Periodic Reporting for period 4 - Immune-senescence (Dual targeting of senescence and tumor immunity for cancer therapy)
Período documentado: 2021-01-01 hasta 2021-06-30
Cancer immunotherapy is emerging as a relevant approach to treat several types of cancer. We have previously found that tumor-infiltrating myeloid cells can support the growth of prostate cancer and trigger treatment resistance. Therefore, a better characterization of the prostate tumor microenvironment and tumor immune response may contribute to the identification of novel immunotherapies in prostate cancer. In Task1 we will characterize the secretome and immune landscape of prostate tumors driven by different genetic alterations. In Task 2 of the proposal, we will assess the mechanisms that promote the recruitment and activation of tumor-associated macrophages (TAMs) and myeloid cells. We will focus on a novel class of secreted factors identified in Pten; Trp53 null tumors that are responsible for M1-M2 macrophages skewing and that can be targeted for cancer therapy. In Task 3 of the proposal we will assess whether the tumor secretome transmits senescence to immune cells. In Task 4 of the proposal, we will focus on tumor-infiltrating B cells (TILBs) in order to characterize their function in different Pten null mouse models. Final objective of this proposal is to identify and develop monoclonal antibodies directed towards senescence tumor cells. These antibodies will be tested in pre-clinical trials (Task 5) to assess whether they improve tumor clearance during TIS.
The major achievements of this project are:
a) Characterisation of the secretome and immune response of prostate tumors
We have performed polysome profiles analysis, in prostate tumors to identify upregulated secreted factors. Through bioinformatics analysis we found 3 upregulated ligand-receptor couples (HGF/MET, Spp1/CD44, Bgn/TLR4) capable to promote the recruitment and increase the immune suppressive function of MDSCs. Given that the release of Hgf, Spp1 and Bgn by tumor cells is regulated at the translational level, we used the MNK1/2 inhibitor eFT508 to inhibit MDSCs recruitment. eFT508 showed a strong anti-tumor activity. This work provides a new therapeutic strategy that combines translation inhibition with standard of therapy to restore immune surveillance in prostate cancer by targeting MDSCs.
b) Defining the mechanism leading to M1-M2 macrophages polarization in prostate cancer.
Using the method described above, we have found that CXCL1, CXCL2, CXCL 3, and CXCL5 are strongly upregulated in prostate tumors and that TAMs express the CXCR2 receptor both in murine and human prostate cancers. We have found that CXCL2 promotes TAM M2-like polarization. Inhibition of the CXCR2 receptor in these TAMs blocked their polarization towards M2-like, enhancing their tumor-suppressive capability. We identified TNF as the factor capable of inducing senescence in tumors treated with the CXCR2 antagonists (Di Mitri D., Cell Reports, 2019).
c) To assess whether the secretome of prostate tumors induces "senescence" in tumor-infiltrating immune cells.
Using FACS and single-cell analyses in prostate tumours, we have found a population of senescent PMN-MDSCs that express the TREM2 receptor. These cells, named senescent-like MDSCs (sMDSCs), are characterized by a prolonged half-life, up regulation of senescence markers, and increased immune suppressive activity compared to canonical-MDSCs. By using the p16-3MR model, we have found that selective elimination of these cells can promote tumor suppression and reactivation of immune surveillance in prostate cancer. Mechanistically, we have found that APOE released by prostate tumor cells can promote senescence in MDSCs by binding to TREM2. Therapeutically, we have found that HDAC inhibitors treatment can selectively remove these cells by down regulating TREM2 and abrogating its downstream signalling.
d) Identify novel senescence biomarkers by developing novel antibodies.
We have performed proteomic analysis in senescent vs. non-senescent epithelial tumor cells sorted out the tumors by using the C12FDG FACS staining. The proteomic analysis allowed us to identify 3 plasma membrane proteins unregulated in senescent cells. Top of the list was Nectin-2 , a plasma membrane protein that can be recognised by DNAM-1, TIGIT, and PVRIG. Whilst activation through DNAM- enhances NK cell-mediated cytotoxicity, TIGIT and PVRIG recognition by Nectin-2 exerts an inhibitory effect on NK and T cells. Of note, in primary prostate cancer, we found that PVRIG is highly expressed in tumor-infiltrating T cells, whereas DNAM-1 and TIGIT are not. Finally, we have found that therapy-induced senescence enhances Nectin-2 levels and that Nectin-2 recombinant protein suppresses T cells function. We will soon test the efficacy of an antibody targeting PVRIG in preclinical trials in combination with TIS. Moreover, we have generated a novel Nectin-2 senolytic immunoliposome that we will use in preclinical trials in vivo.
e) Evaluating whether immunotherapy improve the efficacy of standard of therapy in prostate cancer.
This task aimed to develop the most effective immunotherapy targeting MDSCs to combine with the standard of therapy in prostate cancer. By analyzing both human and murine tumor biopsies from castration-sensitive (HS) and resistant prostate cancer patients (CRPC), we found that MDSCs massively infiltrated tumors from CRPC patients and that MDSCs promote resistance to androgen-deprivation therapies by releasing in the tumor microenvironment IL23. IL23 was the most abundant factor present in the blood of CRPC patients when compared to HS patients. Treatment of different mouse models of CRPC with IL23 antibody synergies with enzalutamide, a standard of therapy in CRPC patients (Calcinotto A. et al, Nature, 2018). This paper has opened a novel exciting perspective for the therapy of CRCP patients, and its relevance has been highlighted in a commentary on the same issue of Nature and additional journals (Galsky, Nature 2018; Bradley CA., Nat Rev Urol. 2018). As mentioned above, we have also completed additional preclinical trials using the MNK1/2 inhibitor eFT508 alone or in combination with standard of therapy in prostate cancer
a) Characterisation of the secretome and immune response of prostate tumors
We have performed polysome profiles analysis, in prostate tumors to identify upregulated secreted factors. Through bioinformatics analysis we found 3 upregulated ligand-receptor couples (HGF/MET, Spp1/CD44, Bgn/TLR4) capable to promote the recruitment and increase the immune suppressive function of MDSCs. Given that the release of Hgf, Spp1 and Bgn by tumor cells is regulated at the translational level, we used the MNK1/2 inhibitor eFT508 to inhibit MDSCs recruitment. eFT508 showed a strong anti-tumor activity. This work provides a new therapeutic strategy that combines translation inhibition with standard of therapy to restore immune surveillance in prostate cancer by targeting MDSCs.
b) Defining the mechanism leading to M1-M2 macrophages polarization in prostate cancer.
Using the method described above, we have found that CXCL1, CXCL2, CXCL 3, and CXCL5 are strongly upregulated in prostate tumors and that TAMs express the CXCR2 receptor both in murine and human prostate cancers. We have found that CXCL2 promotes TAM M2-like polarization. Inhibition of the CXCR2 receptor in these TAMs blocked their polarization towards M2-like, enhancing their tumor-suppressive capability. We identified TNF as the factor capable of inducing senescence in tumors treated with the CXCR2 antagonists (Di Mitri D., Cell Reports, 2019).
c) To assess whether the secretome of prostate tumors induces "senescence" in tumor-infiltrating immune cells.
Using FACS and single-cell analyses in prostate tumours, we have found a population of senescent PMN-MDSCs that express the TREM2 receptor. These cells, named senescent-like MDSCs (sMDSCs), are characterized by a prolonged half-life, up regulation of senescence markers, and increased immune suppressive activity compared to canonical-MDSCs. By using the p16-3MR model, we have found that selective elimination of these cells can promote tumor suppression and reactivation of immune surveillance in prostate cancer. Mechanistically, we have found that APOE released by prostate tumor cells can promote senescence in MDSCs by binding to TREM2. Therapeutically, we have found that HDAC inhibitors treatment can selectively remove these cells by down regulating TREM2 and abrogating its downstream signalling.
d) Identify novel senescence biomarkers by developing novel antibodies.
We have performed proteomic analysis in senescent vs. non-senescent epithelial tumor cells sorted out the tumors by using the C12FDG FACS staining. The proteomic analysis allowed us to identify 3 plasma membrane proteins unregulated in senescent cells. Top of the list was Nectin-2 , a plasma membrane protein that can be recognised by DNAM-1, TIGIT, and PVRIG. Whilst activation through DNAM- enhances NK cell-mediated cytotoxicity, TIGIT and PVRIG recognition by Nectin-2 exerts an inhibitory effect on NK and T cells. Of note, in primary prostate cancer, we found that PVRIG is highly expressed in tumor-infiltrating T cells, whereas DNAM-1 and TIGIT are not. Finally, we have found that therapy-induced senescence enhances Nectin-2 levels and that Nectin-2 recombinant protein suppresses T cells function. We will soon test the efficacy of an antibody targeting PVRIG in preclinical trials in combination with TIS. Moreover, we have generated a novel Nectin-2 senolytic immunoliposome that we will use in preclinical trials in vivo.
e) Evaluating whether immunotherapy improve the efficacy of standard of therapy in prostate cancer.
This task aimed to develop the most effective immunotherapy targeting MDSCs to combine with the standard of therapy in prostate cancer. By analyzing both human and murine tumor biopsies from castration-sensitive (HS) and resistant prostate cancer patients (CRPC), we found that MDSCs massively infiltrated tumors from CRPC patients and that MDSCs promote resistance to androgen-deprivation therapies by releasing in the tumor microenvironment IL23. IL23 was the most abundant factor present in the blood of CRPC patients when compared to HS patients. Treatment of different mouse models of CRPC with IL23 antibody synergies with enzalutamide, a standard of therapy in CRPC patients (Calcinotto A. et al, Nature, 2018). This paper has opened a novel exciting perspective for the therapy of CRCP patients, and its relevance has been highlighted in a commentary on the same issue of Nature and additional journals (Galsky, Nature 2018; Bradley CA., Nat Rev Urol. 2018). As mentioned above, we have also completed additional preclinical trials using the MNK1/2 inhibitor eFT508 alone or in combination with standard of therapy in prostate cancer
When this project started, it was unclear how prostate cancer recruits and controls the function of tumor-infiltrating myeloid cells. Thanks to this ERC project we have analyzed the secretome of prostate cancer driven by different genetic alterations and found a number of factors that are responsible for the recruitment and function of MDSCs and TAMs. This knowledge has allowed us to develop novel immunotherapies to block MDSCs or factors released by these immune cells. We have found that MDSCs promote resistance to androgen-deprivation therapies (ADTs) in prostate cancer by secreting IL23 in the tumor microenvironment (Calcinotto, Nature 2018). We have found that the efficacy of ADT, is enhanced by an antibody that blocks the IL23 receptor. We have started two clinical trials in collaboration with Prof. de Bono team at ICR. In these trials ADT is combined with AZD5069 a compound that blocks the recruitment of MDSCs ( NCT03177187) or with the anti-IL23 antibody Tildrakizumab (NCT04458311). These innovative trials are the first trials assessing the tolerability and efficacy of this class of compounds in prostate cancer.