Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's Disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (Tripartite Motif Family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2 822-982, a flexible interdomain region we designate the "Regulatory Loop" (RL). Phosphorylation of LRRK2 Ser910/935 within LRRK2 RL serves as a molecular switch controlling LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 prevents upregulation of LRRK2 kinase activity by Rab29 and also rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, modulating its cytoskeletal recruitment, turnover, kinase activity, and cytotoxicity.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry–based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911–919, a nine amino acid segment within a flexible interdomain region (LRRK2853–981), which we designate the “regulatory loop” (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2’s association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2’s interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase–dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.

Intratumoral heterogeneity (ITH)—defined as genetic and cellular diversity within a tumor—is linked to a failure of immunotherapy in multiple cancer types and to an inferior anti-tumor immune response even in the absence of therapy. To determine how tumor heterogeneity shapes the immune microenvironment and impacts responses to therapy, we modeled heterogeneous tumors comprised of a pro-inflammatory ("hot") and an immunosuppressive ("cold") tumor population. This was done by mixing two squamous skin carcinoma cell lines that alone give rise to tumors with a reproducible immunologically "hot" or "cold" immune phenotype respectively, and labeling these with YFP or RFP fluorescent tags to enable precise spatial tracking. The resulting mixed-population tumors were made up of a patchwork of distinct regions that could be classified as being comprised of YFP+ ("hot") cells, RFP+ ("cold") cells, or a mixture of YFP+ and RFP+ cells. We find the spatial organization of tumor cells creates a blueprint for the spatial organization and functional activity of infiltrating immune cells, defining the architecture of both tumor-infiltrating T cell and myeloid cell compartments. Using a combination of microdissection techniques and single-cell spatial transcriptomics, we show that YFP regions harbor a higher frequency of Th1 cells and IFNγ+ CD8 T cells compared to RFP regions, whereas immunosuppressive macrophages preferentially accumulate in RFP regions. Total CD4 T cells were also enriched in YFP regions, but by contrast, total CD8 T cells—despite their superior function in YFP regions—were of low abundance throughout the heterogeneous tumors. We identify the chemokine Cx3cl1, produced at higher levels by our "cold" tumors, as a mediator of intratumoral macrophage abundance, and show that overexpression of Cx3cl1 in pro-inflammatory ("hot") tumor cells leads to an increase in immunosuppressive CD206+ macrophages. We further interrogated the impact of this spatial patterning of immune cells on the responses of heterogeneous tumors to checkpoint blockade. We find that combination of PD-1 blockade and CD40 agonist is able to increase the Th1 response in "cold" RFP regions, however the T cell response in "cold" regions remains inferior to "hot" regions and treatment achieves only a modest reduction in tumor growth. Collectively, our results demonstrate that the spatial organization of heterogeneous tumor cells has a profound impact on directing the spatial organization and function of tumor-infiltrating immune cells as well as on responses to immunotherapy.

BackgroundIntratumoral heterogeneity (ITH) is cellular and molecular diversity within a tumor. ITH is linked to failure of immunotherapy in multiple cancer types. One of the suggested mechanisms of this failure is the absence of a productive immune response, which can be driven by the dominance of a tumor population that creates immunosuppressive microenvironment. However, the molecular mechanisms that mediate such dominant immunosuppressive effects and how a dominant immunosuppressive tumor population affects the efficacy of immune checkpoint blockade (ICB) therapy are poorly understood.MethodsWe generated a library of squamous skin cell carcinoma cell lines derived from DMBA+TPA carcinogen-treated mice. Upon transplantation into immunocompetent mice, two cell lines (CF6 and CF9) gave rise to highly and poorly immune-infiltrated tumors, respectively. These two cell lines were tagged with YFP and RFP, respectively, mixed at a 1:1 ratio and injected subcutaneously. The resulting mixed tumors contained a patchwork of distinct regions that are predominantly occupied with YFP (CF6) cells, occupied with RFP (CF9) cells, or are a mixture of YFP and RFP cells. These mixed tumors were microdissected into YFP, RFP, and mixed regions, and the immune infiltrate in each region was analyzed by flow cytometry.ResultsWe found mixed tumors were ”cold” on a whole-tumor level. However, regional analysis showed a higher frequency of total and CD4 T cells in YFP regions compared to RFP regions. In contrast, macrophages exhibited a preferential localization to RFP regions. This suggests that local factors unique to each region drive the immune infiltration patterns of CD4 T cells and macrophages. Interestingly, although CD8 T cells showed a low frequency in all regions, a majority of CD8 T cells from YFP regions produced IFNgamma in response to PMA/Ionomycin stimulation while IFNgamma+ CD8 T cells from RFP regions made up a small fraction. This suggests that the presence of CF9-RFP regions inhibits infiltration of CD8 T cells throughout the tumor, but may not affect the anti-tumoral potential of CD8 T cells that succeed in infiltrating CF6-YFP regions.ConclusionsAn immunosuppressive tumor population rendered our model heterogeneous tumors ”cold” on the whole. Nonetheless, we observed regional patterns in the quantity and quality of immune infiltrates, with local immune infiltration being shaped by the tumor cells present in that region. Understanding how these immune infiltration patterns impact the response of each region to ICB therapy will provide insights into the mechanism by which ITH poses barriers to immunotherapy.

Intratumoral heterogeneity (ITH) – cellular and molecular diversity within a tumor – is linked to failure of immunotherapy in multiple cancer types. A high degree of ITH is associated with poor infiltration of T cells into the tumor and resistance to immune checkpoint blockade (ICB) therapy. To determine how distinct tumor populations within heterogeneous tumors shape the immune microenvironment and how this impacts therapy response, we modeled heterogeneous tumors composed of an RFP-tagged immunosuppressive tumor population and a YFP-tagged pro-inflammatory tumor population. The resulting tumors contained a patchwork of distinct regions with YFP +cells, RFP +cells, or a mixture of YFP +and RFP +cells. Analysis of the immune infiltrates in each region revealed a higher frequency of total CD4 T cells, Th1 cells and IFNg +CD8 T cells in YFP regions compared to RFP regions, whereas macrophages exhibited the opposite pattern. PD-1 blockade and CD40 agonist combinatorial antibody therapy induced an increase in Th1 abundance in RFP regions, but the treatment did not clear the tumors. Together, these results reveal that distinct regional immune infiltration pattens within the tumor are driven by the local tumor cells present in each region, and that the treatment-induced global improvement of Th1 infiltration alone is not sufficient to induce tumor clearance. Moreover, we identified Cx3cl1 as a driver of a dominant immunosuppressive tumor microenvironment, marked by an increase in immunosuppressive CD206 +macrophages and decrease in anti-tumoral neutrophils and monocytes. These results suggest that an immunosuppressive tumor population drives immunotherapy resistance of heterogeneous tumors and Cx3cl1 as a new therapeutic target. Supported by grants from Parker Institute for Cancer Immunotherapy (Project Grant)