ALBERT

Beschreibung: Introduction: We have recently reported that the expression of Krüppel-like factor 4 (KLF4) is upregulated in pediatric Burkitt lymphoma and it was suggested to be a prognostic biomarker for survival (Valencia-Hipolito et al., 2014)*. In addition, preliminary findings have demonstrated that KLF4 is also a resistant factor for drug-induced apoptosis. Rationale: Analysis of the role of KLF4 in resistance may have prognostic and therapeutic implications. Objective: To investigate the molecular mechanism underlying KLF4-induced resistance. Hypothesis: We have reported that the transcription factor Yin Yang 1 (YY1) is overexpressed in hematological malignancies and regulates both drug resistance and immune resistance. In Non-Hodgkin lymphoma (NHL), KLF4 is overexpressed and is under the transcriptional regulation of YY1. Hence, we hypothesized that KLF4 may also regulate drug/immune resistance and its inhibition would reverse the resistance of tumor cells to drug-induced apoptosis. Methods: We have used the NHL cell lines Ramos and DHL4 as models. The expression of KLF4 was assessed by Real Time-PCR and Western. KLF4 inhibition was achieved using the inhibitor Kenpaullone and the inhibition of YY1 and KLF4 was induced by transfection with corresponding siRNAs. Apoptosis was determined by activation of caspase 3 by flow cytometry. Tumor biopsies from pediatric NHL patients were analyzed by IHC. Results: Several NHL human tumor cell lines showed overexpression of KLF4, although at different levels. Treatment with the KLF4 inhibitor, Kenpaullone, resulted in the inhibition of KLF4 expression and the cells were sensitized to Doxorubicin-induced apoptosis. Treatment of tumor cells with YY1 siRNA inhibited both YY1 and KLF4 expressions and the cells were sensitized to drugs-induced apoptosis. Tumor biopsies from patients were divided into two groups, namely, one group with high KLF4 and YY1 and the other group with low levels of KLF4 and YY1. Kaplan Meier analysis revealed that patients with low expression of KLF4 and YY1 responded subsequently to CHOP, whereas patients with high KLF4 and YY1 did not respond to CHOP. Conclusions: The findings revealed the followings: (1) KLF4 overexpression is under the transcriptional regulation of YY1 (2) Inhibition of KLF4 by the chemical inhibitor Kenpaullone, or by YY1 siRNA resulted in the sensitization of tumor cells to drug-induced apoptosis and (3) Patients with NHL whose tumors overexpressed KLF4 and YY1 did not respond to CHOP treatment. Implications: We suggest that the overexpression of KLF4 in NHL may be a novel prognostic biomarker for response to chemotherapy and may also be a therapeutic target. In addition, a recent report by Farrugia M et al (2015)** reported that KLF4 is associated with both BclxL and Mcl-1 and, thus, drug resistance is primarily under the influence of the overexpression of BclxL and Mcl-1. Consequently, since KLF4 is regulated by YY1 and YY1 is in turn, regulated by NF-κB, we suggest the presence of an NF-κB/YY1/KLF4/BclxL/Mcl-1 resistant axis in NHL and gene products in this axis maybe potential novel prognostic biomarkers and therapeutic targets. References: * (Valencia-Hipolito et al 2014, Lek & Lympho 55:1806-1814 ** Farrugia M et al (2015_cell death and disease 19;6:e1699. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Since the discovery and description of microRNAs (miRs), these molecules have become very important, specifically due to their participation in the regulation of proteins and transcription factors involved in the development of cancer. The microRNA-7 (miR-7) has been described as a negative regulator of several proteins involved in cancer such as YY1 and KLF4 in various cancer subtypes. We have recently reported that YY1 and KLF4 play a role in Non-Hodgkin Lymphoma (NHL) and that the expression of KLF4 is regulated by YY1 (Valencia-Hipolito 2014, Morales-Martinez 2019). Therefore, in this study we analyzed the role of miR-7 in NHL through its effect on the negative regulation of YY1 and KLF4 in vitro, and also assessed its expression in clinical specimens from lymphoma patients. Expression of KLF4, YY1 and miR-7 was analyzed used real-time PCR in NHL-B cell lines (Ramos, Raji, DHL4 and 2F7). The expression of KLF4 and YY1 was inverse correlated with miR-7 expression, where Raji has higher miR-7 expression and DHL4 lower, and this correlated with inverse expression of YY1 and KLF4 in both cell lines, respectively. The possible regulation of YY1 and KLF4 by miR-7 was analyzed by using the inducible expression or inhibition of miR-7, (transfection with pre-miR-7 or mimetic miR-7 respectively) as well using reporter-system plasmids containing the 3 'UTR region of YY1 or KLF4. The role of miR-7 in NHL, through the negative regulation of YY1 and KLF4 was determined by chemoresistance/proliferation, and migration assays. The clinical implications of miR-7 in the negative regulation of YY1 and KLF4, were analyzed by ISH and IHC in a TMA with 43 samples of NHL subtypes: DLBCL and Follicular Lymphoma (FL). Our results showed an inverse correlation of miR-7 expression with KLF4 and YY1 expression in B-NHL cell lines, miR-7 is able to regulate the expression of YY1 and KLF4 by direct binding in the 3 'UTR region. Thus, the induced expression of miR-7 inhibited the constitutive expression of YY1 / KLF4, whereas the inhibition of miR-7 correlated with an increase in the expression of YY1 / KLF4. Likewise, the induced expression of miR-7 reverses the chemo resistance and inhibits the migration capacity of NHL-B cell lines. Also, all tumor tissues expressing miR-7 demonstrated a negative correlation with YY1 and KLF4 expression, which was more significant in the FL. Additionally the expression of miR-7 in FL was associated with clinical outcome. Our results show for the first time that miR-7 has a role in NHL through the negative regulation of YY1 and KLF4. These results confirm YY1 and KLF4 as possible therapeutic targets through the regulation of miR-7. References: -Valencia-Hipόlito A, et al Expression of KLF4 is a predictive marker for survival in pediatric Burkitt lymphoma. Leuk Lymphoma. 2014;55(8):1806-14 -Morales-Martinez M, Valencia-Hipolito A, Vega GG, Neri N, Nambo MJ, Alvarado I, Cuadra I, Duran-Padilla MA, Martinez-Maza O, Huerta-Yepez S, Vega MI. Regulation of Krüppel-Like Factor 4 (KLF4) expression through the transcription factor Yin-Yang 1 (YY1) in non-Hodgkin B-cell lymphoma. Oncotarget. 2019;10(22):2173-2188. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Krüppel-like factor 4 (KLF4) is a member of the KLF zinc-finger containing transcription factor family. Supporting evidence has established that KLF4 is either an oncogene or a tumor suppressor. Reported studies have indicated the involvement of KLF4 in the regulation of apoptosis, proliferation, and differentiation of B cells and B-cell malignancies. In contrast to adult lymphomas and solid tumors, recently, we have shown in a TMA the overexpression of KLF4 in pediatric NHL tumor tissues. The KLF4 overexpresion predicted unresponsiveness to CHOP treatment. In addition, we have also reported that the transcription factor Yin Yang 1 (YY1) is overexpressed in B-NHL and is a prognostic factor. We hypothesized that the coexpression of KLF4 and YY1 may result from the transcriptional regulation of KLF4 by YY1. This hypothesis was tested in various experimental designs both in cell lines and tumor tissues derived from patients. Analysis of the B-NHL cell line Ramos revealed that both KLF4 and YY1 are overexpressed compared to normal B cells. The transfection of Ramos with siRNA YY1 showed significant inhibition of KLF4. In silico analyses of the KLF4 promoter identified the presence of four putative binding sites for YY1. We confirmed that the -126 site as the binding site for YY1 by CHIP analysis. We also used a reporter system of the KLF4 promoter and mutated the putative binding site for YY1 (-126) and confirmed it as an important site for the regulation of KLF4. The co-expressions of KLF4 and YY1 were examined in TMA of pediatric lymphomas and showed by IHC that all of the tumor tissues exhibited a positive correlation of the expressions of KLF4 and YY1 and the correlation was markedly significant in the Burkitt subtype. These correlations were consistent with the bioinformatics analyses due by ONCOMINE in several data base. KLF4 acts as a transcriptional activator of epithelial genes and as a repressor of mesenchymal genes. In addition, KLF4 suppresses the extrinsic apoptotic pathway by inhibiting the activation and cleavage of caspases (7, 9, and 3). Thus, the overexpression of KLF4 in lymphoma may be responsible, in part, in the pathogenesis, malignancy, and drug resistance. The putative function of KLF4 was examined by the use of chemical inhibitors for KLF4 (Kenpaullone) and inhibition of KLF4 resulted in the inhibition of cell proliferation and the spontaneous induction of apoptosis. The present findings suggest that both KLF4 and YY1 are prognostic biomarkers for pediatric lymphoma and are also potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3499 Rituximab (chimeric anti-CD20 mAb) (Rtx) has been successfully used in the treatment of patients with B-cell non-Hodgkin's lymphomas (B-NHLs). The combination treatment with chemotherapy results in achieving high response rates and prolongation of survival. However, a subset of patients does not initially respond to treatment and many responding patients relapse and no longer respond to further treatments. Currently, there are no alternative therapies for resistant patients. The mechanism of resistance in vivo is not clear. However, we have explored a potential mechanism by developing in vitro several clones of Rtx-resistant (RR) variants for several B-NHL cell lines and characterized their properties. Briefly, unlike the parental wild-type, the RR clones express CD20 but no longer respond to treatment with Rtx or combination with cytotoxic drugs. Further, these clones overexpress the activity of several survival/anti-apoptotic pathways [1]. It is not known whether chemical modification of Rtx might be necessary to exert its activity and signaling on the RR clones. Hence, a recent report demonstrated that a fusion protein consisting of Rtx and human IFN-α (anti-CD20-hIFN-α) exhibited superior activity over Rtx, IFN-α, or combination of Rtx and IFN-α, and exhibited significant anti-proliferative and apoptotic effects in vitro with several B-NHL cell lines and in vivo an anti-tumor xenograft response [2]. These findings prompted us to investigate the effect of anti-CD20-hIFN-α on the RR clones. We hypothesized that anti-CD20-hIFN-α may exert an anti-proliferative and apoptotic effects on the RR clones and may also synergize when used in combination with chemotherapy. In this study, we used the B-NHL line Ramos (Burkitt) and 2F7 (AIDS-related) and their respective Ramos RR1 and 2F7 RR1 clones as models. We examined the effects of anti-CD20-hIFN-α and Rtx on the wild-type and RR clones following treatment with IgG isotype control, Rtx, anti-CD20-hIFN-α, CDDP (10 mg/ml) and Treanda® (Bendamustine) (5 mg/ml), as well as combinations. Treatment of 2F7 with single agents alone had no cytotoxic effect; however, treatment with the combination of Rtx and CDDP or Treanda® or anti-CD20-hIFN-α plus CDDP or Treanda® resulted in significant cytotoxicity. Treatment of Ramos resulted in similar findings observed with 2F7, however, the anti-CD20-hIFN-α alone was significantly cytotoxic to Ramos cells. Importantly, whereas treatment of 2F7 RR1 or Ramos RR1 with Rtx or Rtx plus CDDP or Treanda® had no cytotoxic effects (as expected), the treatment with the anti-CD20-hIFN-α alone had significant cytotoxicity and synergy was observed when used in combination with CDDP or Treanda®. In all of the above experiments, the level of cytotoxicity was a function of the antibody concentration used (range 10–30 μg/ml). The mechanism by which anti-CD20-hIFN-α signals the RR clones for cytotoxicity and sensitization was examined. Preliminary findings show that treatment of the RR clones with anti-CD20-hIFN-α inhibits the activity of p38MAPK survival pathway and also inhibits the anti-apoptotic gene products, Bcl-2/BclXL and upregulates the pro-apoptotic expression of Bax. These findings established, for the first time, that modification of Rtx by fusion with IFN-α was cytotoxic on the RR clones and synergized with chemotherapy. The findings also show, unlike Rtx that, anti-CD20-hIFN-α signals the RR cells and inhibits survival/antiapoptotic pathways leading to direct cytotoxicity and chemo-sensitization. The molecular signaling mediated by anti-CD20-hIFN-α on the cell membrane of RR cells leading to inhibition of survival pathways will be presented. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: CD40 is a member of the TNF super family, which is expressed mainly in B cells, dendritic cells and macrophages, and the interaction with its ligand CD154 is essential for the development of the immune response. The activation or selective inhibition of this pathway is part of various therapeutic strategies for the treatment of diseases with inflammatory or immune origin, as well as various types of tumors. The fusion protein OmpC-CD154 expresses 10 amino acids of CD154 (Ser149-Trp140) and is capable of binding to CD40, inducing apoptosis and inhibiting proliferation in NHL cell lines. In this study we demonstrate that the fusion protein OmpC-CD154 induced apoptosis and inhibition of proliferation in several B-NHL cell lines. In addition, we analyzed the mechanisms involved in the induction of apoptosis and the inhibition of proliferation. Our results by Western blot demonstrate that OmpC-CD154 induced degradation of Bcl-6 by a MAPK p38 activation dependent mechanism. Pre-treatment of Ramos and Raji cell lines with a specific chemical p38 inhibitor reverted the effect of OmpC-CD154 in proliferation. Our results showed that the fusion protein can also inhibit the constitutive activation of both the canonical and non-canonical NF-kB pathways, and this inhibition was associated with the activation of the TRAFs pathway, as it promotes TRAF2 phosphorylation, and the decreasing of the expression of TRAF3. All these effects were greater than those observed with recombinant human CD154 on B-NHL cell lines. The present findings establish that a CD154 peptide fused with a protein exhibits significant cytotoxicity on B-NHL cells. Current studies are evaluating fusion proteins with CD154 peptide that are not antigenic in vivo and that have significant anti-tumor effects, both direct and on the host immune response, with minimal toxicity. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction: The standard treatment of B-NHL consists of rituximab in combination with CHOP (RCHOP) and results in a significant clinical response. Rituximab inhibits cell-proliferation and inhibits cell survival/anti-apoptic signaling pathways. A subset of patients does not initially respond and a subset of responding patients develops resistance to RCHOP. The genetic engineering of a fusion protein, α-CD20-hIFN-α, was found to be active in the rituximab-resistant B-NHL cell lines. Objective: To investigate the underlying mechanism by which α-CD20-hIFN-α signals in the resistant lines. Hypothesis: We hypothesized that the treatment with the α-CD20-hIFN-α may result in the cooperation of both α-CD20 and hIFN-α and their interactions with corresponding receptors that will result in overriding α-CD20 blocked cell signaling. Methods: Rituximab-resistant cell lines, R-2F7 and R-Ramos, were used as models. Cell signaling was determined by western. Sensitivity to drug-induced apoptosis was done by activation of caspase 3 by flow cytometry. Results: Treatment of the R lines with α-CD20-hIFN-α resulted in the inhibition of cell growth and sensitization to doxorubicin-induced apoptosis. Treatment with single agents alone or combination was not effective. Treatment with the α-CD20-hIFN-α resulted in the inhibition of the NFκB and the p38 MAPK pathways. In addition, the hIFN-mediated signaling pathway, namely, PKC-d, was also inhibited by the α-CD20-hIFN-α.The role of PKC-d in drug sensitization was corroborated by the use of the specific inhibitor, Rotterin, which reversed the drug sensitization by α-CD20-hIFN-α and doxorubicin Conclusion: The ability of the α-CD20-hIFN-α to inhibit cell survival and anti-apoptotic pathways, that was not achieved with single agents or combination, suggested that there may be a crosslinking of the CD20 and hIFN-α receptors by α-CD20-hIFN-α and results in triggering the cells via both receptors and inhibiting intracellular survival pathways and sensitization to drug apoptosis. Clinical Implication: The findings also suggest the potential therapeutic application of the combination of α-CD20-hIFN-α and drugs for the treatment of patients resistant to RCHOP. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 2647 We have reported in B-NHL cell lines that the p38 MAPK was constitutively activated and was involved in the regulation of tumor cell resistance to cytotoxic drugs. Further, inhibition of this pathway reversed drug resistance. Based in these findings we hypothesized that the activation of the p38 MAPK pathway in patients with B-NHL may be associated with unresponsiveness to cytotoxic drug therapy. This study was designed to test this hypothesis. Eighty patients with Diffused Large B Cell Lymphoma (DLBCL) were used for analysis. Freshly derived tumor tissues from these patients were obtained from biopsies prior to any treatment. Tissue microarrays were prepared and examined by immunohistochemistry for the expression of both p38 MAPK and phosphorylated p38 MAPK (active). The antibodies were tested for specificity. The frequency of stained cells

Beschreibung: Abstract 5184 Krüppel-like factor 4 (KLF4) is a transcription factor expressed in a variety of tissues in humans and has been implicated in several physiologic processes including development, differentiation, and tissue homeostasis. KLF4 is a bi-functional transcription factor that can either activate or repress transcription depending on the target gene. Human KLF4 is a protein of 470 amino acids with a 55 kDa. It contains three C-terminal C2H2-type zinc fingers that bind DNA. It is divided into three separate domains, namely, an N-terminal activation domain, a central repression domain and a C-terminal DNA-binding domain. For instance, KLF4 acts as a tumor suppressor gene in several cancers (colon, gastric, esophageal, bladder, and NSCLC) or as an oncogene (laryngeal carcinoma, squamous cell carcinoma, ductal carcinoma of the breast). However, the role of KLF4 in hematologic malignancies is still poorly understood. Reported studies in leukemia suggested that KLF4 may be a tumor suppressor. The goal of this study was to investigate the expression and the clinical significance of KLF4 in B-Non-Hodgkin's lymphomas (B-NHLs). Both B-NHL cell lines and patient-derived tumor tissues (TMA) were examined by western blot and immunohistochemistry, respectively. The expression of KLF4 was calculated based on the intensity and the percentage of the area stained, and scoring was corroborated by two pathologists. The complete absence of KLF4 expression was considered as KLF4 negative. Normal peripheral blood mononuclear cells expressed low levels of KLF4, in contrast, there was a significant overexpression of KLF4 in Ramos and Raji (Burkitt's lymphoma) and 2F7 (AIDS lymphoma) B-NHL cell lines. However, the DHL4 (DBLCL) cell line showed similar expression to normal cells. Among the 73 childhood lymphomas studied, 13/23 (57%) of lymphoblastic lymphoma, 7/20 (35%) of large B-cell lymphoma, 4/4 (100%) of anaplastic large cell lymphoma and 5/6 NHL not otherwise specified were KLF4 positive. Noteworthy, 18/18 (100%) Burkitt's lymphoma was KLF4 positive. In addition, the nuclear expression of KLF4 was significantly higher in Burkitt's lymphoma (n=18) compared to the remaining subtypes (lymphoblastic lymphoma, n=23, large B-cell lymphoma n=20 and others). All patients were treated with chemotherapy and the majority of the patients that were KLF4 positive had a stage 3–4 disease. Analysis of the EFS demonstrated that patients' tumors that were KLF4 negative had significantly higher EFS as compared to tumors that were KLF4 positive. Likewise, there was significant prolongation of survival in patients with tumors that were KLF4 negative. We suggest that the expression of KLF4 and poor response to chemotherapy may be attributed to its role in resistance via its regulation by the resistance factor Notch31. In contrast, the absence of KLF4 and good response to chemotherapy may be due to shifting p53 activity from cellular repair to cell death2. The present findings demonstrate that KLF4 may be considered as an oncogene in Burkitt's lymphoma and subsets of other types of lymphoma. The findings also suggest that the expression of KLF4 may be a potential prognostic factor, though, this need to be validated in a large cohort of patients. We propose that KLF4 may be a therapeutic target in patients with B-NHL lymphomas. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Introduction: ALK+ anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5) resulting in overexpression of NPM-ALK oncoprotein, which activates many oncogenic pathways. The Jab1 (c-Jun activation domain-binding protein-1), initially discovered as a c-Jun co-activator, represents the fifth component of an evolutionary highly conserved 8-subunit protein complex, named COP9 signalosome (CSN). The Jab1/Csn5 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via the CDK inhibitor p27. Recent evidence suggests that Jab1/Csn5 is also involved in immune checkpoint regulation through PD-L1 stabilization by inhibition of PD-L1 proteasomal degradation. Recently, we reported that the protein levels of Jab1/Csn5 are the highest in ALCL as compared to other peripheral T-cell lymphomas (PTCL) and significantly correlate with PD-L1 levels in PTCLs (Drakos et al, HemaSphere 2019; (3):p596, EHA abstract). In this study, we hypothesized that NPM-ALK may regulate Jab1/Csn5 expression and thus contributes to cell proliferation as well as PD-L1/PD1 immune checkpoint regulation. Methods: The in vitro system included 4 ALK+ (Karpas 299, DEL, SUPM2, SUDHL1) and 2 ALK- (Mac1, Mac2a) ALCL cell lines as well as murine Ba/F3 parental and Ba/F3 clones stably transfected with NPM-ALK (Ba/F3-NPM-ALK), EEF1G (Ba/F3-EEF1G-ALK) or control plasmid (Ba/F3-MIG). Transient transfections with Jab1/CSN si-RNAs and STAT3 si-RNAs were also performed in ALCL cells. In addition, ALCL cells were treated with ALK (Crizotonib) or STAT3 (XIII) inhibitors. Two animal models were used in the study: 1) in the ex vivo model, Karpas-299 clones stably transfected with Jab1/CSN5 shRNA constructs were generated and injected in both thighs of SCID-beige immunocompromised mice. The Jab1/CSN5 shRNA and the control mice were followed for tumor development and their tumors were measured and analysed by immunohistochemistry. 2) in the patient derived xenograft (PDX) model of ALK+ ALCL, the mice were treated with the ALK inhibitor Ceritinib or control vehicle and were monitored for changes in tumor characteristics over two weeks. Tumor specimens were taken at early time points (24, 48, and 72 hrs) following treatment in order to obtain viable tumor cells for protein analysis. Results: Jab1/Csn5 was substantially upregulated in the Ba/F3-NPM-ALK and Ba/F3-EEF1G-ALK stable clones as compared to paternal or control Ba/F3-MIG clones. Jab1/Csn5 upregulation was associated with high STAT3 activation (Tyr705-phosphorylation) and increased PD-L1 gene expression in this system. Inversely, inhibition of ALK activity was associated with STAT3 de-activation and decreased protein levels of Jab1/Csn5 and PD-L1 in ALK+ ALCL cells. Knocking down STAT3 by siRNA or inhibition of its activity by the XIII inhibitor resulted in decreased levels of Jab1/Csn5 protein in both ALK+ and ALK- ALCL cell lines. The SCID-beige mice that received Jab1/CSN5-shRNA clones showed significant delay in tumor development and longer survival as compared to control mice, which was associated with significantly decreased PD-L1 protein levels and p27 upregulation in the tumor cells (xenografts). Treatment of the PDX mice with Ceritinib, a potent next generation ALK inhibitor, resulted in substantial tumor necrosis after 72 hrs and decreased tumor size at day 7 post-treatment. At earlier time points, de-activation (de-phosphorylation) of ALK kinase and STAT3 was observed in the Ceritinib-treated mice, which was linked to variably lower levels of Jab1/CSN5 and PD-L1 proteins as compared to control mice. Conclusion: Jab1/CSN5 is a novel downstream target of the NPM-ALK oncogenic kinase that regulates its expression, at least in part, through STAT3 activation. The Jab1/CSN5-mediated stabilization of PD-L1 can be efficiently inhibited by Ceritinib in preclinical animal models of ALK+ ALCL. Disclosures Österborg: BeiGene: Research Funding; Janssen: Research Funding; Abbvie: Research Funding; Kancera AB: Research Funding; Gilead: Research Funding. Vega:National Cancer Institute, national Institutes of Health: Other: Grant Funding-R01CA222918.

Beschreibung: Key Points Novel crosstalk between SMO and NF-κB representing additional level of NF-κB regulation independent of genetic constitutive activation. SMO activates NF-κB by recruiting Gαi and Gα12 to activate PKCβ/CARMA1 and assembling CARMA1/BCL10/MALT1/TRAF6 to SMO.