Periodic Reporting for period 1 - P70-IMMUNEBREAST (Tumor compartment-specific effects of P70S6K in early triple negative breast cancer: regulation of antitumor immune response and therapeutic implications)
Reporting period: 2021-01-01 to 2022-12-31
Breast cancer is currently the most frequently occurring cancer worldwide and it shows significant death rates and economic impact. Its incidence is progressively increasing every year. Breast cancer comprises a heterogeneous group of tumors that shows a very diverse biological and clinical behaviour. Triple negative breast cancer (TNBC) is an aggressive subtype which accounts for up to 15% of the new breast cancer cases. It is characterized by the lack of expression of the three classical markers associated to breast cancer. This absence of clear ´disease-defining´ biomarkers, as well as its molecular heterogeneity, constitutes a major problem for finding the appropriate therapeutic options for TNBC patients. In fact, despite the improvement in outcomes achieved in other breast cancer subtypes, the prognosis of TNBC is still dismal: up to 40-50% of the new patients diagnosed with early TNBC will experience distant relapse and death due to their disease.
Recently, thanks to a taxonomic effort of the hosting group, TNBC has been re-classified on the basis of the activation status of six protein kinases, which in turn constitute novel targets for this disease. Notably, it was found that the functional status of those kinases informs about the risk of relapse of early TNBC patients. One of those kinases is the 70-kDa ribosomal protein S6 kinase (P70S6K), which was associated with an adverse clinical outcome. Likewise, other studies have also shown that increased levels of activation of P70S6K in breast cancer patients correlate with worse prognosis, reduced disease-free survival and increased metastasis.
Preliminary data from the host group suggest that mechanisms of immunosuppression or immune-exclusion are triggered in tumors with high levels of phopho-P70S6K (p-P70S6K, the active form of the kinase). It raises the possibility that inhibition of the activity of P70S6K may relieve local immunosuppression and improve the outcome of patients. Thus, this project was focused on understanding the role of P70S6K in regulating the lymphocyte compartment on TNBC. Specific objectives to be addressed are: (1) To characterize the immune infiltrate at a single cell level among the different TNBC subtypes defined by the levels of p-P70S6K; and (2) To describe the role of P70S6K shaping the immune responses in TNBC and the potential therapeutic benefit of the P70S6K blockade.
Recently, thanks to a taxonomic effort of the hosting group, TNBC has been re-classified on the basis of the activation status of six protein kinases, which in turn constitute novel targets for this disease. Notably, it was found that the functional status of those kinases informs about the risk of relapse of early TNBC patients. One of those kinases is the 70-kDa ribosomal protein S6 kinase (P70S6K), which was associated with an adverse clinical outcome. Likewise, other studies have also shown that increased levels of activation of P70S6K in breast cancer patients correlate with worse prognosis, reduced disease-free survival and increased metastasis.
Preliminary data from the host group suggest that mechanisms of immunosuppression or immune-exclusion are triggered in tumors with high levels of phopho-P70S6K (p-P70S6K, the active form of the kinase). It raises the possibility that inhibition of the activity of P70S6K may relieve local immunosuppression and improve the outcome of patients. Thus, this project was focused on understanding the role of P70S6K in regulating the lymphocyte compartment on TNBC. Specific objectives to be addressed are: (1) To characterize the immune infiltrate at a single cell level among the different TNBC subtypes defined by the levels of p-P70S6K; and (2) To describe the role of P70S6K shaping the immune responses in TNBC and the potential therapeutic benefit of the P70S6K blockade.
In order to achieve the proposed objectives, a combination of immunohistochemistry, multiparametric flow cytometry, gene expression analysis and in vivo experiments, among other studies, were performed.
Aim 1 involved the characterization of the tumor immune infiltrate into the two distinct TNBC subtypes, defined by their level of p-P70S6K. Refined image analyses were performed from tumor samples of 341 TNBC patients. TIL infiltration was evaluated and a reduced stromal TILs (sTILs) percentage was found in those patients exhibiting hyperactivation of the kinase. Further classification of tumor lymphocytes showed that B cell abundance was lower in high p-P70S6K samples. Additionally, a decreased CD8/CD4 ratio was found in high p-P70S6K samples. Moreover, positive correlations were found between the levels of p-P70S6K and CD4 and Foxp3 expression, and CD8/CD4 and CD8/Foxp3 ratios. Overall, these analyses allowed knowing the lymphocytes dysregulated in TNBC patients with high/low p-P70S6K tumor levels.
Aim 2 explored the role of P70S6K in tumor immunogenicity and T cell responses in TNBC. Initial part of this objective was focused on assessing the potential dual role of P70S6K on tumor and TIL compartments in TNBC. First, to know the mechanisms by which hyperactivation of tumor P70S6K could influence tumor immunogenicity, several analyses were completed by in vitro blockade of P70S6K activity in different TNBC cell lines. These analyses included the study of: molecules involved in presentation of tumor antigens to T cells, expression of immune checkpoint inhibitors or release of molecules supporting in T cell recruitment into the tumor. A heterogenous response was obtained from these experiments depending on cell line and inhibitor used. Second, the effect of P70S6K inhibition on T cells was studied. Ex vivo cultures of T cells from tumor or spleen were performed under basal or stimulated conditions and in the presence/absence of inhibitors of P70S6K. Results suggested that inhibition of P70S6K activity has a negative effect on anti-tumoral T cell responses, while promoting suppressive immune responses.
The second part of Aim 2 included the evaluation of the therapeutic effect of P70S6K blockade in preclinical models of TNBC and its potential synergies with current immunotherapies. Pharmacological inhibition of P70S6K was tested in vivo in different mouse models of TNBC. Results revealed that inhibition of P70S6K activity significantly delayed tumor growth and increase survival of mice wearing tumors over-expressing p-P70S6K.
Altogether, these in vitro and in vivo experiments laid the foundation for forthcoming elucidation of the role of P70S6K on tumor immunogenicity and T cell responses in TNBC.
Throughout the length of the project, results found in the above-mentioned experiments were available to the scientific community and public by different channels. Public had the opportunity to know the project in different outreach activities, such as the European Researchers´ Night or the XI Feria Madrid es Ciencia 2022. Moreover, the project has been published in a report entitled ´Breast cancer: an aggressive variant triggers a hunt for cures´ (published in Horizon, The EU Research & Innovation Magazine on 9th December 2022 and in EL PAIS on 20th December 2022; and posted at CNIO twitter). Likewise, data was presented to scientific colleagues through diverse ways: labmeeting seminars, CNIO Progress Report seminars, Clinical Research Programme seminars and other internal seminars. Importantly, data from this project was published in an Open Access peer-review article entitled ´Tumor P70S6K hyperactivation is inversely associated with tumor-infiltrating lymphocytes in triple-negative breast cancer´ (Clin Transl Oncol. 2022. doi: 10.1007/s12094-022-03006-3).
Aim 1 involved the characterization of the tumor immune infiltrate into the two distinct TNBC subtypes, defined by their level of p-P70S6K. Refined image analyses were performed from tumor samples of 341 TNBC patients. TIL infiltration was evaluated and a reduced stromal TILs (sTILs) percentage was found in those patients exhibiting hyperactivation of the kinase. Further classification of tumor lymphocytes showed that B cell abundance was lower in high p-P70S6K samples. Additionally, a decreased CD8/CD4 ratio was found in high p-P70S6K samples. Moreover, positive correlations were found between the levels of p-P70S6K and CD4 and Foxp3 expression, and CD8/CD4 and CD8/Foxp3 ratios. Overall, these analyses allowed knowing the lymphocytes dysregulated in TNBC patients with high/low p-P70S6K tumor levels.
Aim 2 explored the role of P70S6K in tumor immunogenicity and T cell responses in TNBC. Initial part of this objective was focused on assessing the potential dual role of P70S6K on tumor and TIL compartments in TNBC. First, to know the mechanisms by which hyperactivation of tumor P70S6K could influence tumor immunogenicity, several analyses were completed by in vitro blockade of P70S6K activity in different TNBC cell lines. These analyses included the study of: molecules involved in presentation of tumor antigens to T cells, expression of immune checkpoint inhibitors or release of molecules supporting in T cell recruitment into the tumor. A heterogenous response was obtained from these experiments depending on cell line and inhibitor used. Second, the effect of P70S6K inhibition on T cells was studied. Ex vivo cultures of T cells from tumor or spleen were performed under basal or stimulated conditions and in the presence/absence of inhibitors of P70S6K. Results suggested that inhibition of P70S6K activity has a negative effect on anti-tumoral T cell responses, while promoting suppressive immune responses.
The second part of Aim 2 included the evaluation of the therapeutic effect of P70S6K blockade in preclinical models of TNBC and its potential synergies with current immunotherapies. Pharmacological inhibition of P70S6K was tested in vivo in different mouse models of TNBC. Results revealed that inhibition of P70S6K activity significantly delayed tumor growth and increase survival of mice wearing tumors over-expressing p-P70S6K.
Altogether, these in vitro and in vivo experiments laid the foundation for forthcoming elucidation of the role of P70S6K on tumor immunogenicity and T cell responses in TNBC.
Throughout the length of the project, results found in the above-mentioned experiments were available to the scientific community and public by different channels. Public had the opportunity to know the project in different outreach activities, such as the European Researchers´ Night or the XI Feria Madrid es Ciencia 2022. Moreover, the project has been published in a report entitled ´Breast cancer: an aggressive variant triggers a hunt for cures´ (published in Horizon, The EU Research & Innovation Magazine on 9th December 2022 and in EL PAIS on 20th December 2022; and posted at CNIO twitter). Likewise, data was presented to scientific colleagues through diverse ways: labmeeting seminars, CNIO Progress Report seminars, Clinical Research Programme seminars and other internal seminars. Importantly, data from this project was published in an Open Access peer-review article entitled ´Tumor P70S6K hyperactivation is inversely associated with tumor-infiltrating lymphocytes in triple-negative breast cancer´ (Clin Transl Oncol. 2022. doi: 10.1007/s12094-022-03006-3).
Given the successful achievement of the project, it can be considered that data generated can positively impacts on both the scientific community and the society. Results have contributed of knowledge in breast cancer field and provide to scientific community a valuable data to be considered for improving TNBC clustering. Moreover, lymphocyte profiling described in the context of P70S6K hyperactivation could offer new possibilities to further explore immune responses in TNBC. Combination of clustering based on P70S6K pattern and immune profiling could be taken into consideration in future for planning new personalized therapies for TNBC patients. As TNBC incidence increases every year and life expectance is poor for these patients, new knowledge generated in this field could potentially greatly impact by making improvements in current therapeutic indexes. It would benefit to the health and wellbeing of women and, by extension, the society as a whole.