Overlapping Stromal Alterations in Myeloid and Lymphoid Neoplasms.

Myeloid and lymphoid neoplasms share the characteristics of potential bone marrow infiltration as a primary or secondary effect, which readily leads to hematopoietic insufficiency. The mechanisms by which clonal malignant cells inhibit normal hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) have not been unraveled so far. Given the pivotal role of mesenchymal stromal cells (MSCs) in the regulation of hematopoiesis in the BM niche it is assumed that MSCs also play a relevant role in the pathogenesis of hematological neoplasms. We aimed to identify overlapping mechanisms in MSCs derived from myeloid and lymphoid neoplasms contributing to disease progression and suppression of HSPCs to develop interventions that target these mechanisms. MSCs derived from healthy donors (n = 44) and patients diagnosed with myeloproliferative neoplasia (n = 11), myelodysplastic syndromes (n = 16), or acute myeloid leukemia (n = 25) and B-Non-Hodgkin lymphoma (n = 9) with BM infiltration and acute lymphoblastic leukemia (n = 9) were analyzed for their functionality and by RNA sequencing. A reduced growth and differentiation capacity of MSCs was found in all entities. RNA sequencing distinguished both groups but clearly showed overlapping differentially expressed genes, including major players in the BMP/TGF and WNT-signaling pathway which are crucial for growth, osteogenesis, and hematopoiesis. Functional alterations in healthy MSCs were inducible by exposure to supernatants from malignant cells, implicating the involvement of these factors in disease progression. Overall, we were able to identify overlapping factors that pose potential future therapeutic targets.

Molecular and epigenetic ex vivo profiling of testis cancer-associated fibroblasts and their interaction with germ cell tumor cells and macrophages.

Germ cell tumors (GCT) are the most common solid tumors in young men of age 15 - 40. In previous studies, we profiled the interaction of GCT cells with cells of the tumor microenvironment (TM), which showed that especially the 3D interaction of fibroblasts (FB) or macrophages with GCT cells influenced the growth behavior and cisplatin response as well as the transcriptome and secretome of the tumor cells, suggesting that the crosstalk of these cells with GCT cells is crucial for tumor progression and therapy outcome. In this study, we shed light on the mechanisms of activation of cancer-associated fibroblasts (CAF) in the GCT setting and their effects on GCT cells lines and the monocyte cell line THP-1. Ex vivo cultures of GCT-derived CAF were established and characterized molecularly and epigenetically by performing DNA methylation arrays, RNA sequencing, and mass spectrometry-based secretome analysis. We demonstrated that the activation state of CAF is influenced by their former prevailing tumor environment in which they have resided. Hereby, we postulate that seminoma (SE) and embryonal carcinoma (EC) activate CAF, while teratoma (TER) play only a minor role in CAF formation. In turn, CAF influence proliferation and the expression of cisplatin sensitivity-related factors in GCT cells lines as well as polarization of in vitro-induced macrophages by the identified effector molecules IGFBP1, LGALS3BP, LYVE1, and PTX3. Our data suggests that the vital interaction of CAF with GCT cells and with macrophages has a huge influence on shaping the extracellular matrix as well as on recruitment of immune cells to the TM. In conclusion, therapeutically interfering with CAF and / or macrophages in addition to the standard therapy might slow-down progression of GCT and re-shaping of the TM to a tumor-promoting environment. Significance: The interaction of CAF with GCT and macrophages considerably influences the microenvironment. Thus, therapeutically interfering with CAF might slow-down progression of GCT and re-shaping of the microenvironment to a tumor-promoting environment.

New synergistic combination therapy approaches with HDAC inhibitor quisinostat, cisplatin or PARP inhibitor talazoparib for urothelial carcinoma.

Urothelial carcinoma (UC) urgently requires new therapeutic options. Histone deacetylases (HDAC) are frequently dysregulated in UC and constitute interesting targets for the development of alternative therapy options. Thus, we investigated the effect of the second generation HDAC inhibitor (HDACi) quisinostat in five UC cell lines (UCC) and two normal control cell lines in comparison to romidepsin, a well characterized HDACi which was previously shown to induce cell death and cell cycle arrest. In UCC, quisinostat led to cell cycle alterations, cell death induction and DNA damage, but was well tolerated by normal cells. Combinations of quisinostat with cisplatin or the PARP inhibitor talazoparib led to decrease in cell viability and significant synergistic effect in five UCCs and platinum-resistant sublines allowing dose reduction. Further analyses in UM-UC-3 and J82 at low dose ratio revealed that the mechanisms included cell cycle disturbance, apoptosis induction and DNA damage. These combinations appeared to be well tolerated in normal cells. In conclusion, our results suggest new promising combination regimes for treatment of UC, also in the cisplatin-resistant setting.

The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Characterization of the dehydrogenase-reductase DHRS2 and its involvement in histone deacetylase inhibition in urological malignancies.

BACKGROUND: Being implicated during tumor migration, invasion, clonogenicity, and proliferation, the nicotinamide adenine dinucleotide (NAD)/-phosphate (NADP)-dependent dehydrogenase/reductase member 2 (DHRS2) has been considered to be induced upon inhibition of histone deacetylases (HDACi). In this study, we evaluated the current knowledge on the underlying mechanisms of the (epi)genetic regulation of DHRS2, as well as its function during tumor progression. METHODS: DHRS2 expression was evaluated on mRNA- and protein-level upon treatment with HDACi by means of qRT-PCR and western blot analyses, respectively. Re-analysis of RNA-sequencing data gained insight into expression of specific DHRS2 isoforms, while re-analysis of ATAC-sequencing data shed light on the chromatin accessibility at the DHRS2 locus. Further examination of the energy and lipid metabolism of HDACi-treated urologic tumor cells was performed using liquid chromatography-mass spectrometry. RESULTS: Enhanced DHRS2 expression levels upon HDACi treatment were directly linked to an enhanced chromatin accessibility at the DHRS2 locus. Particularly the DHRS2 ENST00000250383.11 protein-coding isoform was increased upon HDACi treatment. Application of the HDACi quisinostat only mildly influenced the energy metabolism of urologic tumor cells, though, the analysis of the lipid metabolism showed diminished sphingosine levels, as well as decreased S1P levels. Also the ratios of S1P/sphingosine and S1P/ceramides were reduced in all four quisinostat-treated urologic tumor cells. CONCLUSIONS: With the emphasis on urologic malignancies (testicular germ cell tumors, urothelial, prostate, and renal cell carcinoma), this study concluded that elevated DHRS2 levels are indicative of a successful HDACi treatment and, thereby offering a novel putative predictive biomarker.

Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR.

Ciliary neurotrophic factor (CNTF) activates cells via the non-signaling α-receptor CNTF receptor (CNTFR) and the two signaling ß-receptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). The CNTF derivate, Axokine, was protective against obesity and insulin resistance, but clinical development was halted by the emergence of CNTF antibodies. The chimeric cytokine IC7 used the framework of interleukin (IL-)6 with the LIFR-binding site from CNTF to activate cells via IL-6R:gp130:LIFR complexes. Similar to CNTF/Axokine, IC7 protected mice from obesity and insulin resistance. Here, we developed CNTF-independent chimeras that specifically target the IL-6R:gp130:LIFR complex. In GIL-6 and GIO-6, we transferred the LIFR binding site from LIF or OSM to IL-6, respectively. While GIO-6 signals via gp130:IL-6R:LIFR and gp130:IL-6R:OSMR complexes, GIL-6 selectively activates the IL-6R:gp130:LIFR receptor complex. By re-evaluation of IC7 and CNTF, we discovered the Oncostatin M receptor (OSMR) as an alternative non-canonical high-affinity receptor leading to IL-6R:OSMR:gp130 and CNTFR:OSMR:gp130 receptor complexes, respectively. The discovery of OSMR as an alternative high-affinity receptor for IC7 and CNTF designates GIL-6 as the first truly selective IL-6R:gp130:LIFR cytokine, whereas GIO-6 is a CNTF-free alternative for IC7.

Stromal alterations in patients with monoclonal gammopathy of undetermined significance, smoldering myeloma, and multiple myeloma.

ABSTRACT: The hallmark of multiple myeloma (MM) is a clonal plasma cell infiltration in the bone marrow accompanied by myelosuppression and osteolysis. Premalignant stages such as monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic stages such as smoldering myeloma (SMM) can progress to MM. Mesenchymal stromal cells (MSCs) are an integral component of the bone marrow microenvironment and play an important role in osteoblast differentiation and hematopoietic support. Although stromal alterations have been reported in MM contributing to hematopoietic insufficiency and osteolysis, it is not clear whether alterations in MSC already occur in MGUS or SMM. In this study, we analyzed MSCs from MGUS, SMM, and MM regarding their properties and functionality and performed messenger RNA sequencing to find underlying molecular signatures in different disease stages. A high number of senescent cells and a reduced osteogenic differentiation capacity and hematopoietic support were already present in MGUS MSC. As shown by RNA sequencing, there was a broad spectrum of differentially expressed genes including genes of the BMP/TGF-signaling pathway, detected already in MGUS and that clearly increases in patients with SMM and MM. Our data may help to block these signaling pathways in the future to hinder progression to MM.