Ubiquitin-Specific Protease 6 mRNA Lipid Nanoparticles Ignite Antitumor Immunity and Suppress Tumorigenesis in Ewing Sarcoma

Abstract

Ewing sarcoma is an aggressive pediatric cancer that has remained refractory to current therapeutics. Immunotherapy has been unsuccessful in Ewing sarcoma, largely due to poor understanding of how its immune tumor microenvironment is regulated. We recently demonstrated that ubiquitin-specific protease 6 (USP6) can remodel the Ewing sarcoma immune landscape to engender an antitumorigenic tumor microenvironment. USP6 expression in Ewing sarcoma cells enhances surface expression of immunostimulatory ligands and receptors and induces production of multiple chemokines, driving recruitment and activation of tumor-suppressive immune lineages, including NK cells. We sought to harness this multifaceted immunostimulatory function into a novel therapeutic by delivering in vitro transcribed USP6 mRNA via ionizable lipid nanoparticles (LNP). Treatment of Ewing sarcoma cells with USP6 mRNA in vitro is capable of inducing the aforementioned antitumorigenic and immunostimulatory responses. In addition, USP6 mRNA-treated Ewing sarcoma cells elicit cytolytic activation of primary human CD8+ and CD4+ T lymphocytes and NK cells in vitro. Intratumoral delivery of USP6 mRNA LNPs suppresses growth of Ewing sarcoma xenografts, coincident with increased immune infiltration and activation. We further demonstrate that USP6 mRNA is capable of igniting an immunostimulatory program in other cancer types (including acute myeloid leukemia, melanoma, prostate cancer, head and neck cancer, and osteosarcoma) in vitro and suppressing acute myeloid leukemia xenograft growth in vivo. Treatment with USP6 mRNA LNPs was well-tolerated, with no observed gross toxicity. Together, these preclinical studies provide proof-of-concept for the immunogenic and antitumorigenic efficacy of USP6 mRNA LNPs and support its promise as a novel immunotherapeutic in diverse cancer types.

Figure 1:. Design and characterization of IVT USP6 mRNA.

A) Design of IVT HA-USP6 mRNA. Elements are detailed in text; pseudo-uracil, y; and 5-methylcytidine, m5C. B,C) RD-ES, A673, or TC-71 ES cell lines were transfected with the indicated μg of HA-USP6 mRNA or molar matched GFP mRNA using Lipofectamine Messenger Max. Flow cytometry was used to quantify (B) percent of live cells expressing GFP or HA-USP6 (by intracellular staining using anti-HA), and (C) cell viability (within the entire sample) using Zombie UV vital stain. D) RD-ES were transfected with HA-USP6 (0.5 or 1.0 μg) or molar matched GFP mRNA, lysed the following day, and subjected to immunoblotting as indicated. For PARP, full length (closed triangle) and cleaved product (open triangle) are highlighted. E,F) RD-ES or A673 cells were transfected with HA-USP6 mRNA (0.5μg), molar equivalent GFP mRNA, or not transfected (NT). On the indicated days, live cell number and percent death were quantified by flow cytometry using 7AAD stain; p values are provided for USP6 vs GFP transfected samples. All experiments were performed at least 3 times, with technical duplicates for each sample.

Figure 2:. IVT USP6 mRNA recapitulates paracrine and cell autonomous immunostimulatory effects in ES in vitro.

RD-ES were transfected with HA-USP6 mRNA (0.5μg in A/B, or the indicated amount in C) or molar-matched GFP mRNA, or not transfected (NT). After 24h, cells were harvested for: (A) RNA isolation and RT-qPCR, (B) ELISA to quantify CXCL10 and CCL5 protein in the conditioned medium, or (C) flow cytometry to monitor surface expression of the indicated markers. In C, GFP and HA-USP6 transfected cells were identified by expression of GFP or anti-HA, respectively. Surface expression of the indicated markers was quantified in the transfected (GFP⁺ or HA⁺) and non-transfected (GFP⁻ or HA⁻) subpopulations. Functional relevance of each factor is described in Supplementary Figure S2. Experiments in A and C were performed at least 3 times; for B, samples were taken from 4 independent experiments, with duplicate or triplicate samples from each.

Figure 3:. USP6 mRNA-treated ES cells induce activation of immune lineages from PBMCs.

RD-ES cells were transfected with HA-USP6 mRNA (0.5μg) or molar-matched GFP mRNA. Cells were washed after 6h to remove residual mRNA that was not taken up, then co-cultured with primary human PBMCs for 1 or 3 days. Surface expression of the indicated markers was examined. Activation (CD69) and degranulation (CD107a) were examined in (A) NK cells and (B) CD8+ and CD4+ T lymphocytes. C) CD40 and HLA-DR were examined in classical and intermediate monocytes. Lineages were gated as follows: NK cells (Viable-> CD45+-> FSC/SSCLow-> CD3--> CD56Bright/Dim/CD16Bright/Dim); CD8+ T cells (CD45+-> FSC/SSCLow, CD3+-> CD4-CD8+); CD4+ T cells (CD45+-> FSC/SSCLow, CD3+-> CD4+CD8−); classical monocytes (CD45+-> FSC/SSCHigh> CD14++CD16−); and intermediate monocytes (CD45+-> FSC/SSCHigh-> CD14++CD16+). Results are from at least 3 independent experiments, with each sample examined in singlicate for each experiment.

Figure 4:. IT injection of USP6 mRNA LNPs induces immune infiltration and suppresses ES tumor growth.

A) Schematic showing synthesis of in vivo-grade mRNA LNPs (left), dynamic light scattering to measure similar sizes of the GFP and USP6 mRNA LNPs (middle), cryo-transmission electron microscopy (inset) to confirm multilamellar structure of USP6 LNPs, and TNS assay on the USP6 LNPs to demonstrate a pKa of ~6.06 (right). B, C) Nude mice bearing subcutaneous A673 tumors between 400–500 mm³ were injected once intratumorally with PBS, or LNPs encapsulating USP6 (15μg) or equimolar GFP mRNA. Tumors were excised after 1, 2, and 3 days (n=3 for each treatment group for each timepoint). B) RNA was isolated from tumors, and expression of GFP and USP6 was quantified by RT-qPCR. C) Flow cytometry was performed on the 9 USP6 mRNA-injected tumors (3 each from Days 1, 2 and 3), quantifying the immune infiltrate (CD45⁺ cells among total live cells). Percentage of CD45⁺ cells was plotted vs. USP6 expression level (CT value normalized against GAPDH). D-G) RDES were subcutaneously xenografted into nude mice; when tumors reached 150–200 mm³, mice were randomly enrolled into one of four treatment groups: PBS, USP6 mRNA LNPs (5μg or 15μg), or GFP mRNA (molar matched to 15μg) (n= 8–9 per group). Intratumoral injections were repeated every 4–5 days until terminal tumor volume (1250 mm³) was reached, or 26 days after the initial injection (whichever occurred first). Total number of doses ranged from 3–6 depending on tumor growth rate. D) Average tumor volume for each cohort and associated p values (E); F) percent of mice whose tumors have yet to reach terminal volume; and G) individual tumor growth curves.

Figure 5:. IT USP6 mRNA LNPs induce infiltration and activation of multiple immune lineages with no adverse effects.

A-D) A673 were subcutaneously xenografted into RAG2^−/− mice, and tumors injected with LNPs carrying USP6 (15μg; n=5) or molar matched GFP mRNA (n=6) as in Figure 4D. A) Tumor growth curves for individual mice. B) Masses of tumors, animals, livers, and spleens at necropsy were recorded. C) RNA was isolated from tumors, tumor-adjacent muscle, spleen and liver. RT-qPCR was performed on tumor and tissues to quantify expression of USP6 (left), and on tumors to quantify expression of human CXCL10 and CCL5, and murine IFNA and IFNG (right; normalized against GAPDH). D) RT-qPCR was performed on tumors for the indicated genes. E) Flow cytometry was performed on tumor digests to quantify levels of total immune cells and the indicated lineages (% of each is relative to total live cells). F) Nude mice were bilaterally xenografted subcutaneously with A673 cells (2.5E6 cells/site). When tumors became palpable (~200mm³), mice were randomly assigned to cohorts in which one tumor (denoted primary tumor) was treated intratumorally with PBS (n=2) or mRNA LNPs (USP6 (15μg; n=6) or molar-matched GFP; n=5); injection was repeated after 4 days. Volumes of treated primary tumors and untreated “distal” tumors at the indicated timepoints is shown.

Figure 6:. USP6 mRNA triggers immunostimulatory effects and suppresses tumorigenesis in acute myelocytic leukemia (AML).

A,B) MV4;11 AML cells were transfected with molar-matched GFP or HA-USP6 mRNA; NT, not transfected. A) Samples were subjected to RT-qPCR to monitor expression of the indicated chemokines. B) Flow cytometry was performed to quantify surface expression of the indicated markers, examining the HA⁺ and HA⁻ populations in the HA-USP6 transfected samples, and the GFP⁺ (G⁺) and GFP⁻ (G⁻) populations in the GFP transfected samples. C-G) Nude mice were xenografted subcutaneously with MV4;11 AML cells. When tumors reached 300–400 mm³, animals were randomly enrolled into treatment cohorts, and injected IT every 4 or 5 days with PBS, USP6 mRNA LNPs (15μg), or molar matched GFP mRNA LNPs (n= 8–9 per group). Animals were sacrificed when tumors reached terminal volume (1250 mm³). or after 30 days (whichever occurred first). C) Average tumor volume in each cohort and p values. D) Percent of mice remaining that have yet to reach terminal volume. USP6 mRNA LNP-treated mice were subdivided into subcohorts USP6-a and USP6-b, with the latter showing greater suppression of tumor growth. E) Growth curves for individual tumors. F/G) Tumors were digested and immune lineages and indicated markers were quantified by flow cytometry. Lineages were defined as follows: neutrophils (Ly6g+Ly6c+); macrophages (F4/80+); inflammatory monocytes (Ly6c^high Ly6g−); classical monocytes (Ly6c⁺Ly6g−), and NK cells (NKp46+).