Innovative therapeutic strategies to enhance the prognosis of pediatric high-grade gliomas (pHGGs)

Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), pose a formidable challenge with dismal survival rates among infants. Despite exhaustive efforts by the scientific community to enhance the prognosis for these children, clinical trials have consistently fallen short in demonstrating improved outcomes. In this critical context, innovative therapeutic strategies that comprehensively address the intricate tumor microenvironment are urgently required.

Oncolytic viruses are emerging as a compelling avenue for brain tumor treatment due to their favorable clinical safety profiles, tumor cell specificity, ability to trigger antitumor immune responses, and others. Among these, the oncolytic adenovirus Delta-24-RGD (also known as DNX-2401 in clinics) has exhibited feasibility and a degree of efficacy in a cohort of newly diagnosed DMG patients. However, room for improvement remains, and combination therapy holds potential. ONC201, a promising small molecule, has shown encouraging therapeutic results in early-phase clinical trials targeting H3K27M-mutant DMGs. The positive responses observed in independent multisite clinical trials prompted the initiation of a phase 3 study (NCT05580562), currently enrolling participants. Notably, this study excludes patients with wild-type tumors or those arising in the pons, leaving an opportunity to optimize the therapeutic impact of this drug.

Thus, our lab decided to elucidate whether the combination of these two promising therapies - Delta-24-RGD and ONC201 – enhances their individual therapeutic effect in preclinical models of pHGGs, including DMGs. We leveraged all available techniques and expertise within the lab for this project. Initially, we examined potential negative interactions between ONC201 and Delta-24-RGD, measured by anti-hexon-based titration. Remarkably, co-treatment with ONC201 did not compromise Delta-24-RGD’s replication capacity in vitro, suggesting improved therapeutic responses. Next, we delved into characterizing the antitumor effect of the combination and its mechanism of action. Viability assays revealed that the combination treatment induced a synergistic or additive antitumor effect in all cells tested, regardless of their H3 mutation status. To gain insights into the underlying mechanisms, we conducted seahorse stress tests, DNA damage immunofluorescence, and mitochondrial and bulk RNAseq analyses. Our findings indicated that transcriptomic and metabolic changes produced by each agent were conserved when both drugs were combined. Notably, this combination significantly increased nuclear DNA damage. In vivo assessments using mouse models of pHGG and DMG confirmed the efficacy of the Delta-24-RGD/ONC201 combination, improving the responses of each agent alone. Last, analyses of the tumor immune infiltration (through RNAseq, multiplexed immunofluorescence, and flow cytometry) showed that the combination reshaped the tumor microenvironment towards a proinflammatory phenotype. We observed an increase in both lymphoid and myeloid lineage in the combined treatment versus either single treatment. Interestingly, Delta-24-RGD/ONC201 produces a higher activation in CD8+ T cells and higher activation and proliferation in CD4+ T cells. Furthermore, tumor cells showed a less proliferative and more differentiated phenotype.

Overall, the encouraged results of this study provide a strong rationale for exploring the Delta-24-RGD/ONC201 combination in the treatment of pHGGs and DMGs, regardless of tumor location or mutation status, and support the design of clinical approaches.

Immune responses to herpes zoster vaccination in patients

Winning this award would have a profound impact on both my personal and professional development, as well as significantly advance my research in the field of rheumatology. On a personal level, this award would be a powerful recognition of my dedication and hard work in studying immune-mediated rheumatic diseases (IMRDs). It would boost my confidence and motivation, reinforcing my commitment to making meaningful contributions to the field.

Additionally, the award would provide me with increased visibility within the medical and scientific community, opening doors to new opportunities for collaboration and mentorship. This recognition would serve as a pivotal milestone in my career, encouraging me to continue striving for excellence and innovation in my research endeavors. Professionally, winning this award would facilitate the advancement of my research by providing essential resources and support. The financial backing and recognition associated with the award would enable me to expand my study on the immunogenicity of vaccines in patients with IMRDs. Specifically, it would allow me to enhance the scope and depth of my research, incorporating more advanced techniques and larger, more diverse patient cohorts. This, in turn, would lead to more comprehensive and robust findings, ultimately contributing to a better understanding of how immunosuppressive therapies impact vaccine responses in these patients.

Furthermore, the award would elevate the significance of my research within the broader scientific community. It would highlight the importance of optimizing vaccination strategies for patients with IMRDs, emphasizing the need for tailored approaches to enhance their immune protection. The insights gained from my study could inform clinical guidelines and influence public health policies, ultimately improving the quality of care and outcomes for patients with rheumatic diseases.

In summary, winning this award would be transformative for my personal and professional development, empowering me to continue my research with renewed vigor and greater resources. The recognition and support would not only enhance my career but also contribute significantly to the advancement of knowledge in the field of rheumatology. This, in turn, would have a lasting impact on patient care and public health, underscoring the vital role of immunogenicity research in improving the lives of those with immune-mediated rheumatic diseases.

The combination of local irradiation with fibroblast activation protein (FAP)-targeted 4-1BB agonist for solid tumors

My lab has expertise on cancer immunotherapy and radioimmunotherapy, focusing their research on combinatorial strategies for translational immunotherapy. In that regard, my PhD project is about the combination of local irradiation with fibroblast activation protein (FAP)-targeted 4-1BB agonist for solid tumors. 

In recent years, cancer immunotherapy with PD-1 and CTLA-4 immune checkpoint blockade with monoclonal antibodies (mAb) has been the breakthrough of the decade in cancer treatment. And radiotherapy, a standard of care in clinic, has also been tested in combination with these agents preclinically, with promising results. Thus, we foresee an opportunity in discovering new radioimmunotherapy combinations and study the immune-mediated effect of radiotherapy in tumor biology. In that sense, we hypothesized irradiation can enhance FAP-4-1BBL mediated antitumoral efficacy. 

We first studied the effect of irradiation on FAP expressing cells, which are mainly cancer associated fibroblasts (CAF), within tumor microenvironment (TME). We observed that irradiation, due to its double-edge sword capacity, enhances FAP expression in tumors making it a more immunosuppressive environment. However, our approach is to use this disadvantage from irradiation for our benefit, targeting 4-1BB costimulatory signal into tumor infiltrating lymphocytes (TIL) by administrating a bispecific 4-1BB agonist with a FAP binding moiety. After this observation, we proceeded with in vivo studies and we showed that there was a synergy with RT and FAP-4-1BBL in primary tumor (treated) control and delayed growth of secondary tumors (untreated). We also deciphered the mechanism mediating antitumoral effect (CD8+ T-cells, IFN-I, IFN- and 4-1BB expression). To translate this data into clinic, we analyzed human colorectal tumor biopsies from pre and post-RT and observed and increased FAP and CD8+ T-cell expression, agreeing with our preclinical observations. These last results open up the opportunity to consider this combination on a clinical trial. 

Regarding lab techniques, we performed many molecular biology assays such as ELISA, RT-PCR, flow cytometry, immunohistochemistry and imnmunofluorescence studies (IHC and IF, respectively), ELISPOT, Luminex (cytokine array assay), RNAseq, RNA extraction, retrotranscription. Lots of mice study as well, therefore, cell culture was also performed. For CAF immortalization in cell culture many other techniques such as transfection with lipofectamine, western blot, protein extraction, plasmid modification with enzyme restrictions and then ligation, cloning, DNA digestion and electrophoresis among other techniques were used. 

Currently, the project is under review for journal publication in a competitive journal (IF 13). I will present it as poster or oral abstract in the international cancer immunotherapy congress (SITC) in Houston, Texas on November. I also received best oral abstract award in a national congress of cancer research (Calatayud, April 2024). In addition, we are under discussion with the pharmaceutical company that owns the molecule (FAP-4-1BBL) for further continuation with a clinical trial in our clinic. 

An alternative based on organoids, 3D models that reproduce the characteristics of the Glioblastoma tissue, allowing us to study with them and thus avoid or reduce the use of animals

The most common primary intracranial malignant tumor in adults is glioblastoma, which is a very aggressive and invasive tumor. Its high lethality makes the development of new therapies and study models very important. Currently, the use of animals in research is still essential to understand biological processes, diseases, and the development of new therapies. Genetically modified mouse models and xerografts of tumor material derived from patients are used to study glioblastoma. The use of these animals has many ethical and legal implications, making it essential to search for alternatives that allow the use of animals to be avoided, or, if this is not possible, the reduction of the number of individuals and the refinement of the methods used.

In this work we propose an alternative based on organoids, 3D models that reproduce the characteristics of the tissue, allowing us to study with them and thus avoid or reduce the use of animals. These models are more manageable and allow a large number of experiments to be carried out compared to in vivo models. Our objective in this project is the development of organoids for the study of gliomagenesis and their application in the discovery of new therapies. For this, we start from a monolayer culture of several cell lines (U-87 MG, T731 MG and C-19MG), which induce the formation of neurospheres, neural differentiation and the formation of organoids. Our results show that both cells of human and murine origin have the capacity to form organoids, showing growth differences between lines, reproducing the behavior they have when they are cultured in monolayer. The tests carried out show cells of different sizes and morphologies, with greater cell density in the periphery. There is expression of glial, neuronal and dedifferentiation lineage markers, so there are phenotypic changes with respect to the initial cells due to dedifferentiation processes and subsequently differentiation and structuring.

These results demonstrate the ability to obtain a 3D model of these cell lines that reproduces the tissue that cells form in the body, allowing their study and understanding for the development of new therapies, as well as their use to test new drugs. This allows the number of animals used in later stages to be reduced, thus reducing ethical and legal implications.