Titolo della tesi: Exploring the role of Notch as MDSC inducer in T-ALL: from inside to outside of tumor cell
In the complex world of tumor microenvironment (TME) a prominent role in supporting cancer progression is played by MDSCs, immuno-suppressive cells of myeloid origin able to inhibit host immune-responses. The effects of MDSCs on tumor progression and their potential definition as a therapy target have been extensively investigated in several cancers, with the notable exception of ‘T-cell acute lymphoblastic leukemia (T-ALL)’. T-ALL is an aggressive hematologic cancer characterized by aberrant proliferation of immature thymocytes, often linked to Notch receptor mutations, and with no resolutive cure, to date.
Preliminary data from our lab, obtained in a murine model of Notch3-dependent T-ALL (N3tg mice), describe for the first time that N3tg T-ALL cells can induce in trans the expansions of CD11b+Gr-1+ MDSCs. We also proved that the human Notch3-dependent T-ALL cell line, ‘TALL-1’ can induce the expansion of CD11b+CD33+CD14+HLADRlow/neg MDSCs in PBMCs from healthy donors, via a mechanism that involves the Notch3/IL-6 axis.
However, the most frequent causative event in T-ALL patients is represented by activating mutations of the Notch1 gene. Thus, my first aim was to further corroborate the importance of this pathway in mediating the induction/expansion of MDSCs in T-ALL context. To this end, I extended the findings obtained above by testing the ability of the Notch1 mutated human cell line ‘KE-37’. I report that KE-37 cells, similarly to TALL-1, drive the expansion of functional CD11b+CD33+CD14+HLADRlow/neg MDSCs in co-culture with PBMCs from healthy donors, through a mechanism that depends on both Notch1 and IL-6 signaling. Indeed, the exposure to the Notch signaling inhibitors ‘γ-secretase’ or blocking anti-IL-6 antibodies inhibits MDSC induction in the co-cultures above.
Furthermore, as second aim of my PhD thesis, I have tried to individuate possible mediators of Notch pathway in inducing in trans MDSCs. To this regard, the downstream constitutive activation of canonical NF-κB pathway (p65/p50) is an important proliferative arm of Notch in T-ALL tumor cells. In this context, we previously generated double mutant mice harboring conventional knock-out mutation of p50/NF-κB1 on the N3-tg background (N3tg/p50−/− mice) and demonstrated that the absence of p50 deeply influence T-ALL outcome and immune-environment. Notably, double-mutant mice display a massive expansion of CD11b+Gr-1+ cells, even more pronounced than in N3tg mice, and I demonstrated here that these myeloid cells are induced in trans by N3tg/p50−/− immature T cells, both in vitro and in vivo. Intriguingly, I also proved, by performing conventional “MDSC suppression assays”, that these CD11b+Gr-1+ cells expanded in the N3tg/p50−/− mice are functional MDSCs, also more suppressive than the N3tg counterpart, suggesting that NF-κB could represent an important regulator of Notch signaling in inducing MDSCs in T-ALL.
The high prevalence of Notch receptor mutations in T-ALL has led to the development of therapies aimed at the direct inhibition of this oncogenic signaling. However, it is now clear that an effective cure for cancer cannot be limited only in the correction of the molecular aberrations confined to the neoplastic cell but must also destroy the interaction that tumor cell creates with its microenvironment. In this context, one of the most extensively studied pathway is the PD-1-PD-L1 inhibitory immune checkpoint. Given the clinical impact that PD-1/PD-L1 checkpoint inhibitors have had in the recent years in many types of cancers and since there are missed information regarding the influence of this pathway in T-ALL context, as third aim, I decided to start investigating the expression of PD-1 and PD-L1 molecules in the TME of our Notch3-dependent T-ALL murine model. Of interest, I evidenced a significant higher expression of PD-L1 on MDSCs and of PD-1 on Natural Killer cells (NKs), in N3tg mice with respect to wt controls. Further, in parallel with tumor progression, N3tg NKs experience alterations in their absolute numbers, as well as in their cytotoxic activity. Though preliminary, my results would suggest that MDSCs in T-ALL context may hinder the anti-tumor activity of NKs via the PD-1/PD-L1 interaction and could represent a potential therapeutic target to restore anti-cancer immune response in T-ALL.