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The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase–associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition–induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. Significance: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19− K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA–specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467–80. ©2018 AACR.

Natural Killer (NK) cells play an essential role in cancer surveillance and have a unique capability of spontaneous cytotoxicity against cancer cells. The human NK cell repertoire is functionally diversified through a tightly regulated differentiation process characterized by an early transition from CD56bright to CD56dim NK cells, followed by coordinated changes in expression of inhibitory receptors, including NKG2A and killer cell immunoglobulin-like receptors (KIR). The acquisition of self HLA class I binding KIRs during NK cell differentiation tunes the cytotoxic potential of NK cells in a process termed education, characterized by increased loading of granzyme B in dense core granules. Although NK cell differentiation and education are critical determinants of the functional potential of the cell, little is known about how these events shape the migratory behavior of NK cells. To mediate appropriate and directed immune response against cancer, NK cells must be capable of migration to the tumor site. This process is mediated by chemokines, which guide cell migration by binding to their specific receptors. For example, in multiple myeloma, CXCR3 and CCR5 ligands (MIG, IP-10, and MIP-1a) are significantly upregulated in the bone marrow compared to healthy controls, affecting the composition of immune cells in the tumor microenvironment. In order to delineate the homing patterns of distinct NK cell subsets, we used high-dimensional flow cytometry combined with functional assays to map the NK cell chemokine receptor expression and migratory behavior. We screened resting and cytokine/feeder cell stimulated peripheral blood NK cells for the expression of a panel of 20 chemokine receptors (A). Based on CD56, CD57, NKG2A, and KIR expression, NK cells were divided into 6 phenotypically and functionally distinct subsets that were ordered according to their differentiation status (B). We found that the expression of CX3CR1, CXCR1, CXCR2, and CMKLR1 gradually increased during differentiation, whereas the expression of CXCR3, CCR7, and CCR5 was lower in more differentiated NK cells. CXCR4, CCR4, and CCR2 expression was relatively uniform across all subsets. Interestingly, CCR1 and CXCR6 were expressed mainly on less differentiated NKG2A+ CD56dim NK cells (B). Next, we stratified the chemokine receptor expression on mature KIR+ NK cells based on the expression of self (educated) or non-self KIR (uneducated). Educated NK cells expressed CXCR1, CX3CR1, CCR5, and CMKLR1 at higher levels than the uneducated NK cells. Conversely, CXCR3 was expressed at lower levels on educated NK cells (C). No difference was observed for CXCR2 expression. To determine whether the observed differences in chemokine receptor expression translate into altered chemokine responsiveness between the subsets, we combined the transwell system with multicolor flow cytometry. We found that the chemokine-induced migration capability of NK cells correlated closely with the expression level of corresponding chemokine receptor, leading to subset specific responses to various chemokine gradients (D). The present results show that peripheral blood NK cell chemokine receptor profile changes in a coordinated fashion during NK cell differentiation and is further influenced by the expression of self-specific KIR. Interestingly, receptors which expression declines during NK cell differentiation (CCR5, CCR7, and CXCR3) are commonly associated with adaptive T cell responses to viruses, whereas receptors that are upregulated along the differentiation axis (CXCR1, CXCR2, CX3CR1, CMKLR1) are typical for neutrophils and macrophages as a part of the innate immune response. Thus, our results suggest that NK cell differentiation and education processes together shape the NK cell migratory capabilities to promote homing of the most functional NK cell subsets to the site of inflammation and serve as the first line of defense in the immune response to pathogens and tumors. Figure Disclosures Malmberg: Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

CD37 is a tetraspanin expressed prominently on the surface of B cells. It is an attractive molecular target exploited in the immunotherapy of B cell-derived lymphomas and leukemia. Currently, several monoclonal antibodies targeting CD37 as well as chimeric antigen receptor-based immunotherapies are being developed and investigated in clinical trials. Given the unique role of CD37 in the biology of B cells, it seems that CD37 constitutes more than a docking point for monoclonal antibodies, and targeting this molecule may provide additional benefit to relapsed or refractory patients. In this review, we aimed to provide an extensive overview of the function of CD37 in B cell malignancies, providing a comprehensive view of recent therapeutic advances targeting CD37 and delineating future perspectives.

The BCL-2 inhibitor venetoclax has only limited activity in DLBCL despite frequent BCL-2 overexpression. Since constitutive activation of the B cell receptor (BCR) pathway has been reported in both ABC and GCB DLBCL, we investigated whether targeting SYK or BTK will increase sensitivity of DLBCL cells to venetoclax. We report that pharmacological inhibition of SYK or BTK synergistically enhances venetoclax sensitivity in BCL-2-positive DLBCL cell lines with an activated BCR pathway in vitro and in a xenograft model in vivo, despite the only modest direct cytotoxic effect. We further show that these sensitizing effects are associated with inhibition of the downstream PI3K/AKT pathway and changes in the expression of MCL-1, BIM, and HRK. In addition, we show that BCR-dependent GCB DLBCL cells are characterized by deficiency of the phosphatase SHP1, a key negative regulator of the BCR pathway. Re-expression of SHP1 in GCB DBLCL cells reduces SYK, BLNK, and GSK3 phosphorylation and induces corresponding changes in MCL1, BIM, and HRK expression. Together, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination.

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

The primary hurdle in non-Hodgkin lymphoma (NHL) treatment is the resistance to the recommended R-CHOP regimen which is composed of anti-CD20-targeted immunotherapy, Rituximab or RTX, and multiple chemotherapeutic drugs: cyclophosphamide, doxorubicin, vincristine and prednisone. In many patients, the resistance is correlated to the reduced level of CD20 antigen on the surface of tumor B cells. While testing drugs that overcome this resistance, we sought of testing whether Salinomycin, a drug that preferentially act on cancer cells over healthy cells, and selectively target cancer stem cells, is able to improve the sensitivity of NHL to Rituximab. Herein, we investigated its effect on NK cell- and complement-dependent cytotoxicity on NHL cells and its mechanism of action. Methods: Antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and degranulation assays were used to assess the sensitivity of NHL cell lines and primary cancer cells upon treatment with salinomycin plus rituximab. Serum and PBMCs of human healthy donors were used as source of complement and Natural Killer cells, respectively. Flow cytometry was used to quantify cell death rate and cell surface protein levels. RNA-seq and qPCR were used to identify and confirm the differentially expressed genes upon salinomycin treatment. GSEA, an online resource of the Broad Institute MA, was used to screen the differentially expressed genes and major signaling pathways. Results and Conclusions: In addition to previously reported ability of Salinomycin to kill cancer stem cells, our data show that the sub-lethal doses of Salinomycin greatly increased surface CD20 (protein target for Rituximab therapy) on both lymphoma cell lines and primary CLL samples. Lymphoma cell death induced by rituximab and mediated by complement and NK cell cytotoxicities was significantly increased upon treatment with salinomycin. Transcriptomic analysis of cells treated with salinomycin identified a sets of differentially expressed genes (including CD20-encoding gene) and markedly activated/affected pathways that are potentially involved in enhancing the sensitivity of Rituximab-treated NHL cells. The anticipated signaling pathways influencing NK cell activity will be further examined. These data indicate that salinomycin is an interesting therapeutic agent when combined with therapeutic anti-CD20 monoclonal antibodies. This particular combination would ensure higher NK cell-mediated cytotoxicity and offset the NHL resistance to R-CHOP regimen. Aknowledgements: National Science Centre (NCN), ID: 2016/23/B/NZ5/02622; Ministry of Science and Higher Education, ID: DI2014007344. Citation Format: Abdessamad Zerrouqi, Nina Miazek-Zapala, Anna Torun, Piotr Zapala, Jakub Golab, Magdalena Winiarska, Beata Pyrzynska. Salinomycin expands the cytotoxicity of both anti-CD20 monoclonal antibodies and natural killer cells towards non-Hodgkin lymphomas [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 LB-261.

Introduction Dasatinib is a potent small molecule kinase inhibitor targeting BCR-ABL kinase - oncogenic driver in Philadelphia chromosome-positive (Ph+) cases of chronic myelogenous leukemia (CML). In addition to BCR-ABL kinase, it also targets a broad array of other kinases, affecting not only leukemia cells but also immune cells. Just one hour after dasatinib oral administration a rapid increase of NK, NKT, T and B cells is observed in peripheral blood. Dasatinib has been also shown to influence NK cell cytotoxicity, however, the results are discordant. Some groups observe potentiation of NK cell cytotoxic activity while others strong inhibitory effects. These inconsistencies may be explained by differences in in vitro protocols used to study this phenomenon. Aim Our study aims to investigate dasatinib influence on immune cells in whole blood assays resembling physiological conditions observed in patients. In particular, we want to investigate dasatinib effect on NK cell mobilization, degranulation, and anti-tumor immunity. In light of clinical and pre-clinical studies involving dasatinib and the importance of NK cells in cancer, it is crucial to establish the mechanisms and kinetics of dasatinib immunomodulatory activity. Methods Before and one hour after first dasatinib administration peripheral blood was collected from CML patients. Collected whole blood was directly added to the target K562 cell line. After co-incubation, erythrocytes were lysed, cells were stained with a panel of monoclonal antibodies and analyzed with flow cytometry. A relative increase in lymphocyte count was determined by Trucount Tubes (BD). Dasatinib effect on NK cells in vitro was studied with degranulation and cytotoxicity assays using NK cells isolated from healthy volunteers PBMCs. Dasatinib at clinically relevant concentrations (20-200mM) was used to assess its effect on NK cell degranulation, cytotoxicity, cytokine, and chemokine production with flow cytometry upon staining with anti-CD107a, TNF-α, IFN-γ and CCL-4 monoclonal antibodies. For in vivo experiments C57BL/6 mice were inoculated with EL4 tumor cell line stably expressing luciferase and human CD20. 3 days after tumor inoculation mice were treated with dasatinib or vehicle intraperitoneally (i.p.) in a dose of 30 mg/kg. To monitor tumor growth, mice were injected with luciferin and imaged using the IVIS system. Results In agreement with previous reports, we confirm NK, NKT, T and B cell count increase in peripheral blood after dasatinib administration. To evaluate how dasatinib influences NK cell cytokine and chemokine production we stimulated NK cells with K562 cell line. Production of major proinflammatory cytokines secreted by NK cells, TNF-α and IFN-γ, was inhibited by dasatinib treatment. Production of MIP-1β (CCL4), a chemokine secreted by NK cells attracting a broad spectrum of immune cells to inflammation sites, was also profoundly decreased. According to our findings, dasatinib presence during the cytotoxicity assay, in a dose-dependent manner, inhibits NK cell cytotoxicity. However, 24-hour dasatinib pretreatment increases their cytotoxic potential. To better mimic the physiological conditions we used whole blood degranulation assay which closely resembles patient settings, including dasatinib concentration. One hour after dasatinib intake we observed a potent inhibitory effect of dasatinib on NK cell degranulation. Additionally, we observed a shift in NK cell subpopulations - dasatinib present during degranulation assay decreases CD16⁻ NK cell number. Finally, we evaluated the influence of high-dose dasatinib treatment on tumor rejection in mice. Mice treated with dasatinib exhibit significantly increased tumor growth compared with vehicle-treated mice. Conclusions Using whole blood degranulation assay and in vitro degranulation and cytotoxicity assays we report that dasatinib effect on NK cell cytotoxicity is dose- and time-dependent. Our results indicate that dasatinib has a dual effect on NK cell degranulation and affects other NK cell functions including cytokine production and migration. Further studies are needed to evaluate the significance of these findings. Disclosures No relevant conflicts of interest to declare.

Aminolevulinic acid (ALA) is an endogenous metabolite normally formed in the mitochondria from succinyl-CoA and glycine. Conjugation of eight ALA molecules yields protoporphyrin IX (PpIX) and finally leads to formation of heme. Conversion of PpIX to its downstream substrates requires the activity of a rate-limiting enzyme ferrochelatase. When ALA is administered externally the abundantly produced PpIX cannot be quickly converted to its final product - heme by ferrochelatase and therefore accumulates within cells. Since PpIX is a potent photosensitizer this metabolic pathway can be exploited in photodynamic therapy (PDT). This is an already approved therapeutic strategy making ALA one of the most successful prodrugs used in cancer treatment.

Rituximab, a therapeutic anti-CD20 monoclonal antibody, is effective against B-cell malignancies, when combined with chemotherapy, such as cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP therapy). However, in case of 30-40% patients the resistance to rituximab-based therapy appears to be a serious concern. One of the major mechanisms of resistance relies on the reduced level of CD20 antigen on the surface of tumor B-cells. While exploring the molecular mechanisms affecting the level of CD20, we found that transcription factor FOXO1 is a negative regulator of MS4A1 expression, the gene encoding CD20 antigen. Using the CRISPR/Cas9 genome-editing technology, we disrupted the loci of FOXO genes in lymphoma cell lines and discovered that ablation of FOXO1 (but not FOXO3) was sufficient for upregulation of the surface level of CD20 by 3-fold. Consistently, the complement-dependent cytotoxicity (CDC), induced by rituximab, was significantly improved in cell clones with abrogated expression of FOXO1, but not FOXO3. Importantly, the treatment with pharmacological inhibitor of FOXO1 activity, AS1842856, resulted in increased levels of CD20 on the surface of both lymphoma cell lines and patients-derived chronic lymphocytic leukemia (CLL) cells cultured ex vivo. In order to verify our findings in the animal model, we inoculated SCID mice intravenously with Raji cells, what resulted in the development of lymphoma-like tumors. We demonstrated that mice treated systemically with rituximab, administered at a dose of 10 mg/kg, survived longer when inoculated with sgFOXO1-transduced Raji cells as compared with mice inoculated with control Raji cells (median survival 49 days versus 29 days, respectively). These results confirmed that FOXO1 ablation in lymphoma cells resulted in higher efficacy of rituximab treatment in vivo. Taken together, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab-induced cytotoxicity and indicate that FOXO1 inhibitors could be exploited for the therapeutic purposes, in combination with anti-CD20 monoclonal antibodies. Novel FOXO1 inhibitors with improved potency and selectivity are however urgently needed for further exploration of our discoveries in the near future. Acknowledgments: NCN, Poland (grant no: 2013/11/B/NZ5/03240) and H2020 (STREAM project, CSA action, grant no: 692180). Citation Format: Beata Pyrzynska, Abdessamad Zerrouqi, Michal Dwojak, Giulia Morlino, Piotr Zapala, Nina Miazek, Agnieszka Zagozdzon, Kamil Bojarczuk, Malgorzata Bobrowicz, Marta Siernicka, Marcin M. Machnicki, Joanna Barankiewicz, Ewa Lech-Maranda, Przemyslaw Juszczynski, Dinis Calado, Jakub Golab, Magdalena Winiarska. FOXO1 is transcriptional regulator of malignant B-cell surface antigen CD20, the target for therapeutic monoclonal antibodies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B17.

Introduction: Vascular endothelial growth factor receptor type 2 (VEGFR2) expression is one of the most prominent biomarkers of the tumor-associated neovasculature. Moreover, VEGFR2 can be aberrantly expressed on the surface of tumor cells. We have previously described the development of V21-DOS47, an immunoconjugate composed of the VHH-portion of a camelid single domain anti-VEGFR2 antibody (V21H4) and jack bean urease, which converts endogenous urea into ammonia and hydroxyl ions. V21-DOS47 is the second in a class of antibody-urease drugs. The first, L-DOS47, is currently in clinical testing for non-small cell lung cancer. In this study, we identified tumor cells with VEGFR2 expression, tested V21-DOS47 binding to tumor cells, and investigated in vivo activity in both immunocompetent and immunodeficient mice. Methods: Flow cytometry experiments were performed with biotinylated V21H4 antibody and anti-biotin fluorochrome-conjugated secondary antibody. Detection of urease by flow cytometry was performed with an anti-urease antibody followed by incubation with a fluorochrome-conjugated secondary antibody. Western blotting was used for the assessment of protein expression. For in vivo experiments, VEGFR2-overexpressing derivatives of human MDA-MB-231 and murine 4T1 breast cancer cell lines were generated. Balb/c or nude mice were inoculated with tumor cells: 2.5 x 105 4T1 or 1x106 MDA-MB-231, respectively, on Day 0 of each experiment. Intravenous injections of V21-DOS47 at a dose of 10 µg/kg were started on Day 3 and continued on Days: 5, 7, 9 and 11. Results: After screening human tumor cell lines for VEGFR2 expression by RT-PCR and Western blotting, we determined that two triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) expressed the highest levels of VEGFR2. Binding of V21H4 to the cell surface of both cell lines was confirmed by flow cytometry. In vivo studies were conducted using both immunodeficient (with human MDA-MB-231-hVEGFR2 cells) and immunocompetent (with murine 4T1-hVEGFR2 cells) mice. In the syngeneic/immunocompetent model of Balb/c mice implanted with 4T1-hVEGFR2 cells, but not wild-type 4T1 cells, the antitumor effect of V21-DOS47 was significant and long-lasting. However, the results obtained from the initial experiment in the MDA-MB-231-hVEGFR2 model suggest that the antitumor effect of V21-DOS47 is transient in immunodeficient mice. Conclusions: Our data show successful targeting of the DOS47 platform to human VEGFR2 expressed on tumor cells. Our in vivo data indicate that the antitumor activity of V21-DOS47 is enhanced in immunocompetent mice, which suggests that the immune system is a significant component of the antitumor activity of the V21-DOS47 immunoconjugate. Citation Format: Angelika Muchowicz, Anna Bujak, Beata Pyrzynska, Justyna Karolczak, Abdessamad Zerrouqi, Magdalena Ozga, Lukasz Szewczyk, Baomin Tian, Wah Wong, Marni Uger, Katarzyna Poplawska, Dorota Gierej, Pawel Wisniewski, Heman Chao, Magdalena Winiarska, Radoslaw Zagozdzon. Targeting urease to human VEGFR2 elicits antitumor activity in triple-negative breast cancer models [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 3919.

Lack of remission or early relapse remains a major clinical concern in diffuse large B-cell lymphoma (DLBCL), with a third of patients failing to respond to current regimens or relapsing with resistant disease. Genome and transcriptome sequencing studies have identified FOXO1 as one of the recurrent targets for somatic mutations in DLBCL associated with patient short survival. These mutations enhance FOXO1 nuclear localization and activity on target genes. Herein, we determine the role FOXO1 (wild-type and mutated) on the expression of the rituximab-target, CD20. The effect of active mutated FOXO1 on CD20 expression was determined upon inhibition of AKT, exogenous expression of active myristolated AKT, and mutated FOXO1 at its N-terminal region. FOXO1 binding activity to CD20 promoter was assessed using various assays combined with an in silico analysis of a publicly available Chip-Seq data. Our results show that the activation of FOXO1 using AKT inhibitors (MK-2206 and GDC-0068) led to a significant decrease of CD20 transcript and protein levels, which resulted in a significant resistance to rituximab-mediated complement-dependent cytotoxicity (CDC). Consistently, the overexpression of either wild-type or mutated FOXO1 repressed CD20 expression. Unlike the activity of exogenous wild-type FOXO1 that can be inhibited by AKT1, the activity of mutated FOXO1-AAA (Thr24, Ser256 and Ser319 mutated to Ala and that cannot be phosphorylated) markedly inhibits CD20 expression. Furthermore, CD20 levels remained high and unchanged by the DNA-binding defective mutant of FOXO1-H215R, suggesting a FOXO1-mediated regulation of CD20 transcription through FOXO1 binding to CD20 promoter, as confirmed by ChIP, EMSA, and the analysis of ChIP-Seq data using anti-FOXO1 antibody. These results show that FOXO1 mutants that are insensitive to AKT1 kinase activity effectively suppress CD20 expression in comparison to wild-type FOXO1, through a binding to CD20 promoter. In addition, the disruption of FOXO1 with CRISPR/Cas9 genome editing resulted in CD20 upregulation and improved the cytotoxicity induced by rituximab and survival of mice with sgFOXO1 tumors. Consistently, in primary samples pharmacologic inhibition of FOXO1 activity upregulated surface CD20 levels. In summary, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab-induced cytotoxicity. In addition, our findings indicate that the genetic status of FOXO1 together with its transcriptional activity needs further attention while designing rituximab-based regimens in the therapy of selected lymphomas. Supported by National Science Center, Poland (NCN#2013/11/B/NZ5/03240), STREAM project (Horizon 2020,#692180). Citation Format: Abdessamad Zerrouqi, Beata Pyrzynska, Michal Dwoyak, giulia Morlino, Piotr Zapala, Nina Miazek, Jakub Golab, Magdalena Winiarska. FOXO1 determines non-Hodgkin lymphomas response to anti-CD20-based therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B51.

Systemic chemotherapy generally has been considered immunosuppressive, but it has become evident that certain chemotherapeutic drugs elicit immunogenic danger signals in dying cancer cells that can incite protective antitumor immunity. In this study, we investigated whether locoregionally applied therapies, such as melphalan, used in limb perfusion for melanoma (Mel-ILP) produce related immunogenic effects. In human melanoma biopsies, Mel-ILP treatment upregulated IL1B, IL8, and IL6 associated with their release in patients' locoregional sera. Although induction of apoptosis in melanoma cells by melphalan in vitro did not elicit threshold levels of endoplasmic reticulum and reactive oxygen species stress associated with danger signals, such as induction of cell-surface calreticulin, prophylactic immunization and T-cell depletion experiments showed that melphalan administration in vivo could stimulate a CD8+ T cell–dependent protective antitumor response. Interestingly, the vaccination effect was potentiated in combination with exogenous calreticulin, but not tumor necrosis factor, a cytokine often combined with Mel-ILP. Our results illustrate how melphalan triggers inflammatory cell death that can be leveraged by immunomodulators such as the danger signal calreticulin. Cancer Res; 75(8); 1603–14. ©2015 AACR.

B-cell receptor pathway inhibitors affect CD20 levels and impair antitumor activity of anti-CD20 monoclonal antibodies

Anti-CD20 mononclonal antibodies (mAbs) have a well-established role in the treatment of B-cell lymphoid malignancies. In addition to classic Fc-dependent mechanisms, including antibody-dependent and complement-mediated cytotoxicity, the so-called type II mAbs induce direct cell death. It has been shown that obinutuzumab, without Fc-crosslinking agents or effector cells, triggers non-apoptotic, lysosomal-dependent programmed cell death (PCD). The mechanism of PCD is characterized by actin reorganization, followed by permeabilization of the lysosomal membrane and subsequent generation of reactive oxygen species (ROS) through NADPH oxidase. Although, mechanisms of PCD are well-described, little is known about factors influencing sensitivity of malignant B-cells to obinutuzumab-mediated direct cell killing. Strategies to improve PCD could be potentially exploited to eliminate malignant cells, which are refractory to conventional immunotherapy. In this study, we aimed to investigate the influence of lysosomotropic agent, chloroquine, on the efficacy of obinutuzumab-mediated cytotoxicity. As PCD is dependent on lysosomal destabilization, we hypothesized that combination of obinutuzumab with lysosome-destabilizing agent would result in increased cell death. In our study, we used a Burkitt lymphoma Raji cell line that is widely employed as a model to assess the efficacy of obinutuzumab. Raji cells were incubated with obinutuzumab, alone or in combination with increasing concentrations of chloroquine, followed by annexin V/PI staining. Chloroquine, significantly increased direct cell death induced by obinutuzumab, without being toxic alone. Those observations were further corroborated by cell staining with other viability dies - TO-PRO-3 iodide and 7-AAD.The efficacy of the tested combination was completely abrogated by cytochalasin D - an inhibitor of actin polymerization and concanamycin A - inhibitor of vacuolar ATPases that activate acidic vacuoles including lysosomes, suggesting that chloroquine and obinutuzumab share a common mechanism of action. Since chloroquine has been reported to promote ROS generation, we analyzed the production of ROS with DCFDA staining. We observed that chloroquine potentiated the oxidative effect of obinutuzumab. Consistently, the effect of the combination was completely abrogated by a cell-permeable ROS scavenger - Tiron. As obinutuzumab has been shown to induce ROS production via activation of NADPH oxidase, we investigated the influence of NADPH oxidase inhibitor - diphenylene iodonium (DPI) on the combination's efficacy. DPI only partially reversed the effect of obinutuzumab, while strongly decreasing the effect of the combination. Altogether, we show for the first time that chloroquine sensitizes cell to lysosomal cell death induced by obinutuzumab. The results of our study provide a strong rationale for combining obinutuzumab with lysosomotropic agents. These findings may have particular importance given the fact that another lysomotropic agent - siramesine has recently been shown to induce selective cytotoxicity in chronic lymphocytic leukemia (CLL). Disclosures No relevant conflicts of interest to declare.

Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.

Histone deacetylases inhibitors (HDACi) have recently emerged as potent antitumor treatment modality. They are currently tested in many phase I, II and III clinical trials as single agents as wells as in combination schemes. They have demonstrated promising antitumor activity and favorable clinical outcome. Histone deacetylases (HDACs) are involved in the process of epigenetic regulation of gene expression. Epigenetic changes are believed to be crucial for the onset and progression of cancer and have recently gained remarkable attention. Since epigenetic regulation of gene expression is a reversible process, targeting histone deacetylases provides a good rationale for anticancer therapy. The acetylation status of histones regulates the organization of chromatin and the access of transcription factors. Moreover, functions of many non-histone proteins are controlled by acetylation. The broad and complicated influences of HDACi on various molecular processes may account for the observed pleiotropic effects. In this review we summarize recent advances in the understanding of biology of HDACs and mechanism of action of their inhibitors.

The therapeutic strategies currently used in B cell malignancies include the treatment with monoclonal antibodies (rituximab and ofatumumab) directed against CD20 antigen. These antibodies specifically eliminate B cells by triggering indirect effector mechanisms of the immune system, like complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. In many patients, the reduced level of CD20 antigen on the surface of tumor B cells leads to the resistance to anti-CD20 therapy. The aim of this study was to explore the molecular mechanisms governing the regulation of CD20 expression in lymphoma cells as a potential explanation of the resistance to rituximab/ofatumumab therapy. We previously observed that CD20 mRNA expression is significantly affected by the SRC family inhibitors. Here, we report that also PTEN tumor suppressor is a negative regulator of CD20 expression. To uncover the transcriptional mechanisms governing the CD20 expression we employed the construct encoding the promoter region of CD20 cloned upstream of the firefly luciferase gene. Overexpression of wild-type PTEN (but not the phosphatase-deficient mutant) strongly affected the promoter activity and the expression of CD20, leading to decreased binding of rituximab and ofatumumab and increased resistance of tumor cells to complement-dependent cytotoxicity. Using the truncated versions of the CD20 promoter we identified a particular region (-313/-198) as the major region sensitive to PTEN overexpression. We found that the negative regulation of CD20 promoter activity by PTEN was mediated by inhibition of AKT signaling. We observed that the overexpression of constitutively active AKT1 (CA-AKT1) overcame the negative effect of PTEN and sensitized cells to rituximab/ofatumumab treatment. The results of our studies indicate that PTEN status in tumor cells should therefore be considered when analyzing the mechanisms of resistance of B cell malignancies to anti-CD20 therapies. Citation Format: Beata Pyrzynska, Kamil Bojarczuk, Marta Siernicka, Michal Dwojak, Malgorzata Bobrowicz, Nina Miazek, Piotr Zapala, Agnieszka Zagozdzon, Jakub Golab, Magdalena Winiarska. PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-242. doi:10.1158/1538-7445.AM2015-LB-242

HDAC (histone deacetylase) inhibitors belong to a novel class of antitumor agents widely explored in hematology. So far two of them - romidepsin and vorinostat have been registered in cutaneous T-cell lymphoma, while panobinostat awaits FDA decision concerning its registration in multiple myeloma. Several other agents are currently being tested in clinical trials in lymphoma and myeloma patients and preliminary results suggest that they may be most effective when used in combinations with other anti-cancer drugs. Reports from others (Sugimoto et al. Biochem Biophys Res Commun 2009 Shimizu et al. Leukemia 2010) and the results from our group show that HDAC inhibitors augment the efficacy of anti-CD20 monoclonal antibodies (mAbs) in complement-dependent cytotoxicity (CDC) by up-regulating CD20 level. In our previous reports we have shown that HDAC6 isoform is involved in the regulation of CD20 degradation and trafficking. Since the efficacy of mAbs depends not only on the level of target molecule but is also regulated by the expression of complement regulatory proteins (mCRPs) - CD46, CD55 and CD59, we sought to investigate the influence of HDAC inhibitors on the expression of those molecules. Moreover, given the fact that HDAC inhibitors have been reported to regulate NK cells activity, we investigated their influence on antibody-dependent cell-mediated cytotoxicity (ADCC) which is one of the mechanisms of action of mAbs in vivo. The purpose of this study was to thoroughly investigate the influence of HDAC inhibition on the level of surface molecules – CD20 and CD38 – targets already used and tested in clinical trials in immunotherapy of B-cell malignancies as well as the expression of mCRPs. Using non-selective pan-inhibitors as well as selective inhibitors of particular HDAC classes and isoforms we sought to show the inhibition of which HDAC enzyme is the most effective in potentiating the outcome of mAbs based anti-tumor therapies. Since the results of clinical trials show that the use of HDAC inhibitors has several non-specific adverse effects, we hypothesize that selective inhibition of particular isoforms may considerably diminish these toxicities. Using flow cytometry we performed experiments assessing the level of surface CD20 CD38 and mCRPs – CD46, CD55 and CD59. We used established lymphoma, chronic lymphocytic leukemia (CLL) and myeloma cell lines as well as primary cells isolated from the patients suffering from CLL. We observed that the use of HDAC pan-inhibitors – trichostatin A (TsA) and vorinostat (Fig.1A) and selective HDAC6 inhibitors – tubacin and ACY1215 (ricolinostat) (Fig.1B) considerably improves the outcome of anti-CD20 mAbs – rituximab and ofatumumab in CDC in several B-cell lymphoma cell lines and in CLL primary samples (Fig. 1C). In our experiments we show that selective inhibition of HDAC6 in Raji cells does not influence the expression of mCRPs (Fig. 1D). However, we observed that pre-incubation with tubacin considerably increases the expression of CD38 (Fig. 1 D). Therefore, in our further studies we aim at expanding our observations to primary samples from multiple myeloma patients, especially since novel anti-CD38 mAbs are currently intensively explored in the therapy of this malignancy. Moreover, using flow cytometry we determined the influence of HDAC inhibition on degranulation of NK cells isolated from healthy donors in ADCC in vitro assay with rituximab and ofatumumab. Contrary to the reports of others, we observed no negative regulation of NK cells activity and their ability to kill target tumor cells. Therefore, since HDAC inhibitors seem to influence mainly CDC, we investigated the influence of specific HDAC inhibitors on the level of mCRPs. Those results suggest that specific inhibition of HDAC3 (Fig. 1E) and HDAC1/2 (Fig. 1F) are effective therapeutic strategies as they considerably decreased the expression of complement inhibitors and concomitantly increased the level of CD20 antigen. However, further studies including silencing of particular HDAC isoforms with shRNA are needed to confirm these findings. Altogether, our results strongly suggest that HDAC inhibitors potentiate the outcome of mAbs used in hematological malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Small molecule Bruton's tyrosine kinase (Btk) inhibitors are extensively studied in preclinical investigations and clinical trials in the treatment of hematological malignancies derived from B-cells. Ibrutinib, due to its safety and efficiency, has been recently approved for the treatment of patients with Chronic Lymphocytic Leukemia (CLL) who received at least one prior therapy. Moreover, another Btk inhibitor AVL-292 is currently tested in clinical trials in patients with B Cell Non-Hodgkin's Lymphomas, CLL and Waldenstrom Macroglobulinemia. Despite a huge therapeutic potential of Btk inhibitors we have recently demonstrated that both natural killer (NK) cells cytotoxicity and degranulation are significantly impaired upon ibrutinib treatment (Bojarczuk et al., Leukemia 2014). Since NK cells are effectors of innate immune system capable to kill tumor cells directly and in the mechanism of antibody dependent cell-mediated cytotoxicity (ADCC), which constitutes one of the major mechanism of monoclonal antibodies widely used in hematooncology, in our ongoing studies we are focused to determine in details the influence of various Btk inhibitors on NK cells cytotoxicity, degranulation, cytokine secretion and expression of activatory/inhibitory receptors. All experiments were performed fully in vitro using human primary NK cells isolated from PBMC of healthy donors as well as NK92 and NK92.CD16 cell lines. To determine cytolytic activity of NK cells CFSE/PI flow cytometry assay was used. Cytokine secretion and NK cells degranulation, evaluated as the expression of CD107a on the surface of NK cells, were determined with flow cytometry upon incubation with target cells for 4 h. Phenotype of NK cells pre-incubated with tested compounds was determined with flow cytometry using antibodies conjugated with fluorochromes. The initial results of our studies show that various Btk inhibitors differentially regulate NK cells antitumor activity. Ibrutinib and AVL-292 which covalently bind to cysteines at the position 481 significantly inhibit NK cells cytotoxicity. Interestingly, we have observed that pre-incubation of NK cells with ibrutinib as well as AVL-292 results in down-regulation of CD25 and CD69. This effect was not observed upon treatment of NK cells with CGI-1746, a reversible Btk inhibitor which blocks phosphorylation of BTK in Y551 and Y223. Expression of NK cells activatory receptors such as NKp30, NK44, NKp46, NKG2D, DNAM-1 and CD16 remained unchanged. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

CD20, an integrate membrane protein expressed on the surface of normal and malignant B-cells is widely used as a molecular target for monoclonal antibodies (mAbs) in the therapy of non-Hodgkin’s lymphomas and chronic lymphocytic leukemia (CLL). Accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional and its up-regulation would result in increased efficacy of anti-CD20 mAbs. CD20 antigen has been reported to be regulated epigenetically e.g. by histone deacetylases (HDACs). The results of our preliminary experiments show that use of non-selective HDAC inhibitors as well as blocking the activity of a single HDAC isoform - HDAC6 leads to up-regulation of CD20 protein in B-cell lymphoma cell lines and increase of the efficacy of therapy with anti-CD20 mAbs. Since HDAC6 is engaged mainly in the acetylation of non-histone substrates and the observed up-regulation of CD20 molecule does not seem to rely on transcriptional mechanisms we postulate that HDAC6 is engaged in processes of CD20 trafficking or/and degradation. CD20 being a membrane bound protein is most probably undergoing endocytosis. However, this process and the role of HDAC6 in its regulation has not been explored so far. The aim of this study was to understand how the inhibition of HDAC6 activity influences CD20 level in normal and malignant B-cells. We wanted to determine the mechanisms underlying this phenomena. This study required use of B-cell lymphoma cell lines as well as lymphocytes infected with Epstein-Barr virus and normal B- lymphocytes. Several HDAC pan-inhibitors and HDAC6 inhibitors were tested. To assess the membrane level of CD20 antigen, FITC-anti-CD20 staining was performed followed by cytometric analysis. The influence of HDACi on total level of CD20 protein was assessed in Western blotting. The complement-dependent cytotoxicity (CDC) assay was performed using rituximab and ofatumumab as well as human serum as a source of complement. Cell cytotoxicity was assessed by propidium iodide staining followed by cytometric analysis. The influence of HDAC inhibition on the transcription of CD20 was examined by qRT-PCR using SyBR Green and hydrolysis probes. The activity of CD20 promoter after inhibition of HDAC was assessed in Dual Luciferase Assay. The colocalisation of CD20 with other proteins that may influence its trafficking/degradation was assessed using immunocytochemistry with specific antibodies and observed under confocal microscope. The results of our study strongly suggest that combining HDACi with anti-CD20 antibodies can be an effective therapeutic modality for patients suffering from B-cell malignancies. Extensive experiments aiming at determining what factors are engaged in the regulation of CD20 by HDAC6 are planned. Disclosures: No relevant conflicts of interest to declare.

Background Anti-CD20 monoclonal antibodies (mAbs) are widely used in the treatment of non-Hodgkin's lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Combining new agents with already used anti-CD20 mAbs seems to be a reasonable approach to further improve current therapeutic options. It seems that signaling via the aberrantly activated B-cell receptor (BCR) plays a key role in the pathogenesis of certain types of B-cell tumors. Blocking BCR pro-survival pathway holds a great therapeutic potential in both NHL and CLL. Several trials are currently being conducted to investigate the effects of combination of BCR-targeting agents with anti-CD20 mAbs–based therapies. To improve these therapeutic approaches in a planned manner it will be utterly important to decipher actual mechanisms of interactions between BCR-targeted therapies and anti-CD20 mAbs in established in vitro models. Aims The aim of this study is to elucidate the role of BCR signaling pathways in the regulation of CD20 levels in B-cell-derived tumor cells and antitumor activity of anti-CD20 mAbs. Methods The project is undertaken fully in in vitro settings in the models of human lymphoma cells as well as primary cells from patients with B-cell tumors. Cells are pre-incubated for 48h with inhibitors of BCR signaling (SYK, BTK, PI3K, AKT, PLC-γ, PKC, mTOR, ERK 1/2) and subsequently tested using flow cytometry for their susceptibility to antitumor activity of anti-CD20 mAbs. Membrane level of CD20 antigen is assessed with FITC-conjugated anti-CD20 antibody staining, total level of CD20 protein is assessed in Western blotting. Transcription processes are analyzed with qPCR, ChIP and EMSA. Moreover, stably transduced lymphoma cells with silenced or constitutively active proteins of interest are employed. Results The results of our preliminary experiments show that blocking BCR network at many stages of the signaling cascade with specific chemical inhibitors or selective shRNA-mediated silencing of SYK or BTK results in considerable down-regulation of CD20 level as determined with flow cytometry. Moreover, a 48-hour incubation with BCR inhibitors leads to a substantial impairment of antitumor activity of anti-CD20 mAbs. Selected inhibitors of BCR signaling considerably decrease CD20 protein level in total cellular lysates as analyzed using Western blotting. In Raji cells incubated with selected BCR inhibitors quantitative real-time PCR shows a significant decrease in CD20 mRNA level. Noteworthy, washout experiments showed that surface CD20 reaches level of control after 96 h from the time that inhibitors were eliminated from the culture media. Studies performed on cell line expressing constitutively active AKT showed up-regulation of CD20 levels at both levels of protein and mRNA. Moreover, constitutively active AKT protects cells from BCR inhibitors-induced decrease of surface CD20. Summary/conclusions Blocking BCR complex network on nearly every step of signal initiation and propagation considerably down-regulates CD20 levels what might have extremely important consequences for the anti-cancer therapy that is based on the use of anti-CD20 mAbs. These studies should provide us with extensive knowledge on the biology of CD20 protein and pathways involved in CD20 regulation. In light of our recent experiments therapeutic combinations of BCR inhibitors and anti-CD20 mAbs-based modalities should be rationally and consciously introduced into clinic in optimized therapeutic schemes. We hypothesize that results of our experiments may lead to identification of the most beneficial therapeutic modalities and schedules that would improve the quality of life of patients suffering from B-cells originating tumors. Disclosures: No relevant conflicts of interest to declare.

Abstract 2742 The treatment of chronic myeloid leukemia (CML) was revolutionized by the introduction of tyrosine kinase inhibitors (TKIs) to the clinical practice. However, there is still a significant group of patients who due to drug resistance do not fully benefit from the therapy. Among various reasons of drug resistance, the changes of intracellular concentration of TKIs caused by the activity of drug transporters may have profound effect on clinical activity of the drug. Intracellular concentration of imatinib is a result of: drug influx into the cell and drug efflux out of the cell. The latter is mediated by ATP-binding casette (ABC) transporters - MDR/ABCB1 and BCRP/ABCG2. According to the recent knowledge modulation of membrane cholesterol level may change the activity of ABC transporters by changing their conformation. Hence we decided to evaluate potential effect of statins, widely used hypocholesterolemic drugs, on antileukemic activity of imatinib in cell lines transformed with BCR/ABL oncoprotein and primary human CML CD34+ cells. In a set of experiments involving cytotoxic assays (i.e. clonogenic assay, XTT, trypan blue exclusion assay) we revealed that lovastatin, a member of statin family, is able to enhance imatinib cytotoxicity both in established cell lines (32Dcl3 BCR/ABL+, K562) as well as in primary CML CD34+ cells obtained from patients in various stages of the disease, including those clinically resistant to imatinib with no detectable ABL kinase domain mutations. In the same time, lovastatin alone and in combination with imatinib did not influence BCR/ABL negative cells (32Dcl3 wild type, HL-60, CD34+ cells obtained from healthy blood donors). As measured using specific, fluorescent-labeled substrates for ABC transporters (rhodamine-123 and Bodipy-FL-prazosin respectively), lovastatin significantly inhibits MDR/ABCB1- and BCRP/ABCG2-mediated efflux capacity. Similar effects were observed for other statins. The addition of cholesterol to the samples previously incubated with lovastatin completely reversed inhibitory activity of statins. To confirm these results we examined direct changes of intracellular concentration of imatinib itself using radiolabeled 14C-imatinib. In these settings, statins caused 2–3-fold increase in intracellular imatinib concentration in murine and human cell lines (32Dcl3 BCR/ABL+, K562) and also in primary CML CD34+ cells including clones resistant to imatinib (Figure 1). Statins did not influence initial concentration of imatinib what may suggest that their effects on imatinib concentration are not mediated by changes in OCT-1 activity. In addition, statins did not influence expression of MDR/ABCB1 and BCRP/ABCG2 drug transporters measured using qPCR nor ABC proteins levels measured using Wester blotting. Combination of imatinib and lovastatin induced cell cycle arrest and increased percentage of apoptotic leukemic cells measured using flow cytometric analysis after propidium iodide staining. Lovastatin also reduced imatinib-induced phosphorylation of the adaptor protein CrkL. In summary, we show that statins are able to increase therapeutic efficacy of imatinib through the inhibition of drug efflux mediated by MDR/ABCB1 and BCRP/ABCG2 transporters. Our results suggest that statin-mediated depletion of cholesterol in the cell may change conformational status of the efflux pumps, including MDR/ABCB1 and BCRP/ABCG2 pumps involved in the transport of imatinib, and in that way modulate intracellular concentration of the drug. The addition of statins may become attractive treatment modality for the selected group of patients in chronic phase and blast crisis.Fig. 1Statins increase intracellular concentration of imatinib in BCR/ABL-positive cells.Fig. 1. Statins increase intracellular concentration of imatinib in BCR/ABL-positive cells. CML-CP, CML-BP–CD34+ cells obtained from CML patient in chronic phase or blast crisis respectively; *p Disclosures: No relevant conflicts of interest to declare.

Abstract 3722 Anti-CD20 monoclonal antibodies (mAbs) (rituximab or ofatumumab) are being successfully used in the treatment of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). They exert antitumor effects by triggering indirect effector mechanisms of the immune system, such as activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, upon crosslinking with secondary antibodies, anti-CD20 mAbs can induce cell death. It is frequently underscored that CD20 expression levels in various B-cell tumors is relatively constant. However, accumulating evidence indicates that CD20 can be modulated at transcriptional, posttranscriptional, and even posttranslational levels. Moreover, it has been clearly shown in vitro that CDC efficacy of anti-CD20 mAbs clearly depends on CD20 expression. We have previously observed that statins impair detection of CD20 in non-Hodgkin lymphoma cells and impair rituximab-mediated CDC and ADCC (Winiarska et al. PLoS Med 2008). Statins are inhibitors of cholesterol synthesis and decrease production of prenyl groups (farnesyl and geranylgeranyl PPi), which are necessary for posttranslational modification of approximately 1% of cellular proteins. In experiments aimed at elucidation of the molecular mechanisms of statin-mediated modulation of CD20 we observed that neither geranylgeranyl transferase (GGT) nor farnesyl transferase (FNT) inhibitors could mimic statins effects. On the contrary, prenyltransferase inhibitors improved rituximab-mediated CDC. Therefore, we decided to investigate in more detail the interaction of prenyltransferase inhibitors and anti-CD20 mAbs. In the initial experiments we evaluated the effects of three different farnesyl transferase inhibitors as well as three different geranylgeranyl transferase inhibitors. Among all FNT and GGT inhibitors, L-744,832 seemed to produce the most significant influence on both rituximab-mediated CDC and CD20 levels (Figure). Moreover, L-744,832 significantly increased rituximab-mediated CDC in 3 out of 5 primary tumor cell cultures isolated from patients with NHL or CLL. Therefore, L-744,832 was selected for further more systematic studies. Interestingly, in Raji cells L-744,832 did not improve rituximab-mediated ADCC and only at the highest non-toxic concentrations it sensitized to rituximab+crosslinking antibody-mediated cytotoxicity. In 10 out of 17 (58.8%) primary lymphoma/leukemia cells L-744,832 increased CD20 expression by at least 20% as measured with flow cytometry. Moreover, we observed that upon L-744,832 treatment CD20 is up-regulated in Raji cells at both mRNA as well as protein level. Experiments aimed at investigation of FTI influence on proteasome activity as well as CD20 endocytosis and shedding revealed that L-744,832 influences CD20 levels independently from its posttranslational regulation. To verify whether modulation of CD20 levels by L-744,832 results from specific inhibition of farnesyltransferase or is an off-target effect of this compound we performed FNT B subunit (FNTB) knock-down experiments that resulted in increased CD20 levels by almost 60%. Incubation of Raji cells with a transcription inhibitor cycloheximide completely prevented L-744,832-mediated increase of CD20 levels in WB. Therefore, a chromatin immunoprecipitation assay was performed to determine whether inhibition of FNT activity is associated with binding of transcription factors to the promoter of cd20 gene. These studies revealed that L-744,832 promotes binding of PU.1 and Oct2, but not TFE3 to target DNA sequences within cd20 promoter in Raji cells. To conclude, our studies indicate for the first time that CD20 expression can be modulated by prenyltransferase inhibitors. While inhibition of FNT activity significantly up-regulates expression of CD20, the influence of GGT inhibitors on this protein is more complex, and requires further studies. Furthermore, pre-incubation of NHL and CLL cells with L-744,832, a FNT inhibitor, potentiates anti-CD20 mAb-mediated activation of the complement-mediated cytotoxicity. Therefore, the combination seems to be promising and its efficacy should be determined in patients with NHL or CLL. Disclosures: Winiarska: Genmab A/S: Research Funding. Golab:Genmab A/S: Research Funding.

Abstract 3723 Monoclonal antibodies (mAbs) targeting CD20 antigen are now routinely used in the treatment of various types of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). Their antitumor action results from the ability to trigger complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, direct cell death can be induced upon crosslinking with secondary antibodies. Previous studies have demonstrated a sigmoidal correlation between CD20 expression level and rituximab-mediated CDC but not ADCC. Next to CD20 expression level also other potential mechanisms of anti-CD20-mediated cytotoxicity have been observed. Among them an increased expression of complement regulatory proteins (CRP), such as CD46, CD55 or CD59 has been shown to contribute to resistance to mAb-mediated CDC. Antibodies blocking CRP or siRNA that knocks-down CRP expression facilitate rituximab-mediated cytotoxicity. Also in xenograft in vivo models it was shown that antibodies blocking the activity of CD55 and CD59 or a recombinant adenoviral fiber knob protein that cross-links CD46 molecules enhance therapeutic effects of rituximab. Fludarabine, the nucleoside analogue clinically active against CLL and indolent NHL has been shown to act synergistically with rituximab and to down-regulate the membrane expression of CD55 without significantly altering CD20 levels. The aim of this study was to investigate the influence of sorafenib, a multikinase inhibitor on the expression of CRPs in human CD20+ B-cell lymphomas and to evaluate its ability to trigger CDC in combination with rituximab. A 48-h incubation of four different human non-Hodgkin lymphoma cells DoHH2 (Fig. 1A), Daudi, Raji and Ramos with sorafenib resulted in a marked concentration-dependent drop in the surface expression of CD46, CD55 and CD59, but did not affect the levels of CD20. Analysis of primary cells isolated from 5 consecutive patients with CD20+ CLL also revealed a significant drop in the levels of CD46 (5 of 5 patients), CD55 (4 of 5 patients), and CD55 (5 of 5 patients) after a 48 h incubation of tumor cells with sorafenib. Accordingly, with previous reports, sorafenib significantly decreased phosphorylation of STAT3 transcription factors that are involved in the regulation of CRP expression. Incubation of tumor cells with sorafenib lowered the levels of mRNA for all three CRPs, but not that of CD20 indicating that sorafenib-mediated downregulation of CRPs occurs at transcriptional level. Pre-incubation of all four lymphoma cell lines cells with sorafenib at concentrations that down-modulate CRPs levels also significantly sensitized tumor cells to rituximab-mediated CDC (Fig. 1B). Moreover, sorafenib led to increased incorporation of membrane-attack complex (C5b-9) into the membranes of DoHH2 cells. On the other hand, sorafenib did not affect rituximab-mediated ADCC nor apoptosis in DoHH2 cells. Sunitinib, another multi-kinase inhibitor, did not affect rituximab-mediated complement-dependent cytolysis.Figure 1.(A) DoHH2 cells were incubated with either diluent or sorafenib at 1.25, 2.5 and 3.75 μM concentration for 48h. Then, cells (1 × 105/100 μl) were stained for CD20, CD46, CD55 and CD59 and analyzed in a FACSCalibur using Cell-Quest Pro software version 5.2. (B) DoHH2, Daudi, Raji or Ramos cells were incubated with either diluent or sorafenib at appropriate concentrations for 48h. Then, equal numbers of cells (1 × 105/well) were incubated for 1h with serial dilutions (from 1 to 100 μg/ml) of rituximab in the presence of 10% human AB serum as a complement source. Cell viability was measured with a MTT assay. The survival of cells is presented as percentage of corresponding diluent- or sorafenib-pretreated cells without rituximab. *P Altogether, these results indicate that sorafenib, which undergoes clinical trials in patients with NHL and B-CLL might be effectively used in combination with anti-CD20 mAbs. Disclosures: No relevant conflicts of interest to declare.

Anti-CD20 monoclonal antibodies (mAbs) have become the mainstay in the treatment of non-Hodgkin's lymphomas and have shown significant activity in patients with B-cell chronic lymphocytic leukemia. Antitumor action of these antibodies results from triggering of indirect effector mechanisms of the immune system that include activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or phagocytosis. Moreover, some studies indicate direct influence of anti-CD20 mAbs on tumor cells that leads to induction of various types of cell death. Despite the wealth of data on the mechanisms of cytotoxicity that accumulated over the last two decades their relative contribution to the therapeutic outcome is still difficult to predict in individual patients. Elucidation of molecular mechanisms of anti-CD20 mAbs action is necessary to deliver their maximal activity in rationally designed combinations with other therapeutic approaches and to design next generation anti-CD20 mAb with improved ability to eliminate tumor cells.

A number of monoclonal antibodies (mAb) are presently in development or approved for CD20-directed immunotherapy. Ofatumumab, a human IgG1 anti-CD20 mAb, is currently being evaluated in phase III clinical trials for B-CLL and FL, and in phase II clinical trials for rheumatoid arthritis (RA). Rituximab, a chimeric IgG1 anti-CD20 mAb, has been approved for 1st line treatment of CD20-positive B cell lymphomas alone or in combination with chemotherapy, and in RA. Virtually all rituximab-treated patients relapse after single-agent treatment. The clinical efficacy of rituximab might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising anti-lymphoma effects. We studied the influence of statins on ofatumumab- and rituximab-mediated cytotoxicity. Surprisingly, B cells incubated with statins showed decreased rather than increased CD20 mAb-mediated complement-dependent cytotoxicity (CDC) in cell viability assays. However, cell lysis of statin-treated B cells remained higher when using ofatumumab in comparison to rituximab. Statins decreased CD20 immunostaining in flow cytometry but did not affect total cellular CD20 levels when compared to non-treated cells. Incubation of B cells with other cholesterol depleting agents (methyl-β-cyclodextrin (MβCD) and berberine) established that the presence of plasma membrane cholesterol and not lipid rafts may be required for CD20 mAb-mediated CDC. Cholesterol restitution reversed ofatumumab- and rituximab-mediated CDC and CD20 mAb staining. Statin incubation resulted in conformational changes of CD20 and impaired binding of CD20 mAb to the CD20 molecule as observed by atomic force microscopy. Freshly isolated cells of 4 B cell lymphoma patients were treated with the cholesterol-depleting agent MβCD, and CD20 mAb (B9E9) binding and rituximab-mediated CDC were significantly decreased (P

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.

Statins, HMG-CoA reductase inhibitors, are used in the prevention and treatment of cardiovascular diseases owing to their lipid-lowering effects. Previous studies revealed that, by modulating membrane cholesterol content, statins could induce conformational changes in cluster of differentiation 20 (CD20) tetraspanin. The aim of the presented study was to investigate the influence of statins on glucose transporter 1 (GLUT1)-mediated glucose uptake in tumor cells. We observed a significant concentration- and time-dependent decrease in glucose analogs' uptake in several tumor cell lines incubated with statins. This effect was reversible with restitution of cholesterol synthesis pathway with mevalonic acid as well as with supplementation of plasma membrane with exogenous cholesterol. Statins did not change overall GLUT1 expression at neither transcriptional nor protein levels. An exploratory clinical trial revealed that statin treatment decreased glucose uptake in peripheral blood leukocytes and lowered (18)F-fluorodeoxyglucose ((18)F-FDG) uptake by tumor masses in a mantle cell lymphoma patient. A bioinformatics analysis was used to predict the structure of human GLUT1 and to identify putative cholesterol-binding motifs in its juxtamembrane fragment. Altogether, the influence of statins on glucose uptake seems to be of clinical significance. By inhibiting (18)F-FDG uptake, statins can negatively affect the sensitivity of positron emission tomography, a diagnostic procedure frequently used in oncology.

Down-regulation of CD20, a molecular target for monoclonal antibodies, is a clinical problem leading to decreased efficacy of anti-CD20-based regimens. Moreover, a substantial number of patients do not fully benefit from anti-CD20 immunotherapy due to the low CD20 expression. It has been shown that histone deacetylase inhibitors (HDACi), in a transcription-dependent mechanism, regulate CD20 surface level. However, non-specific HDAC pan-inhibitors have considerable number of side effects, therefore a new isoform-specific inhibitors are being developed. Recently, HDAC6-selective inhibitors have emerged in non-Hodgkin lymphoma and chronic lymphocytic leukemia (CLL) potential therapies. Preclinical data show therapeutic potential of HDAC6 inhibitors in combination approach. However, their effect on the efficacy of anti-CD20 immunotherapy has not been studied so far. Therefore, this study aimed to investigate the influence of HDAC6 inhibition on the regulation of CD20 expression. This study was performed in in vitro settings using a panel of established B-cell-derived tumors (Raji, Daudi, Ly-1, Karpas) as well as EBV-transformed cells and CLL primary samples. Commercial HDAC6 inhibitors- tubacin, tubastatin and clinically available ricolinostat (ACY1215) were used. In order to silence HDAC6 expression we used shRNA. The expression levels of surface antigens were assessed by flow cytometry. Total protein level was assessed by Western blotting. The influence of HDAC6 inhibition on transcription of CD20 mRNA was analyzed in qRT-PCR. In order to investigate the stability of CD20 protein a cycloheximide chase assay has been employed. The synthesis of protein de novo was investigated using Click-chemistry. To investigate the impact of HDAC6 inhibition on translation of CD20 mRNA polysomes were isolated. In this study using pharmacological inhibitors and genetic modification we have shown that HDAC6 inhibition significantly increases CD20 level in a panel of B-cell derived human tumor cell lines as well as in primary CLL samples. The observed upregulation of CD20 correlates with sensitization to type I and type II anti-CD20 antibodies. We observed that HDAC6 inhibition increases total CD20 protein level without affecting its mRNA level or promoter activity. As HDAC6 has been shown to regulate protein trafficking and degradation, we performed experiments assessing the influence of HDAC6 inhibition on CD20 endocytosis. We concluded that HDAC6 inhibition does not alter the internalization rate of CD20. In cycloheximide chase assay we showed that CD20 is a rather stable protein and therefore hypothesized that HDAC6 inhibition may be a consequence of increased translation of CD20 mRNA. Using Click-it metabolic labelling we concluded that HDAC6 inhibition significantly increased the production of CD20 de novo. To substantiate our findings on increased translation of CD20 after HDAC6 inhibition, we performed polysomal profiling followed by qRT-PCR on polysomal extracts from Raji cells. We observed no considerable changes in polysomal profiling curves between control and tubacin- pretreated cells further confirming that tubacin did not alter the overall level of protein translation. Importantly, our findings showed a significant increase in CD20 mRNA in polysomal fraction in tubacin-treated group when compared with control cells. Altogether, our results clearly indicate that HDAC6 is involve in posttranscriptional regulation of CD20 and HDAC6 inhibition increases the translational efficiency of CD20 mRNA. Our results strongly support that HDAC6 inhibition can be a therapeutic strategy successfully enhancing the efficacy of anti-CD20 mAbs. Our pre-clinical investigation might be relevant in terms of designing clinical combination studies and support further use of HDAC6 inhibitors in combination with anti-CD20 mAbs. Figure Polysomal profiling. Cytosolic lysates from control and tubacin- pretreated Raji cells were prepared in lysis buffer followed by two centrifugations. Cytosolic proteins were layered onto linear sucrose gradient and sedimented by a 2h centrifugation. The gradients were collected in 15 fractions and absorbance profiles were generated at 254 nm (A). Polysomal fractions were pooled for further RNA extraction and quantitative PCR (B). Figure. Polysomal profiling. / Cytosolic lysates from control and tubacin- pretreated Raji cells were prepared in lysis buffer followed by two centrifugations. Cytosolic proteins were layered onto linear sucrose gradient and sedimented by a 2h centrifugation. The gradients were collected in 15 fractions and absorbance profiles were generated at 254 nm (A). Polysomal fractions were pooled for further RNA extraction and quantitative PCR (B). Disclosures No relevant conflicts of interest to declare.