Despite the remarkable benefits of cancer immunotherapies observed in select cases, many patients and cancers are unresponsive or develop resistance to existing treatments. There is emerging evidence that mucins, large cell-surface glycoproteins which are frequently upregulated in cancer, allow tumors to resist treatment with immunotherapies by engaging an inhibitory glycan-binding receptor on immune cells called Siglec-9. However, the molecular epitopes involved in mucin-Siglec-9 binding remain poorly understood due to historical challenges associated with studying receptor-glycoprotein interactions. This proposal seeks to develop new approaches to interrogate the role of mucins as immunological regulators by building upon high-resolution mucin mass spectrometry and cutting-edge precision glycoprotein synthesis techniques. Specifically, I will develop a coupled immunoprecipitation and mass spectrometry workflow to identify mucin glycopeptide epitopes that interact with Siglec-9 from both recombinant mucins and those derived from diverse human tumor cell lines (Aim 1). In parallel, I will carry out high-throughput screens of synthetic glycopeptide arrays bearing defined glycan structures to comprehensively elucidate the structural determinants of Siglec-9 binding (Aim 2). Finally, I will characterize the binding site of Siglec-9 via co-crystallization with a mucin-mimetic glycopeptide and use insights gained to develop and validate candidate glycopeptide inhibitors of the mucin-Siglec-9 immune synapse (Aim 3). This work will shed light on a previously understudied mechanism of immune evasion exploited by a significant fraction of tumors, with the potential to identify novel cancer immunotherapy agents that could increase the number of patients who benefit from treatment.
Overexpression of mucins, highly glycosylated cell-surface proteins, is a frequently observed cancer phenotype associated with disease aggressiveness and poor patient outcomes; however, the functional roles of mucins in cancer are not fully understood. This proposal seeks to use state-of-the-art mass spectrometry and precision glycoprotein synthesis approaches to interrogate an emerging role of mucins as immune checkpoint ligands. This work will shed light on a mechanism of immune evasion exploited by a significant fraction of tumors, with the potential to increase the number of patients who benefit from cancer immunotherapy.
