Cancer immunotherapies are a promising method for treating cancers and directly enhance the antitumor capabilities of a patient's immune system. In particular, therapeutic mRNA cancer vaccines are an attractive strategy as they offer a specificity and safety level that other immunotherapies are unable to achieve. Incorporation of tumor-specific or tumor-associated antigens in mRNA vaccines aims to enhance tumor antigen-specific T cells. mRNA vaccine adjuvants often encode immune stimulating proteins such as proinflammatory cytokines, costimulatory molecules, and pattern recognition receptors or their corresponding agonists. This review summarizes prominent clinical and preclinical therapies utilizing mRNA tumor-associated or tumor-specific antigens and mRNA encoding immunostimulants delivered either as vaccine adjuvants or as monotherapies for the treatment of cancer. The limitations, challenges, and future directions of therapeutic mRNA vaccines are also discussed.

Bezafibrate (BEZ) is a drug used to treat hypertriglyceridemia and its long-term use has been associated with reduced risk of cancer in patients with coronary artery disease. Recent studies uncovered that BEZ is a potent modulator of mitochondrial biogenesis through activation of PGC-1α/PPAR complexes, resulting in modulation of lipid metabolism and fatty acid oxidation. Mitochondria impact virtually all processes linked to oncogenesis, and disruption of normal mitochondrial bioenergetics and oxidative phosphorylation (OXPHOS) occurs early during oncogenesis to change the energy metabolism of cancer cells as well as various cells in the tumor microenvironment (TME). Therefore, we synthesized a BEZ analog (Mito-BEZ) that preferentially localizes to mitochondria, thereby enabling lower doses of Mito-BEZ than BEZ to achieve greater efficacy. Our studies demonstrate that Mito-BEZ is significantly more potent than BEZ at inhibiting LUAD cell growth in vitro and inhibiting lung tumorigenesis in preclinical mouse models. Mito-BEZ was also >200-fold more potent than BEZ at inhibiting both complex I and III in LUAD cells. Furthermore, Mito-BEZ suppresses oxidative metabolism in cancer cells while markedly upregulating mitochondrial function in effector CD8+ T cells, resulting in activation of a potent T cell immune response in the TME. Our results show that Mito-BEZ, with its favorable toxicity profile, exhibited a striking inhibitory effect on lung cancer progression and metastasis by targeting a fundamental difference in metabolic plasticity between cancer cells and effector T cells in the TME.

Lung cancer (LC) is a leading cause of cancer-related deathworldwide, and altered cholesterol metabolism is a hallmark of cancer cells. Acyl-CoA:cholesterol acyltransferase 1(ACAT1), or Sterol O-acyltransferase 1 (SOAT1), isa key cholesterol esterification enzyme. Its overexpression promotes tumorprogression by accumulating cholesterol esters. Inhibition of ACAT1 also potentiatesCD8+ T cells medicated anti-tumor immunity by increasing plasma membranecholesterol level. This study, as the first of its kind, shows the ACAT1/SOAT1 overexpressioncorrelates with poor prognosis in early-stage lung adenocarcinoma (LUAD) patients.Long-term treatment with ACAT1 inhibitor avasimibe suppresses tumorigenesis inboth Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growthfactor receptor (EGFR) mutation-induced LC mouse models without overttoxicity. ACAT1 inhibition reduces tumor cell proliferation, migration, andinvasion and causes G0/G1 cell cycle arrest, while boosting CD8+ T cells'effector function and memory phenotype. Single-cell RNA sequencing reveals thatACAT1 inhibition downregulates cholesterol biosynthesis and central carbon andnitrogen metabolism pathways in tumor cells, while upregulating genes relatedto oxidative phosphorylation and fatty acid oxidation in CD8+ T cells. Finally, avasimibe improves the efficacy of a human EGFR vaccine in preventing LCprogression. These novel findings suggest potential strategies for cancer preventionand therapy.

Triple-negative breast cancer (TNBC) is associated with a poor prognosis due to high recurrence rates and a lack of targeted therapies. Significant challenges in developing efficacious TNBC cancer vaccines are tumor antigen heterogeneity and the risk of antigen-negative variant escape, where target antigen-negative tumor cells can emerge, evading single-antigen vaccine-induced immunity, and drive tumor growth. To address this, we developed TNBCvax, a multi-antigen, multi-peptide vaccine targeting three tumor-associated antigens overexpressed in TNBC: TOP2A, HIF-1α and IGF-1R. The immune preventive effect of TNBCvax was evaluated in both a syngeneic M6 TNBC tumor graft model and the C3(1)/Tag genetically engineered mouse model of TNBC. Our findings demonstrate that TNBCvax significantly reduced tumor development and progression, compared to single-antigen vaccines. TNBCvax induced a robust tumor-associated antigen-specific immune response as evidenced by the increased infiltration of CD3+ T cells, particularly CD8+ T cells, with elevated levels of granzyme B and tumor necrosis factor alpha (TNF-α). TNBCvax was well-tolerated with no significant major organ toxicities, supporting its potential safety in the clinic. In conclusion, TNBCvax offers a promising immunopreventive strategy against TNBC by targeting multiple antigens to provide a broader and more robust immune coverage against TNBC antigens while reducing the risk of antigen-negative variant escape.

Abstract The V-domain Ig Suppressor of T cell Activation (VISTA) is expressed on cells of the myeloid and lymphoid lineages and is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. Furthermore, a tripeptide small molecule antagonist of VISTA, CA170, exhibited potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. We have previously demonstrated that KRAS or EGFR vaccines are effective in preventing lung tumorigenesis driven by either KRAS or EGFR in a pure prevention setting but are less effective in mice carrying existing lesions. Here, we determined if combining an MHC class II-restricted multi-antigen vaccine targeting KRAS or EGFR with CA170 will provide enhanced efficacy in preventing lung cancer progression. Using tetracycline-inducible KRAS and EGFR transgenic mice, we found that lung tumor development was significantly suppressed when CA170, delivered by aerosol inhalation to minimize systemic toxicity, was combined with either KRAS or EGFR MHCII-directed peptide vaccines using a post-initiation model. Flow cytometry and single-cell RNA sequencing (scRNA-seq) revealed that CA170 significantly increases tumor infiltrating CD8+ T cells and enhances effector-memory T cell frequencies and functions of both CD4+ and CD8+ T cells. These changes coincide with significant reductions in G-MDSCs (granulocytic myeloid-derived suppressor cells) and Foxp3+ Treg populations within lung tumors from mice treated with CA170. We found that the KRAS and EGFR preventive vaccines primarily induced the expansion of CD4+ effector T cells. VISTA antagonism by CA170 revealed strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory, and regulatory T cells, and drive an adaptive T cell tumor-specific immune response that enhances the efficacy of the KRAS and EGFR preventive vaccines in transgenic mouse models of lung cancer. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Ming You. Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7297.

The development of chemopreventive strategies with the ability to prevent the progression of lung lesions to malignant cancers would reduce the mortality and morbidity resulting from this deadly disease. Delivery of microRNA (miRNA) by inhalation is a novel method for lung cancer prevention. In this study, we investigated the combined efficacy of aerosolized miR-138-5p and miR-200c miRNA mimics in lung cancer prevention. Combination of the two miRNAs inhibited Benzo(a)pyrene (B((a))P)-induced lung adenomas and N-nitroso-tris-chloroethylurea (NTCU)-induced lung squamous cell carcinomas with no detectable side effects. Using single-cell RNA sequencing (scRNA-seq) and imaging mass cytometry (IMC), we found that both miRNAs inhibited programmed cell death ligand 1 (PD-L1) expression. Our flow cytometry results showed that aerosolized delivery of combined miRNAs increased CD4+ and CD8+ T cells and reduced the expression of programmed cell death protein 1 (PD-1) and T-regulatory cells. Our results demonstrated that the delivery of aerosolized microRNAs targeting PD-L1 can be highly effective in preventing lung cancer development and progression in mice.

supplementary figures S1-3. Supplementary Fig S1: Light photomicrographs of representative adenomas from the lung nodules. Supplementary Fig S2: H & E staining of representative normal lungs. Supplementary Fig S3: Baseline ECAR and OCAR level of H23 and h1299.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has a poor prognosis. TOP2A is a key enzyme in DNA replication and is a therapeutic target for breast and other cancers. TOP2A-specific Th1-promoting epitopes with optimal binding affinity to MHC II were identified using a combined scoring system. The multi-peptide TOP2A vaccine elicited a robust immunologic response in immunized mice, as demonstrated by the significant production of Th1 cytokines from immunized animals’ splenocytes stimulated in vitro with TOP2A peptides. Anti-tumor efficacy of the TOP2A vaccine was demonstrated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to adjuvant control. In a genetically engineered mouse (GEM) model of TNBC, vaccinated animals demonstrated a significant reduction in tumor incidence and average tumor volume compared to adjuvant control. Finally, we examined TCR sequences in CD4 tumor Infiltrating lymphocytes (TIL) from vaccinated mice and found that the TIL contained TCR sequences specific to the three vaccine peptides. These data indicate that our newly developed multi-peptide TOP2A vaccine is highly immunogenic, elicits TILs with vaccine specific TCRs, and is highly effective in preventing and intercepting TNBC development and progression in vivo.

The herb Prunella vulgaris has shown significant immune-stimulatory and anti-inflammatory effects in mouse models. Here, the effects of a novel Prunella vulgaris-containing herbal mixture, PV-1, were examined in several mouse models for cancer, including chemically induced models of lung and oral cancers as well as syngraft models for lung cancer and melanoma. PV-1, consisting of extracts from Prunella vulgaris, Polygonum bistorta, Sonchus brachyotus and Dictamnus dasycarpus, exhibited no toxicity in a dose escalation study in A/J mice. PV-1 significantly inhibited mouse lung tumor development induced by the lung carcinogens vinyl carbamate and benzo[a]pyrene. PV-1 also hindered the induction of oral squamous cell carcinomas in C57BL/6 mice caused by 4-nitroquinoline-1-oxide. Flow cytometry analysis showed that PV-1 increased the numbers of CD8+ tumor-infiltrating lymphocytes (TILs) and increased the production of granzyme B, TNF-α, and IFN-γ by CD8+ TILs. PV-1 also suppressed granulocytic myeloid-derived suppressor cell numbers (g-MDSCs) and improved the anti‐cancer activity of anti-PD‐1 immunotherapy. These results indicate that PV-1 remodels the tumor immune microenvironment by selectively inhibiting g‐MDSCs and increasing CD8+ TILs within tumors, resulting in decreased immune suppression and enhanced cancer chemopreventive efficacy.

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Epidermal growth factor receptor (EGFR) mutations occur in about 50% of lung adenocarcinomas in Asia and about 15% in the US. EGFR mutation-specific inhibitors have been developed and made significant contributions to controlling EGFR mutated non-small cell lung cancer. However, resistance frequently develops within 1 to 2 years due to acquired mutations. No effective approaches that target mutant EGFR have been developed to treat relapse following tyrosine kinase inhibitor (TKI) treatment. Vaccination against mutant EGFR is one area of active exploration. In this study, we identified immunogenic epitopes for the common EGFR mutations in humans and formulated a multi-peptide vaccine (E-mut Vax) targeting the EGFR L858R, T790M, and Del19 mutations. The efficacy of the E-mut Vax was evaluated in both syngeneic and genetic engineered EGFR mutation-driven murine lung tumor models with prophylactic settings, where the vaccinations were given before the onset of the tumor induction. The multi-peptide E-mut Vax effectively prevented the onset of EGFR mutation-driven lung tumorigenesis in both syngeneic and genetically engineered mouse models (GEMMs). Flow cytometry and single-cell RNA sequencing were conducted to investigate the impact of E-mut Vax on immune modulation. E-mut Vax significantly enhanced Th1 responses in the tumor microenvironment and decreased suppressive Tregs to enhance anti-tumor efficacy. Our results show that multi-peptide E-mut Vax is effective in preventing common EGFR mutation-driven lung tumorigenesis, and the vaccine elicits broad immune responses that are not limited to anti-tumor Th1 response.

Lung cancer is the leading cause of cancer death worldwide. Vaccination against EGFR can be one of the venues to prevent lung cancer. Blocking glutamine metabolism has been shown to improve anticancer immunity. Here, the authors report that JHU083, an orally active glutamine antagonist prodrug designed to be preferentially activated in the tumor microenvironment, has potent anticancer effects on EGFR‐driven mouse lung tumorigenesis. Lung tumor development is significantly suppressed when treatment with JHU083 is combined with an EGFR peptide vaccine (EVax) than either single treatment. Flow cytometry and single‐cell RNA sequencing of the lung tumors reveal that JHU083 increases CD8+ T cell and CD4+ Th1 cell infiltration, while EVax elicits robust Th1 cell‐mediated immune responses and protects mice against EGFRL858R mutation‐driven lung tumorigenesis. JHU083 treatment decreases immune suppressive cells, including both monocytic‐ and granulocytic‐myeloid‐derived suppressor cells, regulatory T cells, and pro‐tumor CD4+ Th17 cells in mouse models. Interestingly, Th1 cells are found to robustly upregulate oxidative metabolism and adopt a highly activated and memory‐like phenotype upon glutamine inhibition. These results suggest that JHU083 is highly effective against EGFR‐driven lung tumorigenesis and promotes an adaptive T cell‐mediated tumor‐specific immune response that enhances the efficacy of EVax.

Atovaquone, an FDA-approved drug for malaria, is known to inhibit mitochondrial electron transport. A recently synthesized mitochondria-targeted atovaquone increased mitochondrial accumulation and antitumor activity in vitro. Using an in situ vaccination approach, local injection of mitochondria-targeted atovaquone into primary tumors triggered potent T cell immune responses locally and in distant tumor sites. Mitochondria-targeted atovaquone treatment led to significant reductions of both granulocytic myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment. Mitochondria-targeted atovaquone treatment blocks the expression of genes involved in oxidative phosphorylation and glycolysis in granulocytic-myeloid-derived suppressor cells and regulatory T cells, which may lead to death of granulocytic-myeloid-derived suppressor cells and regulatory T cells. Mitochondria-targeted atovaquone inhibits expression of genes for mitochondrial complex components, oxidative phosphorylation, and glycolysis in both granulocytic-myeloid-derived suppressor cells and regulatory T cells. The resulting decreases in intratumoral granulocytic-myeloid-derived suppressor cells and regulatory T cells could facilitate the observed increase in tumor-infiltrating CD4+ T cells. Mitochondria-targeted atovaquone also improves the anti-tumor activity of PD-1 blockade immunotherapy. The results implicate granulocytic-myeloid-derived suppressor cells and regulatory T cells as novel targets of mitochondria-targeted atovaquone that facilitate its antitumor efficacy.

Top2A is a key enzyme involved in DNA replication and is a therapeutic target for several cancer types including breast cancer. Overexpression of Top2A has been observed in both human and mouse triple-negative breast cancer (TNBC). The present study evaluated both immunogenicity and antitumor efficacy of a newly formulated multi-peptide vaccine targeting multiple epitopes of the Top2A protein. Top2A-specific MHC II epitopes with optimal binding affinity were identified using a combined scoring system, which predicted their potential to elicit a Th1 immune response. The formulated vaccine contained top three Top2A peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. Anti-tumor efficacy of the Top2A vaccine was initially evaluated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to the adjuvant controls. The Top2A peptide vaccine exhibited striking efficacy in a genetically engineered TNBC mouse model (C3(1)/Tag), reducing tumor burden by >90% when compared with adjuvant alone. Splenocytes collected from vaccinated animals showed a robust immunologic response to the immunizing peptides. There were no overt toxicities observed with the Top2A vaccination. To explore potential mechanisms underlying the anti-tumor response induced by Top2A vaccine treatment, scTCR-seq of tumors in both control and Top2A vaccine groups revealed new T cell clones as a consequence of Top2A vaccination. Furthermore, in vitro stimulation of these splenocytes by the vaccinated Top2A peptides resulted in the secretion of cytokines indicative of Th1 responses but with minimal secretion of Th2-related cytokines. Our data indicate that the newly developed multi-peptide Top2A vaccine is immunogenic and efficacious in the prevention of TNBC development and progression in vivo. Citation Format: Sang Beom Lee, Jing Pan, Donghai Xiong, Katie Palen, Bryon Johnson, Jeffrey E. Green, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. Immunoprevention of triple negative breast cancer by TOP2A derived peptide vaccination [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA015.

Combined treatment targeting mitochondria may improve the efficacy of lung cancer chemoprevention. Here, mitochondria-targeted honokiol (Mito-HNK), an inhibitor of mitochondrial complex I and STAT3 phosphorylation, and mitochondria-targeted lonidamine (Mito-LND), an inhibitor of mitochondrial complexes I/II and AKT/mTOR/p70S6K signaling, were evaluated for their combinational chemopreventive efficacy on mouse lung carcinogenesis. All chemopreventive treatments began one-week post-carcinogen treatment and continued daily for 24 weeks. No evidence of toxicity (including liver toxicity) was detected by monitoring serum levels of alanine aminotransferase and aspartate aminotransferase enzymes. Mito-HNK or Mito-LND treatment alone reduced tumor load by 56% and 48%, respectively, whereas the combination of Mito-HNK and Mito-LND reduced tumor load by 83%. To understand the potential mechanism(s) of action for the observed combinatorial effects, single-cell RNA sequencing was performed using mouse tumors treated with Mito-HNK, Mito-LND, and their combination. In lung tumor cells, Mito-HNK treatment blocked the expression of genes involved in mitochondrial complex ǀ, oxidative phosphorylation, glycolysis, and STAT3 signaling. Mito-LND inhibited the expression of genes for mitochondrial complexes I/II, oxidative phosphorylation, and AKT/mTOR/p70S6K signaling in lung tumor cells. In addition to these changes, a combination of Mito-HNK with Mito-LND decreased arginine and proline metabolism, N-glycan biosynthesis, and tryptophan metabolism in lung tumor cells. Our results demonstrate that Mito-LND enhanced the antitumor efficacy of Mito-HNK, where both compounds inhibited common targets (oxidative phosphorylation) as well as unique targets for each agent (STAT3 and mTOR signaling). Therefore, the combination of Mito-HNK with Mito-LND may present an effective strategy for lung cancer chemoprevention.

MicroRNAs are potential candidates for lung cancer prevention and therapy. A major limitation is the lack of an efficient delivery system to directly deliver miRNA to cancer cells while limiting systemic exposure. The delivery of miRNA via inhalation is a potential strategy for lung cancer prevention in high-risk individuals. In this study, the authors investigate the efficacy of aerosolized let-7b miRNA treatment in lung cancer prevention. Let-7b shows significant inhibition of B[a]P-induced lung adenoma with no detectable side effects. Single-cell RNA sequencing of tumor-infiltrating T cells from primary tumors reveals that Let-7b post-transcriptionally suppresses PD-L1 and PD-1 expression in the tumor microenvironment, suggesting that let-7b miRNAs may promote antitumor immunity in vivo. Let-7b treatment decreases the expression of PD-1 in CD8+ T cells and reduces PD-L1 expression in lung tumor cells. The results suggest that this aerosolized let-7b mimic is a promising approach for lung cancer prevention, and that the in vivo tumor inhibitory effects of let-7b are mediated, at least in part, by immune-promoting effects via downregulating PD-L1 in tumors and/or PD-1 on CD8+ T cells. These changes potentiate antitumor CD8+ T cell immune responses, and ultimately lead to tumor inhibition.

Abstract Lung cancer is the leading cause of mortality worldwide. MicroRNAs (miRNAs) are potential candidates for lung cancer therapy. However, a major limitation is the lack of an efficient delivery system to directly deliver miRNA to cancer cells while limiting exposure to healthy cells. In our previous studies on aerosolized let-7b in lung cancer prevention, let-7b showed good inhibition of B[a]P-induced lung adenoma with no side effects. In this study, we found that aerosolized let-7b decreased tumor growth in the LKR13 (KRAS mutant) syngeneic mouse model. Let-7b post-transcriptionally suppresses PD-L1 and PD-1 expression in the tumor immune microenvironment, suggesting that let-7 microRNAs may promote antitumor immunity in vivo. Single cell RNA sequencing (scRNAseq) data showed that let-7b treatment decreased the expression of PD-1 in CD8+ T cells and reduced PD-L1 expression in lung tumor cells. Let-7b treatment also significantly changed the percentages of distinct CD8+ tumor-infiltrating lymphocytes (TIL) states. The proportion of CD8+ T cells mediating anti-tumor functions (EM-like CD8+ TILs) was increased significantly by let-7b treatment compared to control. In contrast, the CD8+ T cell subpopulation that has negative effects on anti-tumor immune response, exhausted CD8+ TILs, was significantly decreased by let-7b. Flow cytometry data showed that Let-7b treatment led to the accumulation of CD8+ T cells, granzyme B+ CD8+ T cells and IFN-γ+ CD8+ T cells in tumors, and a decrease of intratumoral Granulocyte-like myeloid derived suppressor cells (G-MDSC) cells. Our results suggest that the in vivo tumor-suppressive efficacy of let-7 is mediated, at least in part, by immune-promoting effects via down-regulating PD-L1 in tumors and/or PD-1 on CD8+ T cells. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Alberto Izzotti, Ming You. Tumor-suppressive efficacy of let-7b microRNA against lung carcinogenesis is mediated by modulating the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2369.