Person:

Lo, Jennifer

To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome [1,2]. The residues essential for primer annealing are initially locked in intramolecular interactions [3,4,5]; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements [6]. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and (tRNA^{Pro}) primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues—which are exactly complementary—for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNA^{Pro} reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.

Immune checkpoint inhibitors targeting cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) pathways can deliver durable anti-tumor effects. However, a major fraction of metastatic cancer patients fail to respond to checkpoint blockade.

Recent studies suggest that efficacy of checkpoint inhibitors is associated with inflammation in the tumor microenvironment. In this thesis, I demonstrate using genetically-defined murine models that sterile melanomas can be converted into inflamed tumors with improved responses to checkpoint blockade via two independent approaches: introduction of neoantigens and a novel combinatorial therapeutic strategy.

In addition to tumor inflammation, genomic studies have identified elevated numbers of neoantigens, mutated proteins that can serve as targets of immune responses, as potential predictors of clinical benefit. The preponderance of UVR-associated somatic mutations in melanoma has been proposed to play a role in mediating responses to immunotherapy, but model systems to study the contribution of such mutations to anti-melanoma immunity have been lacking. In chapter 2, I present a BrafV600E/Pten-/- syngeneic tumor graft murine model in which melanomas bearing numerous non-synonymous UVB-induced mutations were markedly more inflamed and responsive to PD-1 inhibition than matched parental melanomas.

For the treatment of neoantigen-deficient, poorly-inflamed tumors, in chapter 3 I tested the novel combination of imiquimod, ablative fractional photothermolysis (aFP), and checkpoint inhibitors. In anti-PD-1 resistant models of melanoma and pancreatic adenocarcinoma, addition of imiquimod and aFP produced abscopal tumor regressions with long-term survival in 50-60% of cases. Combination therapy stimulated autoimmunity against wildtype tumor-lineage antigens, suggesting that therapeutic strategies which enhance inflammation and responses against self-antigens may bypass a need for neoantigens and produce major regressions of cancers that are currently refractory to checkpoint blockade in the clinic.

In chapter 4 I show that PD-L1 expression is transcriptionally regulated by the melanocyte lineage factor and oncogene microphthalmia-associated transcription factor (MITF). PD-L1 expression is significantly correlated with MITF copy number in patient melanomas and is induced in skin as part of the MITF-dependent tanning response pathway. Our data suggest that loss of PD-L1 predisposes mice to apparent vitiligo after chronic UVR, suggesting that the UVR-MITF-PD-L1 axis represents a melanocyte lineage-specific mechanism of immune tolerance.