Mechanisms of resistance to anti-GD2 immunotherapy in neuroblastoma
Restricted (Penn State Only)
- Author:
- Liu, Xiaoming
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 12, 2022
- Committee Members:
- Vladimir Spiegelman, Major Field Member
Jeffrey Sundstrom, Outside Unit Member
Hong-Gang Wang, Chair & Dissertation Advisor
Todd Schell, Major Field Member
Edward Harhaj, Outside Field Member
Ralph Keil, Program Head/Chair - Keywords:
- Neuroblastoma
Small extracellular vesicles
Tumor microenvironment
NK cells
anti-GD2 immunotherapy
CRISPR screen - Abstract:
- Anti-GD2 monoclonal antibody immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. The tumor-bound anti-GD2 antibodies recruit immune effector cells to trigger Fc-receptor-mediated killing by both complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). However, 40% of patients fail to respond or develop resistance to the treatment, and the molecular mechanisms by which this occurs remain poorly understood. Tumor-derived small extracellular vesicles (sEVs) have emerged as critical regulators in modulating the response to immunotherapy, while the role of tumor-derived sEVs in modulating the response to monoclonal antibody-based therapy has not yet been investigated. Here, we utilize the syngeneic 9464D-GD2 mouse model to investigate the role of neuroblastoma-derived small extracellular vesicles (sEVs) in developing resistance to the anti-GD2 monoclonal antibody dinutuximab. Strikingly, neuroblastoma-derived sEVs significantly attenuated the efficacy of dinutuximab in vivo. Mechanistically, RNA-sequencing and flow cytometry analysis of whole tumors revealed that neuroblastoma-derived sEVs modulate immune cell tumor infiltration upon dinutuximab treatment to create an immunosuppressive tumor microenvironment that contains more tumor-associated macrophages (TAMs) and fewer tumor-infiltrating NK cells. In addition, neuroblastoma-derived sEVs suppressed splenic NK cell maturation in vivo and dinutuximab-induced NK cell-mediated antibody-dependent cellular cytotoxicity in vitro to provide additional mechanisms to dinutuximab resistance. Importantly, tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion, drastically enhanced the efficacy of dinutuximab in vivo and reversed the immunosuppressive effects of neuroblastoma-derived sEVs. Notably, tipifarnib modulated immature myeloid cells in the bone marrow to impair the formation of CD11b+Ly6ChighLy6Glow cells that are precursors for TAMs. Taken together, these preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate immunosuppression to promote resistance to dinutuximab and suggest that tipifarnib-mediated inhibition of sEV secretion can serve as a viable treatment strategy to enhance the anti-tumor efficacy of anti-GD2 immunotherapy in high-risk neuroblastoma patients. To further investigate molecular mechanisms modulating the resistance to anti-GD2 immunotherapy, we performed in vivo genome-wide screening using the CRISPR/Cas9 gene editing system to understand and dissect the complicated mechanisms that drive resistance to dinutuximab therapy in neuroblastoma using the same syngeneic 9464D-GD2 mouse model. This unbiased in vivo screen demonstrated significant deletions of sgRNAs targeting genes acting as essential regulators of the JAK-STAT and TGF-beta/BMPs signaling pathways in tumors that resist dinutuximab treatment, highlighting that the activation of the JAK-STAT and TGF-beta/BMPs signaling pathways can be a potential mechanism for anti-GD2 immunotherapy resistance in neuroblastoma. Notably, IFNAR1, JAK1, and STAT1 have been identified as the top hits that modulate the resistance to dinutuximab in this loss-of-function in vivo screen, suggesting that the IFNAR1-JAK1-STAT1 signaling cascade may be crucial for developing resistance to dinutuximab compared to other signaling cascades within the JAK-STAT pathway network. Thus, selective targeting of the IFNAR1-JAK1-STAT1 signaling pathway can be a potential therapeutic approach for overcoming the resistance to anti-GD2 immunotherapy in neuroblastoma. Together, our unbiased in vivo genome-wide screening has identified potential mechanisms modulating resistance to anti-GD2 immunotherapy and provides rationales for subsequent translational studies in neuroblastoma. Collectively, these studies investigated both extrinsic tumor microenvironment and tumor-intrinsic mechanisms in modulating resistance to anti-GD2 immunotherapy. As JAK-STAT signaling pathway activation in either tumor cells or tumor-infiltrating immune cells has been associated with the formation of the immunosuppressive microenvironment and tumor immune evasion, respectively, future studies will focus on investigating the potential positive feedback loop between neuroblastoma cells and neuroblastoma microenvironment, which enforces the resistance to anti-GD2 immunotherapy through the sEV-mediated intercellular communications.