EXCEL - Research projects
The role of chronic inflammation in the development of hematologic neoplasms
Although numerous genetic alterations have been described in patients with myeloproliferative neoplasms (MPN) over the past 10 years, the molecular etiology of these heterogeneous diseases is still poorly understood. For example, around 50% of all patients with primary myelofibrosis (PMF) carry a mutation in the JAK2 gene, JAK2V617F, which leads to constitutive activity of this kinase. Recently, the first JAK2 inhibitor, ruxolitinib (Jakavi®), was approved for the treatment of PMF patients. Surprisingly, it shows the same efficacy in both patients with and without JAK2V617F mutation. Since PMF patients have highly elevated serum levels of inflammatory cytokines and ruxolitinib also inhibits JAK1 kinase, it is postulated that an inflammatory milieu in the bone marrow niche, which would be inhibited by ruxolitinib, contributes significantly to the etiology of MPN. In preliminary work, AG Pahl has shown that chronic inflammation in a mouse model (ITCH-/- mice) is sufficient to cause MPN, even without a JAK2 mutation.
Hypothesis: Chronic inflammation of the bone marrow niche, which is sufficient to initiate and maintain myeloproliferation, leads to the accumulation of somatic mutations that cause MPN.
The role of PD-L1 in the therapy of hematologic neoplasms
Acute graft-versus-host disease (GvHD) is a major complication in patients who receive an allo-HCT (hematologic stem cell transplant) due to their leukemia. In this case, transplanted immune cells attack the patient's tissue and can cause damage leading to death. A better understanding of the development of the disease and the cell populations involved could improve the survival of patients after allo-HCT. There is evidence that the gastrointestinal tract is a central site for the development of GvHD. Our preliminary data show that there is a release of proinflammatory adenosine triphosphate (ATP) in the gastrointestinal tract after allo-HCT and that infiltration by various cells of so-called innate immunity can be detected. The extent to which these cell populations contribute to GvHD and influence each other has not yet been clarified. The investigations in the mouse model are to be supplemented by tissue analyses of patients who have undergone allogeneic transplantation and thus evaluate the possible clinical relevance.
Hypotheses: 2 hypotheses will be tested: A: The release of chemokines by the presenting cells leads to the recruitment of donor T cells into the gastrointestinal tract. B: The formation of reactive oxygen compounds by neutrophil granulocytes and the associated tissue damage contribute directly to intestinal GvHD.
JAK/STAT and T-cell receptor signaling pathways as therapeutic targets for the treatment of T-cell lymphomas and paraneoplastic inflammatory phenomena
Peripheral T-cell lymphomas (PTCL-NOS), angioimmunoblastic T-cell lymphomas and Sézary syndromes are highly aggressive malignant diseases with a very poor prognosis. Resistance to standard chemotherapies and the lack of targeted therapeutic approaches result in a 5-year survival rate of less than 30 %. In preliminary work, the Dierks group developed mouse models that recapitulate human T-cell lymphoma diseases (xenografts, transgenic models and bone marrow transplantation models). Among other things, overexpression of the ITK-SYK oncogene in mouse T cells can trigger peripheral T-cell lymphoma with massive paraneoplastic inflammation. The malignant T-cell population in this model, but also in various human xenograft models, exhibits constitutive activation of the JAK/STAT and T-cell receptor (TCR) signaling pathways, which are controlled by mutations, translocations or stimuli from the microenvironment surrounding the lymphoma cells. The JAK/STAT signaling pathway is not only activated in the T cells, but also in the surrounding inflammatory effector cells, such as granulocytes and macrophages, due to massive cytokine secretion. Interestingly, initial experiments in ITK-SYK mice show that inhibition of the JAK/STAT signaling pathway by the tyrosine kinase inhibitor ruxolitinib can suppress the development of lethal disease in an ITK-SYK-driven PTCL model and completely abrogate the inflammatory phenotype. Direct inhibition of granulocytes also leads to suppression of the inflammatory phenotype and to a significant prolongation of survival.
Hypothesis: Inhibition of the JAK/STAT or TCR signaling pathway in patients with PTCL or Sézary syndrome reduces organ infiltration with malignant T cells and improves the inflammatory phenotype and thus represents a promising therapeutic option.
Characterization of the tumor-specific CD8+ T cell response in patients with HCC
HCC is one of the most common malignant tumors worldwide. As most HCCs are detected late and the often underlying liver architecture disorder limits the therapeutic options, treatment of the disease remains difficult and the prognosis unfavorable. The median survival time without therapy is around 6 months. The tumor-specific immune response is thought to play an important role in the development and progression of HCC. In preliminary work, the Thimme working group was able to show that a strong and multispecific CD8+ T-cell response against various tumor antigens is associated with better long-term survival. Interestingly, however, the tumor-specific CD8+ T cell responses show defects in their functions, in particular proliferation and cytokine production, compared to virus-specific CD8+ T cells from the same patients. The mechanisms for tumor-specific CD8+ T cell failure are not yet fully understood, but inhibitory receptors, such as PD-1, which are already being used clinically in phase I and phase II trials in HCC, are thought to play an important role in suppressing the CD8+ T cell response.
Hypothesis: The expression of inhibitory receptors on tumor-specific CD8+ T cell responses contributes to the functional failure of the tumor-specific immune response and represents an important therapeutic approach for the treatment of HCC.
Functional modulation of HCC-specific CD4 T cell responses by OX40 stimulation and PD-1 blockade
Checkpoint inhibition, such as the blockade of PD-1 or CTLA-4 signaling pathways, is already clinical practice in various tumor entities, sometimes with synergistic effects in combination therapy. OX40 (CD134) is a costimulatory molecule that is of crucial importance for an effective immune response. Preliminary work of the Böttler group has shown that OX40 mediates the maintenance of the antiviral CD4 T cell response in a mouse model of chronic viral infection. In particular, follicular T helper cells (Tfh cells), specialized CD4 T cells that provide B cell help, are dependent on OX40 signals for their differentiation and function. Furthermore, unpublished data have shown that simultaneous OX40 stimulation combined with PD-1 blockade leads to a strengthening of the human antigen-specific CD4 T cell response against various viral antigens. Therefore, it should be investigated whether OX40, in combination with PD-1, also represents a possible target structure in the therapy of HCC.
Hypothesis: Stimulation of OX40, in combination with therapeutic check-point blockade, improves the tumor-specific CD4 T cell response in patients with HCC.
Influence of B cells on the tumor-specific CD8+ T cell response in HCC
With regard to the treatment of HCC, surgical resection is a curative treatment option. Due to the central importance of the immunological tumor microenvironment for the long-term prognosis after HCC resection, parallel projects are currently underway to investigate cellular subtypes of tumor-infiltrating immune cells and their specific function. This comprehensive examination of the tumor microenvironment will make it possible to develop specific immunotherapy and also apply it to patients. For this purpose, HCC-infiltrating immune cells will be isolated from surgical specimens and analyzed for antigen expression by flow cytometry. Functional analyses are carried out in co-culture of subtypes of immune cells with HCC tumor organoids. Ongoing projects include the analysis of antigen-presenting cells, functional subtypes of T helper lymphocytes and the further processing of cytotoxic T lymphocytes and cytotoxic cells of the innate immune system. With reference to the cytotoxic activity of CD8+CD62L-KLRG1+CD107a+ effector memory lymphocytes, the mechanisms of immune evasion through the interaction of HCC tumor cells with cytotoxic cells are currently being investigated with the aim of precisely determining the specific checkpoint antigens (PD-1, CTLA-4, GITR, 4-IBB, OX4-O and TIGIT). This involves close cooperation with the P4 and P5 projects.
Hypotheses: A: Specific subsets of HCC-infiltrating B lymphocytes influence the differentiation of CD8+CD62L-KLRG1+CD107a+ effector memory lymphocytes and increase antigen-specific cytotoxicity against HCC. B: Humoral factors produced by B lymphocytes mediate this effect on the differentiation of CD8+CD62L-KLRG1+CD107a+ effector memory lymphocytes.
Abscopal effects of local radiotherapy in combination with immunotherapy in metastatic malignant melanoma and HCC
In recent years, it has become apparent that certain forms of local radiotherapy can have an immunostimulating effect. Preclinical studies have shown that hypofractionated radiotherapy induces tumor-specific T cells in some tumor models. Particularly in combination with immunotherapeutic agents, such as immune checkpoint inhibitors, this can not only improve local tumor control in some cases, but also control distant metastases (abscopal response). The combination with other immunotherapeutic agents also appears promising. However, the optimal radiation dose and fractionation as well as other requirements for optimal immunostimulation are not yet sufficiently known. The Niedermann group has investigated the immunostimulatory effects of hypofractionated radiotherapy in syngeneic mouse melanoma models. In addition, the extent to which hypofractionated radiotherapy and PD-1 checkpoint blockade or bispecific T-cell-recruiting antibodies act synergistically was investigated. Synergistic effects were found in combination with anti-PD-1 antibodies, but not in combination with bispecific T-cell recruiting antibodies. PD-1 and PD-L1-specific PET tracers were developed for non-invasive visualization of the effects of combined radiotherapy and immunotherapy.
Hypothesis: Immunostimulatory and immunomodulatory effects of certain forms of local radiotherapy may improve local and systemic tumor control in malignant melanoma in combination with immunotherapeutics.
Molecular mechanisms of immune-mediated RAS-associated leukoproliferative disease (RALD) and juvenile myelomonocytic leukemia (JMML)
Adult cancers usually exhibit 50 to well over 100 non-synonymous mutations. In contrast, childhood leukemias and tumors show only a few genetic alterations. In juvenile myelomonocytic leukemia (JMML), an aggressive neoplasia of infancy, there is usually only one mutation affecting one of five genes (KRAS, NRAS, NF1, PTPN11 or CBL) on the RAS-MAP kinase signaling pathway. While all alterations lead to hyperactivation of the signaling pathway, the clinical manifestation and recurrence rates after hematopoietic stem cell transplantation of the molecular subtypes are different. In addition to genetic changes, epigenetic modifications play a significant role in the JMML phenotype. For example, the Niemeyer group was able to describe an aberrant DNA methylation pattern of 4 genes that predicts an unfavorable prognosis of the disease. The importance of epigenetic changes for the pathogenesis of JMML is underlined by recent observations showing that a demethylating substance as monotherapy can induce a clinical remission of JMML. In the absence of in vitro culture systems, AG Niemeyer has developed a murine xenograft model in which the epigenetic pattern of JMML stem cells is preserved during serial transplantations.
Hypothesis: The five genetic subtypes of JMML show different epigenetic profiles in the xenograft model and a different extent of depletion of leukemic stem cells after treatment with demethylating substances.
The importance of the tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO) in the primary and secondary resistance of immunotherapy for malignant melanoma
Malignant melanoma can be effectively treated using approaches that aim to specifically stimulate the immune response. The blockade of the inhibitory molecules CTLA-4, PD1/2 or their ligands, as well as the inhibition of the tolerance-inducing enzyme indoleamine 2,3-dioxygenase (IDO) are proven effective pharmacological therapies, especially in combination, and are currently undergoing clinical development/testing. The enzyme IDO degrades the essential amino acid tryptophan in the tissue and thus blocks the proliferation of T cells; in addition, regulatory T cells are produced. These two effects prevent an efficient immune response. IDO-deficient mice treated with anti-CTLA-4 antibodies show significantly slower growth and higher survival rates than wild-type mice. In the mouse model, it is clear that IDO can be produced either by melanomas or by immune cells and has an immunosuppressive effect in both cases. All available data from mice as well as initial phase I studies with an IDO inhibitor in humans suggest that this also applies to melanoma patients. Bubnoff's group was able to show that the expression of IDO in human melanomas correlates significantly with the tumor thickness of the melanoma. IDO expression in melanoma cells and in antigen-presenting cells such as macrophages and dendritic cells in the tumor microenvironment also correlates with the strength of the inflammatory infiltrate around the melanoma. Interestingly, IDO is also seen in very early melanomas at the exact site of tumor development. IDO is therefore involved early, but also constantly in the development of melanoma. The extent to which IDO is causally involved in melanoma development is an open question.
Hypotheses: A: Increasing activity of IDO in serum indicates recurrence. B: The expression of IDO is an essential resistance mechanism in the context of immunotherapy with CTLA-4 or PD-1 inhibitors in melanoma. The production of IDO has a counter-regulatory effect on the therapeutically induced T cell response.
Changes in immunosurveillance in patients with primary immunodeficiency: mechanisms of lymphoma development
Primary immunodeficiencies (PID) are rare diseases of the immune system that provide a unique view of the human immune system due to the underlying monogenetically defined disorders. Frequent tumor development is observed in particular in those PIDs in which the homeostasis of immune cells (proliferation and cell death) is disturbed. This indicates that changes in the same signaling pathways can contribute to susceptibility to infection and autoimmunity as well as to tumor development. The Ehl WG studies patients with autoimmune lymphoproliferative immunodeficiencies (AL-PID), a clinical phenotype associated with germline or somatic mutations in a number of genes, e.g. dominant negative mutations in FAS or activating mutations in PIK3CD, STAT-1 or STAT-3. Somatic mutations in these genes are also found in lymphomas and approximately 10% of AL-PID patients with germline mutations in these genes develop lymphomas. In recent years, the Ehl group has investigated lymphocyte differentiation and cellular programming in patients with Fas defects and identified new gene defects in genome-wide studies. The hypothesis is that proto-oncogenic pathway alterations and the resulting changes in cellular and metabolic programming may also be the basis of benign lymphoproliferation, autoimmunity and immunodeficiency.
Hypothesis: Germline mutations in genes that cause immunodeficiency and autoimmunity through disturbed T-cell homeostasis are often also susceptibility genes for lymphoma, indicating common pathogenicity pathways.
Tumor microenvironment-mediated reduction of T-cell activity through nutrient deprivation
In patients with chronic diseases, e.g. cancer, cachexia often occurs, which is associated with high morbidity and mortality. It has recently been shown that cancer cells secrete factors that lead to a pronounced increase in an energetically useless cycle of fatty acid synthesis and fatty acid oxidation in white adipose tissue. The same tumor-secreted factors lead to increased fatty acid oxidation and cellular stress in muscle cells. These changes are causally involved in cachexia, as it was recently shown that pharmacological inhibition of fatty acid oxidation prevented tumor-associated cachexia in mouse models. A key step in tumor progression is cancer cell-induced modulation of the immune response, which inactivates potentially effective immune cells. Our preliminary work suggests that metabolic changes are integrally involved in this modulation of the immune system. Indeed, "checkpoint blockade" therapy, in which the immune system is reactivated, also revises metabolic changes in effector T cells. Growing and metastatic tumors are infiltrated by either activated macrophages or regulatory T cells (T-regs), which play an important role in tumor progression. In both cell types, fatty acid oxidation is the central metabolically active metabolic pathway. Pharmacologic inhibition of fatty acid oxidation leads to loss of immune function in both macrophages and T-regs.
Hypothesis: The same factors secreted by cancer cells cause both cachexia and inactivation of the immune response through metabolic changes.
EXCEL Research College
Prof. Dr. H. Pahl / Prof. Dr. R. Thimme
+49 (0) 761 270-61060