Role of Stat3 Activation in Cell-Cell Interaction between B-Cell Lymphoma and Macrophages : The in vitro Study

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the United States (US). DLBCL is a heterogeneous lymphoma, including the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, which have different gene expression profiles, oncogenic aberrations, and clinical outcomes (Alizadeh, Nature 2000; Staudt, Adv Immunol 2005). ABC-DLBCL is characterized by chronic active B-cell receptor (BCR) signaling (Davis, Nature 2010), which is required for cell survival. Thus, the BCR signaling pathway is an attractive therapeutic target in this type of B-cell malignancy. Bruton's tyrosine kinase (BTK), which plays a pivotal role in BCR signaling, is covalently bound with high affinity by ibrutinib, a first-in-class BTK inhibitor approved in the US for mantle cell lymphoma and chronic lymphocytic leukemia (CLL) patients (pts) who have received at least one prior treatment, CLL with del17p, and WaldenstršmÕs macroglobulinemia. A recent phase 2 clinical trial of single-agent ibrutinib in DLBCL pts revealed an overall response rate of 40% for ABC-DLBCL (Wilson, Nat. Med 2015); however, responses to single kinase-targeted cancer therapies are often limited by the cellÕs ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway (Nardi, Curr Opin Hematol 2004; Gazdar, Oncogene 2009). The serine/threonine-protein kinase PIM1 is one of several genes exhibiting differential expression in ibrutinib-resistant ABC-DLBCL cells compared with wild-type (WT) cells. We identified and report herein the role of PIM1 in ABC-DLBCL ibrutinib-resistant cells. Methods: PIM1 gene expression was analyzed by RT-qPCR. In vitro, cell viability was assessed in the human ABC-DLBCL cell line HBL-1 after treatment with ibrutinib and/or a pan-PIM inhibitor for 3 days, and the effect on colony formation was determined 7 days post-treatment. PIM1 mutational analysis was performed with clinical tumor biopsy samples from 2 studies, PCYC-04753 (NCT00849654) and PCYC-1106-CA (NCT01325701). PIM1 protein stability was analyzed by treating cells with cycloheximide and examining protein levels at different time points up to 8 hours. Results: Gene expression profiling of ibrutinib-resistant ABC-DLBCL cells revealed an upregulation of PIM1 (15-fold increase compared with WT cells) as well as PIM2 and PIM3. We also found that, compared with single-drug treatment, in vitro cell growth could be synergistically suppressed with a combination of ibrutinib and a pan-PIM inhibitor. This effect was observed in both WT (combination index (C.I.) = 0.25; synergy score = 3.18) and ibrutinib-resistant HBL-1 cells (C.I. = 0.18; synergy score = 4.98). In HBL-1 cells, this drug combination reduced colony formation and suppressed tumor growth in a xenograft model (Figure 1). In 48 DLBCL patient samples with available genomic profiling, PIM1 mutations appeared more frequently in pts diagnosed with ABC-DLBCL compared with GCB-DLBCL (5 out of 6 DLBCL pts with PIM1 mutations were ABC-subtype). 4 of these 5 pts exhibited a poor clinical response to ibrutinib, ie, 80% of ABC-DLBCL pts with PIM1 mutations had progressive disease, compared with only 13 of 26 (ie, 50%) ABC-DLBCL pts without PIM1 mutations. Subsequent characterization of the mutant PIM1 proteins (L2V, P81S, and S97N) confirmed that they were more stable than WT PIM1, suggesting increased protein levels by 2 potential mechanisms (WT PIM1 gene up-regulation or increased mutant PIM1 protein half-life). The impact of these mutations on PIM1 function and ibrutinib sensitivity is under investigation. Conclusions: Ibrutinib-resistant ABC-DLBCL cells have increased PIM1 expression, and synergistic growth suppression was observed when ibrutinib was combined with a pan-PIM inhibitor. PIM1 mutations identified in ABC-DLBCL pts with poor responses to ibrutinib contributed to increased PIM1 protein stability. A better understanding of the role of PIM1 in ibrutinib-resistant ABC-DLBCL tumors could provide a rationale for the design of combination therapies. Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p < 0.01, repeated measures MANOVA adjusted univariate F-test). Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p < 0.01, repeated measures MANOVA adjusted univariate F-test). Disclosures Kuo: Pharmacyclics LLC, an AbbVie Company: Employment. Hsieh:pharmacyclics LLC, an AbbVie Company: Employment. Schweighofer:Pharmacyclics LLC, an AbbVie Company: Employment. Cheung:Pharmacyclics LLC, an AbbVie Company: Employment. Wu:Pharmacyclics LLC, an AbbVie Company: Employment. Apatira:Pharmacyclics LLC, an AbbVie Company: Employment. Sirisawad:Pharmacyclics LLC, an AbbVie Company: Employment. Eckert:Pharmacyclics LLC, an AbbVie Company: Employment. Liang:Pharmacyclics LLC, an AbbVie Company: Employment. Hsu:Pharmacyclics LLC, an AbbVie Company: Employment. Chang:Pharmacyclics LLC, an AbbVie Company: Employment.

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Elevated Serum IL-12 Levels Attenuate Tumor Immunity Via the TIM-3/Gal-9 Axis and Worsen Patient Prognosis in Follicular B-Cell Non-Hodgkin Lymphoma (NHL).

Blood ◽

10.1182/blood.v114.22.2668.2668 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2668-2668

Author(s):

Zhi-Zhang Yang ◽

Steven C. Ziesmer ◽

Anne J. Novak ◽

Toshiro Niki ◽

Mitsuomi Hirashima ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Tumor Immunity ◽

Cd8 T Cells ◽

Cell Lymphoma ◽

Elevated Serum ◽

B Cell Lymphoma ◽

Serum Levels

Abstract Abstract 2668 Poster Board II-644 Interleukin-12 (IL-12) has been demonstrated to induce IFN-g production by T and NK cells and thereby contribute to anti-tumor immunity. However, the administration of IL-12 to boost anti-tumor immunity in B-cell lymphoma has shown no clinical benefit. In fact, clinical trials of IL-12 in combination with rituximab in follicular B-cell lymphoma (FL) showed a lower response rate in patients treated with the combination than in patients treated with rituximab alone (Clin Cancer Res. 2006 15; 12:6056-63). The goal of this study was therefore to determine the role of IL-12 in the antitumor response in B-cell NHL. First, we measured serum levels of IL-12 in patients with untreated FL before treatment with rituximab and normal healthy controls. We found that serum IL-12 levels were elevated in FL patients compared to healthy individuals (median: 0.50 ng/ml, n=30 vs 0.32 ng/ml, n=22; p= 0.03) and that elevated serum IL-12 levels were associated with a poor outcome in these patients when treated with rituximab alone as initial therapy. Using 0.56 ng/ml as a cutoff, patients with serum IL-12 levels of greater than 0.56 ng/ml had a significantly shorter time to progression than patients with IL-12 levels less than 0.56 ng/ml (12 months versus 40 months; p=0.001). To determine the mechanism by which IL-12 may contribute to a poor prognosis, we investigated the role of IL-12 on induction of immune tolerance. First, we found that TIM-3, a member of the T cell immunoglobulin and mucin domain-containing protein (TIM) family that functions to terminate TH1-mediated immunity and promote tolerance, was constitutively expressed on a subset of intratumoral T cells accounting for approximately 15% and 25% of the intratumoral CD4+ and CD8+ T cells, respectively. In contrast, less than 2% of T cells from peripheral blood of normal individuals expressed TIM-3. TIM-3-expressing T cells were distinct from regulatory T cells since CD25+ and Foxp3+ T cells lacked TIM-3 expression. Secondly, we found that TIM-3-expressing CD4+ cells were unable to produce cytokines such as IL-2, IFN-g or IL-17 and that TIM-3-expressing CD8+ T cells failed to produce Granzyme B, IFN-g or IL-2. We also observed that TIM-3-expressing T cells lost the capacity to proliferate in response to TCR activation. These results suggest that TIM-3 expressing CD4+ and CD8+ T cells are functionally exhausted. Thirdly, we observed that TIM-3 expression on T cells could be induced by activation and that IL-12 was the strongest stimulus to induce TIM-3 expression on CD4+ and CD8+ T cells. Finally, we found by immunohistochemistry (IHC) that Galectin-9 (Gal-9), a ligand for TIM-3, was abundantly expressed on lymphoma B cells. In vitro incubation with a stable form of Gal-9 induced apoptosis of CD4+ and CD8+ T cells in a dose dependent fashion. Gal-9-mediated apoptosis of T cells was attenuated by a TIM-3 Fc protein and isolated TIM-3+ T cells exhibited a significantly higher apoptosis rate than TIM-3− T cells in response to Gal-9. These results indicate that, in contrast to the observations in vitro or in vivo in mice, IL-12 actually plays a detrimental role in lymphoma patients. Given the findings that IL-12 strongly induces TIM-3 expression on effector T cells and that the TIM-3/Gal-9 pathway impairs the immune response, we conclude that increased serum levels of IL-12 suppress anti-tumor immunity in follicular lymphoma patients and is associated with a poor prognosis. Disclosures: Witzig: Novartis: Research Funding.

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Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas

JNCI Journal of the National Cancer Institute ◽

10.1093/jnci/djz078 ◽

2019 ◽

Vol 112 (1) ◽

pp. 95-106 ◽

Cited By ~ 3

Author(s):

Miguel Gallardo ◽

Prerna Malaney ◽

Marisa J L Aitken ◽

Xiaorui Zhang ◽

Todd M Link ◽

...

Keyword(s):

B Cell ◽

Rna Binding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Hnrnp K ◽

Large B Cell Lymphoma ◽

Transgenic Lines ◽

Large B Cell

Abstract Background Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown. Methods Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided. Results hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P < .001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (P < .001 for all transgenic lines; Line 171[n = 30]: hazard ratio [HR] = 64.23, 95% confidence interval [CI] = 26.1 to 158.0; Line 173 [n = 31]: HR = 25.27, 95% CI = 10.3 to 62.1; Line 177 [n = 25]: HR = 119.5, 95% CI = 42.7 to 334.2, compared with wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K’s oncogenic potential stems from its ability to posttranscriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in both in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients. Conclusion Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.

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Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma

Blood Advances ◽

10.1182/bloodadvances.2021005682 ◽

2021 ◽

Author(s):

Chiara Pighi ◽

Taek-Chin Cheong ◽

Mara Compagno ◽

Enrico Patrucco ◽

Maddalena Arigoni ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Burkitt Lymphoma ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Conditional Deletion ◽

Frequent Mutations

The expression of BCL6 in B cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase involved in the degradation of BCL6 and is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of Burkitt lymphoma (BL) patients. FBXO11 mutations impaired BCL6 degradation and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of either one or two copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B cell lymphoma onset, providing experimental evidence that FBXO11 is a haplo-insufficient oncosuppressor in B cell lymphoma. In WT and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degrader or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches such as BCL6 degraders and direct MYC inhibition could be exploited as a targeted therapy for BL.

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The Acquirement of Rituximab Resistance Is Associated with a Global Downregulation of CD54 in B-Cell Lymphoma Cells: The Potential Role of Adhesion Molecules in Rituximab Anti-Tumor Activity

Blood ◽

10.1182/blood.v112.11.2607.2607 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2607-2607

Author(s):

Ping-Chiao Tsai ◽

Naveen Bangia ◽

Scott Olejniczak ◽

Francisco J. Hernandez-Ilizaliturri ◽

Myron Czuczman

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Adhesion Molecules ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Aggregation ◽

B Cell Lymphomas ◽

Cell Clustering

Abstract Cell adhesion plays an important role in the cell-cell communication and provides important signals for cell survival, migration, aggregation, or other cell functions. Preclinical studies have been conducted to investigate the expression profiles of different adhesion molecules on the surface of malignant B-cells in an attempt to explain differences in the clinical behavior and patterns of spread between non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Of interest, CLL cells have lower levels of both adhesion molecules and CD20 when compared to follicular lymphomas (FL). Recently, knockout studies had demonstrated that CD26, an adhesion molecule, modified responses to chemotherapy in B-cell lymphomas. It is unclear if the expression of adhesion molecules affects rituximab activity. To this end, we studied the patterns of cell aggregation and expression of adhesion molecules in a panel of rituximab-sensitive (RSCL) and rituximab-chemotherapy lymphoma cell lines (RRCL) that had been extensively characterized by our group (Czuczman S. et al. Clin Cancer Res.2008; 14:1561–70). Homotypic adhesion of B-cells is known to, due to the interaction of ICAM-1(CD54) and LFA-1(CD11a). Expression of CD54 and its ligand CD11a was studied by flow cytometry analysis and polymerase chain reaction (PCR, CD54 only). Patterns of cell aggregation in RSCL and RRCL in resting conditions were studied by inverted light microscopy. To define further the role of CD54 in B-cell aggregation and rituximab activity, RSCL (Raji and RL cells) were exposed to RPMI, rituximab (10mg/ml), isotype (10mg/ml) with or without a blocking anti-CD54 monoclonal antibody (0.25mg/ml) and patterns of cell aggregation were evaluated by inverted light microscopy, and photographs were captured at different time intervals. Experiments were conducted with or without the potent pan-caspase inhibitor Q-VD-OPh and performed in triplicates. Cell death was detected by propidium iodine staining and quantified by flow cytometry. Differences in the expression levels of CD54 were observed in the NHL cells tested. RRCL were found to have lower levels of CD54 at the surface protein and gene level. No differences in the CD11a were observed. RSCL aggregate and form clusters under culture conditions whereas RRCL do not aggregate in vitro. In vitro exposures to rituximab lead to a rapid cell clustering in RSCL. Blocking CD54 using mAbs prevented spontaneous and rituximab induced cell clustering, resulting in a phenotype similar to the RRCL. Of interest, in vitro exposure to anti-CD54 mAb and to a lesser degree rituximab resulted in apoptosis of RSCL, suggesting that cell adhesion is important for survival in B-cell lymphomas. The decrease in cell aggregation following CD54 blocking was not reduced by inhibition of caspase activation suggesting that cell death was not the dominant factor in preventing cell clustering in RSCL. In summary, our data suggests that CD54 is important for B-cell lymphoma cell aggregation and survival. Furthermore, blocking of CD54 appears to abolish the clustering effects of rituximab in vitro. Loss of CD54 is observed in rituximab-chemotherapy cell lines and may disrupt signaling events that control cell proliferation (i.e. pro- or anti-apoptotic proteins) rendering these cells resistant to rituximab and chemotherapy drugs. Ongoing studies in lymphoma severe combined immunodeficiency mice (SCID) are underway to further define the role of CD54 in the progression of B-cell lymphomas and responses to rituximab activity in vivo.

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