Haploidentical γδ T Cells Induce Complete Remission in Chemorefractory B-cell Non-Hodgkin Lymphoma.

The transformation of chronic lymphocytic leukemia to an aggressive lymphoma, called Richter transformation, is often accompanied by resistance to chemotherapy and high mortality. Thus, novel therapeutic strategies are required for the successful treatment of these patients. One possibility is cellular immunotherapy with chimeric antigen receptor T cells. However, the time delay until cells are available and the limited number of effector cells due to the impaired immune system of these patients potentially compromises the efficacy of this approach. Another promising attempt might be the therapy with γδ T cells. Once activated, they exhibit various antitumor effects against several types of malignancies. Furthermore, they can be safely used in an allogeneic setting and can be multiplied in vivo as already demonstrated in clinical studies. In vitro data, in addition, show that the cytotoxicity of γδ T cells can be significantly enhanced by monoclonal antibodies. Here we present a patient, who suffered from Richter transformation and did not respond to several lines of immunochemotherapy. Due to the lack of further therapy options, we conducted an individual therapy with adoptive transfer of haploidentical γδ T cells combined with the application of the monoclonal antibody obinutuzumab. A histologically confirmed complete remission was achieved through this therapy approach, whereby relevant side effects were not seen. This case highlights the potential of γδ T cells and the feasibility of this therapeutic approach for further clinical trials.

First-line Treatment With Bendamustine and Rituximab for Old and Frail Patients With Aggressive Lymphoma: Results of the B-R-ENDA Trial.

The incidence of aggressive B-cell lymphomas increases with age, but for elderly or frail patients not eligible for doxorubicin-containing treatment standard therapy remains to be defined. In this prospective, multicenter, phase-2 B-R-ENDA trial, we investigated the feasibility, toxicity, and efficacy of 8 cycles rituximab combined with 6 cycles bendamustine (BR) in elderly or frail aggressive B-cell lymphoma patients: 39 patients aged >80 years and 29 patients aged 61-80 years with elevated Cumulative Illness Rating Scalescore >6 were included. Progression-free survival (PFS) and overall survival (OS) at 2 years were 45% (95% confidence interval [CI], 28%-61%) and 46% (28%-63%) for the patients age >80, as well 32% (13%-51%) and 37% (17%-57%) for frail patients age 64-80, respectively. In a preplanned retrospective analysis, we found no significant differences in PFS and OS comparing the outcome of the 39 patients age >80 years with 40 patients aged 76-80 years treated with 6xR-CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) and 2 x rituximab in the RICOVER-60 trial (DSHNHL 1999-1, NCT00052936, EU-20243), yet we detected lower rates of infections and treatment-related deaths in the BR-treated patients. We demonstrate that older and frail patients with aggressive B-cell lymphoma who are not able to receive standard CHOP-based therapy can benefit from anthracycline-free therapy as a feasible and effective therapeutic option.

Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands.

The molecular pathogenesis of salivary gland acinic cell carcinoma (AciCC) is poorly understood. The secretory Ca-binding phosphoprotein (SCPP) gene cluster at 4q13 encodes structurally related phosphoproteins of which some are specifically expressed at high levels in the salivary glands and constitute major components of saliva. Here we report on recurrent rearrangements [t(4;9)(q13;q31)] in AciCC that translocate active enhancer regions from the SCPP gene cluster to the region upstream of Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) at 9q31. We show that NR4A3 is specifically upregulated in AciCCs, and that active chromatin regions and gene expression signatures in AciCCs are highly correlated with the NR4A3 transcription factor binding motif. Overexpression of NR4A3 in mouse salivary gland cells increases expression of known NR4A3 target genes and has a stimulatory functional effect on cell proliferation. We conclude that NR4A3 is upregulated through enhancer hijacking and has important oncogenic functions in AciCC.

TGFß1 regulates HGF-induced cell migration and hepatocyte growth factor receptor MET expression via C-ets-1 and miR-128-3p in basal-like breast cancer.

Breast cancer is the most common cancer in women worldwide. The tumor microenvironment contributes to tumor progression by inducing cell dissemination from the primary tumor and metastasis. TGFß signaling is involved in breast cancer progression and is specifically elevated during metastatic transformation in aggressive breast cancer. In this study, we performed genomewide correlation analysis of TGFBR2 expression in a panel of 51 breast cancer cell lines and identified that MET is coregulated with TGFBR2. This correlation was confirmed at the protein level in breast cancer cell lines and human tumor tissues. Flow cytometric analysis of luminal and basal-like breast cancer cell lines and examination of 801 tumor specimens from a prospective cohort of breast cancer patients using reverse phase protein arrays revealed that expression of TGFBR2 and MET is increased in basal-like breast cancer cell lines, as well as in triple-negative breast cancer tumor tissues, compared to other subtypes. Using real-time cell analysis technology, we demonstrated that TGFß1 triggered hepatocyte growth factor (HGF)-induced and MET-dependent migration in vitro. Bioinformatic analysis predicted that TGFß1 induces expression of C-ets-1 as a candidate transcription factor regulating MET expression. Indeed, TGFß1-induced expression of ETS1 and breast cancer cell migration was blocked by knockdown of ETS1. Further, we identified that MET is a direct target of miR-128-3p and that this miRNA is negatively regulated by TGFß1. Overexpression of miR-128-3p reduced MET expression and abrogated HGF-induced cell migration of invasive breast cancer cells. In conclusion, we have identified that TGFß1 regulates HGF-induced and MET-mediated cell migration, through positive regulation of C-ets-1 and negative regulation of miR-128-3p expression in basal-like breast cancer cell lines and in triple-negative breast cancer tissue.

MicroRNA-519a-3p mediates apoptosis resistance in breast cancer cells and their escape from recognition by natural killer cells.

Aggressive breast cancer is associated with poor patient outcome and characterized by the development of tumor cell variants that are able to escape from control of the immune system or are resistant to targeted therapies. The complex molecular mechanisms leading to immune escape and therapy resistance are incompletely understood. We have previously shown that high miR-519a-3p levels are associated with poor survival in breast cancer. Here, we demonstrate that miR-519a-3p confers resistance to apoptosis induced by TRAIL, FasL and granzyme B/perforin by interfering with apoptosis signaling in breast cancer cells. MiR-519a-3p diminished the expression of its direct target genes for TRAIL-R2 (TNFRSF10B) and for caspase-8 (CASP8) and its indirect target gene for caspase-7 (CASP7), resulting in reduced sensitivity and tumor cell apoptosis in response to apoptotic stimuli. Furthermore, miR-519a-3p impaired tumor cell killing by natural killer (NK) cells via downregulation of the NKG2D ligands ULBP2 and MICA on the surface of tumor cells that are crucial for the recognition of these tumor cells by NK cells. We determined that miR-519a-3p was overexpressed in more aggressive mutant TP53 breast cancer that was associated with poor survival. Furthermore, low levels of TRAIL-R2, caspase-7 and caspase-8 correlated with poor survival, suggesting that the inhibitory effect of miR-519a-3p on TRAIL-R2 and caspases may have direct clinical relevance in lowering patient's prognosis. In conclusion, we demonstrate that miR-519a-3p is a critical factor in mediating resistance toward cancer cell apoptosis and impairing tumor cell recognition by NK cells. This joint regulation of apoptosis and immune cell recognition through miR-519a-3p supports the hypothesis that miRNAs are key regulators of cancer cell fate, facilitating cancer progression and evasion from immunosurveillance at multiple and interconnected levels.

Co-Inflammatory Roles of TGFß1 in the Presence of TNFα Drive a Pro-inflammatory Fate in Mesenchymal Stem Cells.

High plasticity is a hallmark of mesenchymal stem cells (MSCs), and as such, their differentiation and activities may be shaped by factors of their microenvironment. Bones, tumors, and cardiomyopathy are examples of niches and conditions that contain MSCs and are enriched with tumor necrosis factor α (TNFα) and transforming growth factor ß1 (TGFß1). These two cytokines are generally considered as having opposing roles in regulating immunity and inflammation (pro- and anti-inflammatory, respectively). Here, we performed global gene expression analysis of human bone marrow-derived MSCs and identified overlap in half of the transcriptional programs that were modified by TNFα and TGFß1. The two cytokines elevated the mRNA expression of soluble factors, including mRNAs of pro-inflammatory mediators. Accordingly, the typical pro-inflammatory factor TNFα prominently induced the protein expression levels of the pro-inflammatory mediators CCL2, CXCL8 (IL-8), and cyclooxygenase-2 (Cox-2) in MSCs, through the NF-κB/p65 pathway. In parallel, TGFß1 did not elevate CXCL8 protein levels and induced the protein expression of CCL2 at much lower levels than TNFα; yet, TGFß1 readily induced Cox-2 and acted predominantly via the Smad3 pathway. Interestingly, combined stimulation of MSCs by TNFα + TGFß1 led to a cooperative induction of all three inflammatory mediators, indicating that TGFß1 functioned as a co-inflammatory cytokine in the presence of TNFα. The cooperative activities of TNFα + TGFß1 that have led to CCL2 and CXCL8 induction were almost exclusively dependent on p65 activation and were not regulated by Smad3 or by the upstream regulator TGFß-activated kinase 1 (TAK1). In contrast, the TNFα + TGFß1-induced cooperative elevation in Cox-2 was mostly dependent on Smad3 (demonstrating cooperativity with activated NF-κB) and was partly regulated by TAK1. Studies with MSCs activated by TNFα + TGFß1 revealed that they release factors that can affect other cells in their microenvironment and induce breast tumor cell elongation, migration, and scattering out of spheroid tumor masses. Thus, our findings demonstrate a TNFα + TGFß1-driven pro-inflammatory fate in MSCs, identify specific molecular mechanisms involved, and propose that TNFα + TGFß1-stimulated MSCs influence the tumor niche. These observations suggest key roles for the microenvironment in regulating MSC functions, which in turn may affect different health-related conditions.

miRNA-1246 induces pro-inflammatory responses in mesenchymal stem/stromal cells by regulating PKA and PP2A.

The tumor microenvironment (TME) has an impact on breast cancer progression by creating a pro-inflammatory milieu within the tumor. However, little is known about the roles of miRNAs in cells of the TME during this process. We identified six putative oncomiRs in a breast cancer dataset, all strongly correlating with poor overall patient survival. Out of the six candidates, miR-1246 was upregulated in aggressive breast cancer subtypes and expressed at highest levels in mesenchymal stem/stroma cells (MSCs). Functionally, miR-1246 led to a p65-dependent increase in transcription and release of pro-inflammatory mediators IL-6, CCL2 and CCL5 in MSCs, and increased NF-κB activity. The pro-inflammatory phenotype of miR-1246 in MSCs was independent of TNFα stimulations and mediated by direct targeting of the tumor-suppressors PRKAR1A and PPP2CB. In vitro recapitulation of the TME revealed increased Stat3 phosphorylation in breast epithelial (MCF10A) and cancer cells (SK-BR-3, MCF7, T47D) upon incubation with conditioned medium (CM) of MSCs overexpressing miR-1246. Additionally, this stimulation enhanced proliferation of MCF10A cells, increased migration of MDA-MB-231 cells and induced attraction of THP-1 monocytic cells. Our data shows that miR-1246 acts as both key-enhancer of pro-inflammatory responses in MSCs and putative oncomiR in breast cancer, suggesting its influence on cancer-related inflammation and breast cancer progression.