Research Programme 5 (RP5) focuses on proof-of-concept preclinical studies in the form of in vivo confirmation of experimental treatment strategies, which are conducted in immunodeficient mice that carry patients’ xenografts, and on the early stages of treatment (phase ½) conducted on patients. What are the main results achieved in the past several years?
Institutions participating in RP5 are the Institute of Pathological Physiology of the First Faculty of Medicine of CU and the 1st Department of Internal Medicine – Haematology of the First Faculty of Medicine of CU and the General University Hospital in Prague (Professor Klener), the BIOCEV centre of the First Faculty of Medicine of CU (Associate Professor Havránek), Thomayer University Hospital in Prague (Associate Professor Boublíková). These institutions nevertheless collaborate with other centres across the entire NICR consortium; examples of such cooperation include the development and testing of polymer-bound drugs undertaken jointly with the Institute of Macromolecular Chemistry of the Czech Academy of Sciences (RNDr. Etrych). Below, we introduce the main results achieved within RP5.
Creation of a database of new preclinical models as a platform for proof-of-concept preclinical studies (phase 2-like murine studies)
Thanks to support from NICR, we have succeeded in significantly expanding the database of new preclinical models of lymphoproliferations, including non-Hodgkin lymphomas and acute lymphoblastic leukaemias. Our database currently includes over 30 new cell lines and over 60 new PDX models (Jakša et al., Laboratory Investigation 2022). In contrast to commercially available cell lines, all the lines we had derived have been validated using whole exome sequencing in direct comparison with the primary tumour cells from which the lines were derived. That is why we call the newly derived lymphoma and leukaemia lines ‘patient-derived lymphoma cell lines’ (PDCL). Thanks to a sufficiently wide database of PDCL and PDX models, we managed to finish some proof-of-concept in vivo studies which mimic phase 2 studies on human subjects but are conduced on immunodeficient mice that carry PDX or PDCL xenografts (phase 2-like proof-of-concept studies). Studies conducted in immunodeficient mice moreover enable subsequent translational research of PDX tumours, for instance after a failure of the texted treatment. The results were presented at the annual congress of the American Society of Hematology (ASH 2024) and related publications are currently either under review or in an advanced stage of preparation (Kazanstsev et al. Blood 2024; Löber et al., Blood 2024).
Mutation and transcriptome analysis of lymphomas, clonal development, and the study of adaptation changes in lymphoma cells after treatment failure
One of the key projects of RP5 is a mutation analysis of lymphomas and adaptation mechanisms of lymphoma cells after treatment failure. The goal of this research is to improve our understanding of the molecular mechanisms responsible for treatment failure and to search for new therapeutic goals. Our greatest success in this context is a publication dedicated to clonal development based on so far the largest set of pairs of samples acquired from patients with mantle cell lymphoma collected at the point of diagnosis and after relapse of the lymphoma, that is, after failure of the first-line treatment that is based on immunochemotherapy (Karolová et al., American Journal of Hematology 2023). Our data from whole exome sequencing (WES) have shown a selection of lymphoma cells with a larger number of genetic aberrations, which participate in the detection of genotoxic tress (mutation TP53, deletion CDKN2A).
In 2024, we focused on a study of genetic changes associated with the clinical course of mantle cell lymphoma. Using whole exome sequencing, we have analysed the samples of patients who either did not respond to treatment or experienced an early relapse and compared them with samples of patients who either did not relapse or relapsed at least seven years post-treatment. We have shown a significantly higher number of mutations and copy number aberrations in patients with early relapse. We have also demonstrated that certain mutations (e.g., NOTCH1/2, deletion CDKN2A) are not present in patients with a long remission after treatment and they can be detected only in the cohort of patients with early relapse. We have presented our preliminary results at ASH 2024, the annual congress of the American Society for Hematology (Karolová et al., Blood 2024).
In the past two years, we have introduced an analysis of lymphoma cells based on single cell RNA sequencing (scRNAseq). We have used scRNAseq to analyse lymphoma cells acquired from patients with a lymphoma before the beginning of treatment and during early relapse, that is, after a failure of immunochemotherapy. We were able to detect already in the diagnostic samples a small percentage of lymphoma cells (lymphoma subclones) that were transcriptionally identical to the majority lymphoma population detected at relapse. Our results thus indicate the presence of a small subclone of resistant lymphoma cells already at the point of diagnosis. We have presented the preliminary results at ASH 2023 (Kazantsev et al., Blood 2023) and the manuscript is currently under review.
The study of adaptation mechanisms that enable the survival of lymphoma cells in deep hypoxia (1% O2)
In collaboration with the Institute of Pathological Physiology of the First Faculty of Medicine of the CU and the BIOCEV of the First Faculty of Medicine, we have completed and published a comprehensive characterisation of lymphoma cells adapted to long-term deep hypoxia (Daumova et al., Cell Death Discovery 2025). Our data have revealed in lymphoma cells adapted to long-term hypoxia complex changes on the level of transcriptome, proteome, and metabolome, and indicated new potential targets for the elimination of thus adapted cells, including new proapoptotic strategies (Dolníková et al, Blood Advances 2024), metabolic inhibitors, or inhibitors of the PI3K-AKT pathway (Bettazová et al., Blood Advances 2024).
New therapeutic approaches to inhibiting the PI3K-AKT signalling pathway
The PI3K-AKT signalling pathway is pathologically hyperactivated in many types of tumours, including lymphomas. That is why there were developed numerous low-molecular inhibitors aimed at various levels of this pathway, but their effectiveness is diminished both by various feedback mechanisms of re-activation of the pathway and by severe side effects. We have developed a new and highly effective therapeutic concept of so-called vertical combination inhibition. It is based on inhibiting one signalling cascade at four levels simultaneously. We have shown that a vertical inhibition of PI3K-AKT signalisation is highly effective in lymphomas as it prevents a compensatory re-reactivation of the pathway and is tolerated in vivo (Kupcova et al., Experimental Hematology and Oncology 2024). In 2024, we have also completed a study which outlines a new therapeutic target in the PI3K-AKT signalling pathway: the PDPK1 kinase, which is among other things responsible for phosphorylation and activation of AKT. Its genetic elimination was universally toxic in all tested lymphoma model lines, and it seems that especially its targeted degradation could be the way forward. These results will be presented this year at ICML 2025, a prestigious international conference focused on malignant lymphomas (Herman et al. Phosphoinositide dependent protein kinase 1 is a key metabolic regulator and potential therapeutic target in lymphoma) and soon thereafter submitted for publication.
A study of using circulating tumour DNA for lymphoma diagnosis and monitoring
Free non-cellular circulating tumour DNA (ctDNA) seems a promising tool for diagnostics and monitoring of response to tumour treatment. We have therefore developed our own specific gene panel for ctDNA sequencing in lymphomas. In collaboration with the Ophthalmology Clinic and 1st Department of Internal Medicine of the First Faculty of Medicine of CU and the General University Hospital, we have shown that circulating tumour DNA is a clearly a future method of choice in the diagnostics of very rare vitreoretinal lymphomas whose diagnosis is otherwise very complicated. An analysis of samples from 15 patients had shown that ctDNA is present in the vitreous body in high concentrations. In all patients, we have detected lymphoma-specific DNA mutations with a high reliability. The results were presented at ASH 2024 (Velasova et al., Blood 2024). We have also confirmed that the initial concentration of ctDNA in the plasma is an independent prognostic factor in patients with the most common type of lymphoma and its decrease in the course of treatment correlates with survival and treatment response (ASH 2024, publication under review). We have published an overview of issues related to ctDNA in two overview articles (Maco et al., Annals of Hematology 2022; Hamova et al., Expert Review of Molecular Diagnostics 2025).
A study of deregulation of signalling cascades in lymphomas
One of the signalling cascades which are active in lymphomas and contribute to tumour growth is the signalling from B cell receptor (BCR). As part of a collaborative effort, we have contributed to elucidating the dominant mechanism of BCR signalling activation in lymphomas by antigen-independent autonomous BCR signalling (Eken et al., Journal of Experimental Medicine 2024). In further studies, we focus on the role of mutations in BCR co-receptor molecules CD79A/B (an overview article Tkachenko et al., International Journal of Molecular Sciences 2024) and on the new role of mutations of EZH2 histone methyl transferase (article under review).
Translational research of testicular tumours
Testicular tumours are rare tumours belonging to the so-called orphan diagnoses. At the same time, they are the most common solid tumours in young adult men and the most common cause of death from malignancy in this population. Unlike the vast majority of other malignancies, there has been no significant progress in their treatment for decades, which is still based on cisplatin-based chemotherapy and fails in some patients. In these so-called platinum-refractory patients, we have identified several molecular aberrations using whole-exome sequencing of tumour samples and circulating tumour DNA that are likely to be associated with disease progression and the development of platinum resistance, the cause of which has not yet been elucidated (manuscript in preparation for publication). We have established broad international collaboration in translational research on testicular tumours and organised the 10th European Testicular Workshop in Prague in October 2024 as the largest international conference dedicated to testicular tumours, attended by most of the world’s experts in this field. Based on the workshop resolution, we are establishing the European Testicular Tumor Network consortium and submitting an application for its support in the European COST Action Call 2025 programme.
Outlook into the future
The mission and challenges of the RP5 programme remain largely the same: The programme will focus on a further expansion of the database of new preclinical models for relevant preclinical research, on testing innovative drugs and treatment strategies on mouse models, and on a better understanding the molecular mechanisms responsible for treatment failure (mechanisms of substance resistance). Our other future goals include a further study of consequences of deregulation of signalling cascades or tumour-specific mutations (BCR signalling, PI3K-AKT signalling, epigenetic regulation) and continued research into the use of ctDNA for diagnosis and monitoring of lymphomas.
Pavel Klener, Ludmila Boublíková, main leaders of RP5