBCL cells, which are addicted to NF-κB and STAT3 survival signaling, DMF treatment efficiently inhibits the activity of the IKK complex and JAK kinases. Interestingly, the BCL-2 specific BH3 mimetic ABT-199 and an inhibitor of ferroptosis suppressor protein 1 synergize with DMF in inducing cell death in DLBCL. Collectively, our findings identify the clinically approved drug DMF as a promising novel therapeutic option in the treatment of both GCB and ABC DLBCL.