Medizinische Klinik
Innere Medizin II
Hämatologie, Onkologie, klinische Immunologie und Rheumatologie

2. Identification of new gene mutations in patients with inherited neutropenia and leukemia using NGS. 

We have identified very high frequency of cooperative acquired mutations in CSF3R and RUNX1 in severe congenital neutropenia patients who overt leukemia.

 3. Understanding the mechanisms of G-CSF-triggered myeloid differentiatio. 

We found that Lymphoid Enhancer-binding Factor 1 (LEF-1) was severely down-regulated in granulocytic progenitors of CN patients, inducing maturation arrest of granulopoiesis at the promyelocyte stage due to defective activation of C/EBPa (Nature Medicine, 2006). We also have demonstrated that LEF-1 is downregulated in CN patients via enhanced ubiquitination and degradation of LEF-1 protein by hyperactivated STAT5 (Blood, 2014).

We also described Hematopoietic Cell-specific Lyn-Substrate 1 (HCLS1) protein as an important palyer in the G-CSFR-triggered granulocytic differentiation of hematopoietic cells in vitro and in vivo.

HCLS1 is highly expressed in human myeloid cells and treatment with G-CSF resulted in the phosphorylation and activation of HCLS1 protein. HCLS1 together with its binding partner HAX1 (HCLS1-associated protein X 1) interacts with the LEF-1 transcription factor, inducing nuclear translocation of LEF-1 and activation of LEF-1 target genes. These events are essential for myeloid differentiation. In patients with severe congenital neutropenia (CN) HCLS1 expression and functions are severely downregulated leading to severe defects in LEF-1 and LEF-1 target genes expression followed by defective granulopoiesis (Nature Medicine, 2012).

We study the mechanisms of down-regulation of HCLS1 in CN patients in contrast with mechanisms and consequences of hyper-activated HCLS1 protein in myeloid leukemia.

 4. Hematopoietic differentiation of iPS cells. 

iPS cells are reprogrammed somatic cells with embryonic stem (ES) cell-like characteristics produced by the introduction of specific transcription factors into somatic cells. ES/iPS cells can help to elucidate the process of normal embryogenesis and differentiation process on several lineages, especially in early stages. On the other hand, it is believed that iPS cell technology, which generates disease-specific pluripotent stem cells in combination with directed cell differentiation. We have established neutrophil differentiation systems from human iPS cells using stroma cell co-culture system (Morishima T, et al. J Cell Physiol. 2011) and serum- and feeder-free monolayer culture system (Morishima, T. et al. Haematologica. 2014). We generated iPS cell lines from a severe congenital neutropenia (SCN) patient with HAX1 gene deficiency and showed that in vitro differentiation of these patient-derived iPS cells recapitulate the hematological phenotype in the patient. Furthermore, we corrected for the HAX1 gene deficiency in patient-derived iPS cells by lentiviral transduction with HAX1 cDNA and successfully reserved the disease presentation in gene-corrected cells (Morishima, T. et al. Haematologica. 2014). These results shows our culture system combined with lentiviral gene transduction will serve as a useful tool to facilitate disease modeling for myeloid cell lineages. We are now investigating the mechanism of normal myeloid cell differentiation and disease pathophysiology in myeloid cell lineage using iPS cell technology.

 5. Post-translational modification of proteins by de-/acetylation and its role in hematopoietic differentiation and leukemogenic transformatio. 

Posttranslational modification (PTM) is crucial for regulating the functions of many eukaryotic proteins. Among the prominent PTMs are phosphorylation; lysine acetylation, ubiquitination and methylation. In the past decade, Lysine acetylation has been found in a variety of proteins but not studied as widespread as phophorylation. Post-translational protein de-/acetylation lead to activation or deactivation of their functions. Besides, Lysine acetylation dependent on the con­text could interplay with other PTMs in an agonistic or antagonistic manner.

We recently identified nicotinamide phosphoribosyltransferase (NAMPT), as an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis. G-CSF treatment of both healthy individuals and congenital neutropenia patients increase Intracellular NAMPT as well as plasma levels. NAMPT activates NAD(+)-dependent Sirtuins which are responsible for post-translational deacetylation of many transcription factors and adaptor proteins (Nature Medicine, 2009).

We also have shown that LEF-1 is deacetylated and deactivated by SIRT1, which is NAD+-dependent protein deacetylase. We also study the effects of LEF-1 de-/acetylation on ubiquitination. We investigate new therapeutic approaches using modulation of LEF-1 acetylation status (treatment with NAMPT, vitamin B3 and NAMPT inhibitor) for improvement/correction of hematopoietic differentiation in healthy individuals, CN, CN/AML and de novo AML patients.

We further study the mechanisms of down-regulation of HCLS1 in CN patients in contrast with mechanisms and consequences of hyper-activated HCLS1 protein in myeloid leukemia. We focus on GCSF-triggered activation of HCLS1 via NAD+/NAMPT/SIRT deacetylation and cross-talk between phosphorylation and de-/acetylation in HCLS1 activation.

 6. In vivo modeling of leukemia and neutropenia in humanized NGS mice 

We are in the process to establish humanized NGS mouse model for leukemia and neutropenia.

 7. Evaluation of the role of IFNß signaling and neutrophils in inflammation and tumorigenesis. The mechanism of type I interferon-mediated polarization of tumor-associated neutrophils in mice and human. 

Neutrophils are the most abundant of all white blood cells and play a key role in host inflammatory responses. Importantly, inflammation has been associated with increased susceptibility for cancer and neutrophils, as a crucial component of this process, play essential role in inflammation-driven tumorigenesis. These cells represent also an independent prognostic marker in a broad variety of neoplasias e.g. high number of intra-tumoral neutrophils in localized as well as metastatic renal cell carcinoma, correlates with a negative prognosis.

The tumor microenvironment represents a special niche that is extremely influencing infiltrating immune cells. The concept of immune cell polarization was first described for macrophages (anti-tumor M1/pro-tumor M2). Recently neutrophil polarization have been postulated, since these cells appear to have diverse functions in the tumor microenvironment including such that promote (N2) or inhibit (N1) tumor growth.

Previously, we could show that significantly elevated numbers of neutrophils accumulate in tumors of mice that lack endogenous type I IFNs. Such TANs do not only efficiently support tumor angiogenesis and growth by up-regulating pro-angiogenic molecules (VEGF and MMP9), but also secrete higher amounts of neutrophil attracting chemokines and display prolonged survival, compared to their WT counterparts. Moreover, we could show that pro-tumor neutrophils efficiently support metastatic processes, due to up-regulation of pro-metastatic proteins, like Bv8, MMP9, S100A8 and S100A9 and inhibition of direct killing of tumor cells by neutrophils. Altogether, our data revealed IFN-β as N1 promoting cytokine.

Here, we add further evidence emphasizing the importance of type I IFNs for neutrophil polarization in tumor microenvironment and reveal possible mechanism responsible for this phenomenon. In Ifnb1-/- mice, we observe a significant down-regulation of anti-tumor neutrophil markers, like ICAM1 and TNF-α. Moreover, neutrophils show reduced formation of NETs, accompanied by lower tumor killing capacity. Under these conditions, massively enhanced neutrophil turnover in combination with accumulation of immature neutrophils is observed. Importantly, therapeutic intervention in mice using low dose IFN-β, induced anti-tumor activation of neutrophils in tumors and in pre-metastatic lungs. Correspondingly, in human melanoma patients undergoing type I IFN therapy, neutrophil anti-tumor characteristics were augmented, suggesting effective outcome of therapy that should be further investigated in order to optimize therapeutic use of type I IFNs.

Elevated neutrophils numbers in Ifnb1-/-mice are probably due to significant upregulation of G-CSF expression in neutrophils from different anatomical compartments in tumor-bearing Ifnb1-/-mice. Consistent with this, significantly elevated serum levels of G-CSF are observed. Importantly, we could recently show that G-CSF induces synthesis of NAMPT, which is a rate-limiting enzyme converting nicotinamide (NA) into NAD+that in turn activates NAD+-dependent protein deacetylases sirtuins (SIRTs). NAMPT serves as an inhibitor of neutrophil apoptosis and as neutrophil chemoattractant (CXCL8 upregulation in human). It is a potent pro-inflammatory factor (upregulation of ROS release) and pro-angiogenic factor (smooth muscle maturation). Our results show not only elevated levels of G-CSF, but also NAMPT and SIRT1 in Ifnb1-/- tumor bearing mice. It correlates with enhanced tumor angiogenesis, growth and metastasis.

Since tumor associated neutrophils (TANs) represent a highly potent therapeutic target, these data highlight the therapeutic potential of interferons and NAMPT inhibitors, suggesting optimization of their clinical use as potent anti-tumor agent.

Proteins have numerous functions in the human body. For example, a specific group called cytokines plays an important role in the production of blood cells. These cells are formed in the bone marrow from stem cells which are regulated by cytokines inducing cell division, cell growth and maturation into mature blood cells. Together with Dr. M. ElGamacy who is jointly working at Max Planck Institute for Biology, and Division of Translational Oncology, at the UKT, the group of Prof. Dr. Julia Skokowa, Head of the Division for Translational Oncology of the Department of Internal Medicine II of the University Hospital Tübingen, aimed to solve the problem of the highly sparse availability of cytokines. They create new cytokines for treating human diseases such as hematopoietic stem cell disorders – inherited and acquired immunodeficiencies and leukemia. Steven Pohl, Communications Specialist at the University Hospital Tübingen, had the opportunity to talk with Prof. Skokowa on new study findings which have recently been published in the renowned scientific journal Nature communications.