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: 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: 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: White blood cell counts (WBC) and absolute neutrophil counts (ANC) are well-established predictors of a patient's risk of fever and infections or febrile neutropenia (FN). Currently, patients at risk of FN due to cancer chemotherapy, idiosyncratic drug-reactions or congenital neutrophil disorders are monitored at hospital or clinic laboratories. For many patients, effective and frequent monitoring is difficult due to the time required and costs of repeated laboratory visits. We herein present the first results for a novel device called Athelas One, a miniature point-of-care hematology analyzer suitable for at home monitoring of WBC and ANC. Methods: Athelas One (A1) is a small cylindrical device with a built-in, single head, light microscope. To determine the WBC and ANC, a small drop of blood (~ 3.5 uL) from a finger stick or an anticoagulated blood sample is drawn by capillary action onto a specially designed microfluidic test strip which creates a stained, precisely dimensioned monolayer of blood cells. The slide is then inserted into the device which scans the test strip and reports the WBC and ANC based on an image analysis process (Computer Vision). We compared results for A1 with a standard laboratory counter [Sysmex XE5000 (SX)]. Results: Initially, A1's reproducibility was demonstrated using 43 blood samples with a wide range of known WBCs (i.e., samples with normal counts, leukocytosis, leukopenia, neutropenia, neutrophilia) run on 4 devices using a single lot of test strips. The A1 results were then compared to SX results using the Passing Bablok Regression with Bootstrap method. These results showed strong linearity and comparability between paired anti-coagulated venous blood samples and the blood samples compared to finger prick capillary blood samples collected almost simultaneously. The slope and intercept indicated a linear relationship, with a 95% confidence for interval slope and intercept containing 1 and 0 respectively. (Comparisons: A1 blood to SX blood: WBC, r=0.998, ANC, r=0.989; A1 capillary to SX blood: WBC, r=0.998, ANC, r=0.97). Table 1 and Figure 1 show a summary of the comparisons. In addition to these results, we have tested 18 samples with WBC 〈 1.0 x 109/L with closely comparable results for A1 and standard hospital counter. Summary: These initial results show that the WBC and ANC can be accurately determined with a finger stick drop of blood and this point-of-care hematology analyzer. Given its small size, ease of use and accuracy, the device is suitable for home monitoring. Disclosures Dale: Athelas, Inc.: Equity Ownership; Amgen: Consultancy, Research Funding; Sanofi-Aventi: Consultancy, Honoraria; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer-Ingelheim: Consultancy; Coherus: Consultancy. Navarro-De La Vega:Athelas, Inc.: Employment. Parthasarathy:Athelas, Inc.: Employment, Equity Ownership. Bodapati:Athelas, Inc.: Employment, Equity Ownership, Patents & Royalties: patent owned by Athelas on the Athelas One device. Virey:Athelas, Inc.: Employment, Equity Ownership, Patents & Royalties: patent owned by Athelas on the Athelas One device. Moffatt:Athelas, Inc.: Employment, Equity Ownership, Patents & Royalties: patent owned by Athelas on the Athelas One device. Tandon:Athelas, Inc.: Employment, Equity Ownership, Patents & Royalties: patent owned by Athelas on the Athelas One device.

Beschreibung: * A.R. and M.L. have contributed equally. INTRODUCTION: Despite great advances in knowledge and treatment in the last decade, multiple myeloma (MM) remains an incurable disease. Several studies suggest its association with infectious pathogens, such as hepatitis C virus (HCV) and Epstein Barr virus (EBV). Here we report on the case of a female MM patient in fourth relapse (after VTD treatment + ASCT+ Lenalodomide maintenance and experimental NK therapy + Lenalidomide and lastly Bendamustine), who achieved stable complete response (CR) once her HCV infection was successfully treated, thus establishing a probable relationship between HCV infection and MM in this patient. METHODS: Serum samples from the patient were analyzed before and after antiviral treatment (Sofusbuvir + Ledipasvir). The HCV burden was determined by RT-qPCR. Additionally, viral loads of EBV and Cytomegalovirus were established by qPCR.The number of tumor plasma clones was determined in bone marrow samples by NGS using the Ion Proton sequencer and a depth of 2000 readings/nucleotide. Monoclonal immunoglobulins (mc Ig) were separated from polyclonal Ig by agarose gel electrophoresis and elution. Mc Ig purity was evaluated by isoelectric focusing and immunodetection with anti-IgG antibodies. Their reactivity to different microbial antigens was determined by means of the multiplex infectious-antigen microarray (MIAA) assay and using the commercial kit INNO-LIA HCV score (Fujirebio). RESULTS: The patient, age 66, presented with ISS IIA stage MM (56.7 g/L mc IgG, 2.7 g/L free lambda chains, 2.9 mg/L b2-microglobulin, 90% plasma cells in the bone marrow, without genetic alterations, and osteolytic lesions). HCV infection, causing hepatic toxicity, was discovered in this patient before MM disease. The patient was in third relapse of MM treatment when she received anti-HCV treatment. After antiviral therapy, the HCV load in the patient's serum decreased to undetectable levels (Fig. 1a). Simultaneously, the patient achieved CR of MM, with minimal residual disease (MRD) negativity, as assessed by multiparameter flow cytometry (MFC). NGS revealed the existence of at least one plasmacytic clone, which became MRD negative after anti-HCV treatment (Fig. 1b). Before and after anti-HCV treatment, the patient's serum samples were reactive against antigens of various viruses and other microorganisms (Fig. 1c). Agarose gel electrophoresis of pre-HCV treatment samples showed one band of mc IgG, which disappeared after anti-HCV treatment (Fig. 1d). The patient's mc IgG was purified (Fig. 1e) and analysis of its specificity of recognition revealed that it targeted the HCV core protein (Fig. 1f). Three years have now passed after HCV treatment, and the patient remains in stable CR of MM. CONCLUSION: In this case of refractory MM where the patient's mc IgG targeted HCV, successful HCV eradication with antivirals Sofosbuvir and Ledipasvir resulted in persistent complete remission of MM as well as of hepatitis C. These results suggest that for HCV-positive individuals, a causal relationship exists between HCV infection and the development of MM, and that MM patients infected with HCV would benefit from early anti-HCV therapy. Disclosures Martinez Lopez: Celgene: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau; Jansen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

Beschreibung: Key Points Elevated Jun signaling promotes lymphoma growth and dissemination to extranodal sites. Jun-regulated genes mediate the interaction of malignant cells with stromal cells and adhesion to extracellular matrix proteins.

Beschreibung: The leukocyte activation marker CD69 is a novel regulator of the immune response, modulating the production of cytokines including transforming growth factor-β (TGF-β). We have generated an antimurine CD69 monoclonal antibody (mAb), CD69.2.2, which down-regulates CD69 expression in vivo but does not deplete CD69-expressing cells. Therapeutic administration of CD69.2.2 to wild-type mice induces significant natural killer (NK) cell–dependent antitumor responses to major histocompatibility complex (MHC) class I low RMA-S lymphomas and to RM-1 prostatic carcinoma lung metastases. These in vivo antitumor responses are comparable to those seen in CD69-/- mice. Enhanced host NK cytotoxic activity correlates with a reduction in NK-cell TGF-β production and is independent of tumor priming. In vitro studies demonstrate the novel ability of anti-CD69 mAbs to activate resting NK cells in an Fc receptor–independent manner, resulting in a substantial increase in both NK-cell cytolytic activity and interferon γ (IFNγ) production. Modulation of the innate immune system with monoclonal antibodies to host CD69 thus provides a novel means to antagonize tumor growth and metastasis.

Beschreibung: Trib1, Trib2, and Trib3 are mammalian homologues of the Tribbles protein family, an evolutionarily conserved group of proteins that can mediate proteasome-dependent degradation. Evidence suggests that these proteins function as adapters, where they recruit E3 ligases and enhance ubiquination of the target protein in order to promote its degradation. To date, increased Trib1 and Trib2 mRNA expression has been shown to correlate with acute myelogenous leukemia (AML) in humans and induces AML in mice; whereas Trib3 has not been associated with AML. In order to understand the effects of Trib family members in hematopoietic cells, we reconstituted mice with hematopoietic progenitors retrovirally expressing Trib1, Trib2, or Trib3. Trib1 and Trib2 mice developed AML whereas Trib3 mice did not. Our previous data suggested that Trib2-mediated degradation of the transcription factor, C/EBPα, is important for leukemogenesis. We now show that Trib1, like Trib2, strongly binds C/EBPα and induces its degradation. In contrast, Trib3 weakly binds C/EBPα and fails to induce its degradation. Consistent with the ability to strongly bind and degrade C/EBPα, Trib1 and Trib2, but not Trib3, block differentiation of myeloid cells. We are currently mapping the domains that account for the differences between Trib1/Trib2 and Trib3 in leukemogenesis. Together, our results strengthen the correlation between Trib-induced C/EBPα degradation and induction of AML. Furthermore, our data show that different Tribbles family members have distinct targets and understanding this specificity may provide opportunities to therapeutically target Tribbles.