Research projects

OPUS13-Principal investigator: Prof. Jakub Golab

Determination of arginase-1 as a potential drug target in the treatment of multiple myeloma and in reducing proteasome inhibitor-mediated cardiotoxicity

 Multiple myeloma (MM) is a tumor originating from the terminally differentiated B cells. It develops mainly in elderly patients and leads to compromised immune system, hypercalcemia, and end-organ damage typically encompassing renal failure, anemia, or bone lesions. Although the therapeutic efficacy of novel drugs for MM has improved over recent years MM remains a largely incurable disease, with a median survival of up to 6 years. Intriguingly, the improvements in anticancer efficacy of novel therapeutic approaches are associated with increased rate of other organ injuries. For example, cardiovascular diseases resulting from  anticancer treatments have now become the second leading cause of long-term morbidity among cancer survivors. Therefore, identification of combinations of therapeutic approaches that would exert potentiated antitumor effects and at the same time would specifically reduce treatment-related toxicity are of paramount importance. We have previously shown that bortezomib, which is a proteasome inhibitor used in the treatment of MM patients, is cardiotoxic. It seems that at least to some extent the bortezomib-associated cardiotoxicity is related to decreased nitric oxide (NO) synthesis in endothelial cells. Our unpublished results indicate that sildenafil, which is triggering NO signaling pathway, is ameliorating bortezomib-induced cardiotoxicity, further confirming potentially beneficial effects of NO. Nitric oxide is produced in enzymatic reaction from an amino acid L-arginine. Coincidentally, L-arginine is also a substrate for arginase-1 (Arg-1), an enzyme involved in L-arginine degradation. In our preliminary studies, we have observed that Arg-1 inhibitor co-developed by our research team is increasing NO concentrations in mice. Therefore, we wish to investigate whether turning off Arg-1 activity might reduce bortezomib-induced cardiotoxicity, by providing increased L-arginine concentrations for NO synthesis. Moreover, the published data as well as our preliminary findings demonstrate that Arg-1 is interfering with the development of effective immune response against various types of tumors. However, the role of Arg-1 has not been extensively studied in MM. Thus, using both genetically engineered models as well as Arg-1 inhibitor, we plan to determine whether Arg-1 is a potentially druggable target in the management of MM. We will investigate whether lack of or pharmacological inhibition of Arg-1 is regulating anti-MM immune response, affecting the rate of MM progression and potentiating antitumor effects of bortezomib. This knowledge is necessary to better understand molecular mechanisms involved in the development of MM-associated immune response. The results of this project might also be helpful in identifying novel targets for cancer treatment, better understanding of the shortcomings and adverse effects observed in cancer patients undergoing cancer treatment and in finding novel areas to be exploited in the fields of immunology, cardiooncology and experimental oncology.

DIAMENTOWY GRANT – Principal investigator: Paweł Matryba

Evaluation of the arginase-1 inhibition on the development of the antigen-specific immune response in the lymph nodes

Tumor immunotherapy has recently become an effective approach, leading to the complete remission in 20-30% of patients. However, majority of the patients remains unresponsive encouraging the research on the potential mechanisms of immunotherapy resistance. One of such mechanisms might be the enzymatic activity of arginase, the L-arginine degrading enzyme. Decreased L-arginine concentration in the tumor microenvironment and/or in tumor-draining lymph nodes inhibits lymphocytes proliferation via downregulation of CD3ζ and CD3ε molecules, crucial signaling components of the antigen-recognizing T-cell receptor (TCR). In our preliminary studies we observed that arginase inhibits T-cells proliferation while arginase inhibitor reverses this effect in cell culture. The main aim of this project is to investigate the effects of arginase-1 inhibitor on the development of the antigen-specific immune response in a fully novel model of transparent lymph nodes. We will develop an optical clearing method resulting in fully transparent lymph nodes which will enable fast, whole organ microscopic examination in 3D. We will use in our studies DsRed fluorescent protein expressing T-cells isolated from transgenic mice to localize these cells in detail and to generate their spatial distribution map in a whole organ not undergoing histological sectioning. In the future, this new method might overcome the limitations associated with tissue sectioning. Moreover, it will allow us to precisely evaluate the immunostimulatory effects of arginase-1 inhibitor in T-cells. Finally, the result of our project will complement the existing evidence supporting the future clinical use of arginase inhibitors in combination with other immunotherapy approaches.

HARMONIA (2015-2020) - Principal investigator: dr Tomasz Stokłosa

Identification of new, clinically relevant genetic and epigenetic aberrations by next-generation sequencing in chronic myeloid leukemia patients with poor disease outcome

Chronic myeloid leukemia (CML) is a model neoplasm, studies on this disease resulted in landmark discoveries and started the era of targeted therapy in oncology; for the first time in CML genetic abnormality was linked to cancer, also for the first time studies on the pathogenesis of this disease  led to the successful introduction of targeted therapy with small molecule tyrosine kinase inhibitors (TKI). Unfortunately, significant number of patients develop resistance to TKI another problem in CML is inability to cure CML due to the intrinsic resistance of small fraction of cells, called leukemic stem cells . Genetic events underlying progression of the disease are still poorly understood. Major aim of the proposed research project is to characterize genetic and epigenetic events which may drive the progression of the disease and which may be responsible for the resistance to targeted therapy with high-throughput sequencing technology. In the next steps selected abnormalities are examined to determine their biological role in disease progression and the experimental approach to target newly discovered abnormalities is performed.

In the proposed project high-throughput, next-generation sequencing is used to describe genomic and epigenomic landscape of CML evolution. Targeted enrichment strategy for initial screening followed by whole-exome enrichment and global methylome analysis is applied. After selection experimental in vitro evaluation of candidate therapeutic targets in various chronic myeloid leukemia models are performed followed by testing of novel experimental compounds to target newly discovered aberrations preselected by earlier in vitro validation.

Although high -throughput sequencing became leading tool in cancer genetics there are several rare neoplasms which still are poorly characterized in the terms of global genomic landscape, such as CML, especially considering genetic events underlying disease progression. Characterization of genetic and epigenetic events which may drive the progression of the disease and which may be responsible for the resistance to targeted therapy may translate not only into better understanding of the molecular pathogenesis of CML but may also help in the future to find new better therapy for patients who do not benefit from TKI and effectively target leukemic stem cells.

HARMONIA grant allows not only to continue long-term, fruitful scientific collaboration (between Dr. Stoklosa and Dr. Skorski) but it helps in reaching new levels of scientific excellence. Synergy between two multidisciplinary teams and complementary fields of expertise and infrastructure gives high probability of important  scientific discoveries with translational potential upon implementation of the proposed project. Exchange of know-how and new methodology by visits of young scientists from Dr. Stoklosa group (mgr inż. Marcin Machnicki and mgr inż. Monika Pępek)  and exchange of ideas to pursue new scientific projects related to pathogenesis of leukemia in the future constitute an added value.

OPUS (2016-2020) - Principal investigator: dr Tomasz Stokłosa

Characterization of aberrant mechanisms of telomere maintenance in chronic myeloid leukemia - the role of POT1 and RAP1 in genomic instability in leukemic stem cells

 Telomerase activity and genomic instability belong to the hallmarks of cancer. However, the role of telomerase complex in genomic instability of human cancers is not fully understood. Chronic myeloid leukemia (CML) represents a model hematological malignancy characterized by increased genomic instability during disease progression and as a major cause of resistance to targeted therapies. Although there are reports describing differential expression of telomerase in different phases of CML, the causative mechanisms between telomere maintenance mechanisms and genetic events underlying progression of the disease are still poorly understood. Interestingly, chromosome changes are accompanied by telomere shortage. Telomeres as nucleoprotein structures located on chromosome ends are key regulators of genomic stability and telomerase is responsible for the maintenance of telomere length.. This important discovery by E. Blackburn, C. Greider and J. Szostak was awarded Nobel Prize in 2009 in physiology and medicine. However, telomeres may be also elongated by recombination-based alternative mechanism and immortal cancer cells are frequently characterized by the lack of telomerase expression/activity. This may suggest that the system regulating telomere homeostasis is more complex than previously thought. Elucidation the mechanisms controlling telomere status is important for better understanding of the causes of chromosome damage as well as for diagnostic and prognostic issues. Proposed research project aims at molecular characterization of the whole telomerase complex in different phases of the disease and in different leukemic cell populations, including leukemia stem cells to identify the potential role in drug resistance and progression of the disease. In the proposed project CML cell lines as well as primary leukemic cells from patients in different phases of the disease will be used to analyze expression of all known members of the telomerase complex at the RNA and protein level. Telomerase activity as well as telomere length will be measured. Different cytogenetic  methods including several modification of fluorescent in-situ hybridization will be applied to assess genomic instability. Also potential influence of different tyrosine kinase inhibitors  on telomerase complex and genomic instability will be evaluated. The results of proposed project should provide complex picture of the role of telomerase complex in CML and give better understanding how telomerase complex and telomere maintenance mechanisms (including alternative lengthening of telomeres) is involved in the pathogenesis of CML. We expect that  with special regard to genomic instability and disease progression this may contribute in the future to find new therapeutic targets in leukemic cells and to design new therapies, especially for advanced phases of the disease with the ultimate goal of eradication of leukemic clone.