Kornelia Polyak

Tumor-suppressive immunity is more evident in early-stage compared to advanced tumors making it the ideal point for cancer interceptive immunotherapies. We previously described that higher peripheral T cell diversity is associated with more pronounced CD8 ⁺ T cell infiltration in ductal carcinoma in situ of the breast, implying a close interaction between peripheral and intratumor immunity. Here, we developed lipid nanoparticle (LNP)-encapsulated messenger ribonucleic acid (mRNA) vaccines expressing the alpha-lactalbumin (LALBA) protein unique to mammary luminal progenitors (LPs) to test whether enhancing immune response by prophylactic vaccination against the putative cell-of-origin of breast cancer suppresses tumorigenesis. Vaccination of outbred Sprague-Dawley rats with N1-methylpseudouridine-modified or unmodified Lalba mRNA-LNP induced different degrees of LALBA-specific and nonspecific immune responses. The vaccination suppressed carcinogen-induced mammary tumorigenesis and improved tumor-free and overall survival without obvious toxicity in the normal mammary glands and other organs. Single-cell transcriptomic analysis revealed that vaccination decreases the frequency of a proliferative LP population in immunoreactive early epithelial hyperplasia. Overall, we provide proof of principle that prophylactic Lalba mRNA-LNP has the potential to suppress the initiation and progression of early breast neoplastic lesions.

Triple-negative breast cancer (TNBC) has poor outcomes and few effective treatments, partly because of epigenetic heterogeneity. We show that the heterochromatin mark H4K20me3 is more variable than H3K9me3 across normal and malignant breast tissues, and that H4K20me3-enriched regions are linked to more open chromatin and active transcription. Pharmacologic inhibition of the H4K20me3 methyltransferases KMT5B and KMT5C with A-196 decreases H4K20me3 and increases H4K20me1 at loci involved in lineage identity, stress responses, and the cell cycle. A-196 suppresses tumor growth in vivo while promoting mesenchymal features and the unfolded protein response (UPR). Multi-omic profiling shows that mesenchymal TNBCs have the lowest H4K20me3 levels and the greatest sensitivity to UPR stress. In basal TNBC, KMT5B&5 C inhibition drives mesenchymal transition and elevated endoplasmic reticulum stress-associated growth arrest via E2F4. Combining A-196 with the HSP90, IRAK4 inhibitors, or STING agonist synergistically reduces TNBC tumor growth. These findings nominate KMT5B&5C as promising therapeutic targets in TNBC.

Supplementary Fig. S1. Analysis of differences between HER2hi and HER2lo cells. Supplementary Fig. S2. HER2-targeted therapy response in HER2 heterogeneous cell models. Supplementary Fig. S3. Characterization of HER2-targeting therapy resistant cells. Supplementary Fig. S4. The effect of co-culture on HER2hi and HER2lo cells. Supplementary Fig. S5. DNA barcoding experiment to compare monoculture and co-culture. Supplementary Fig. S6. Clonal evolution of HER2hi and HER2lo cells upon treatment with neratinib or T-DXd. Supplementary Fig. S7. Characterization of HER2 homogeneous and heterogeneous tumors. Supplementary Fig. S8. The effect of ABCC1 inhibition on response to T-DXd efficacy. Supplementary Fig. S9. The effect of USP9X on HER2 protein levels and response to T-DXd response.

Lists of proteins expressed in HCC1954 and 21PT HER2hi, HER2lo cells in monoculture and in co-culture (mix).

Lists of differentially expressed genes between parental and resistant cells in each model.

Each excel spreadsheet lists markers tested in each of the three cell line models.

Lists of differentially expressed genes between HER2hi and HER2lo cells in each model.

Lists of genes in the CRISPR screen with scores in control and T-DXd treated cells.

Lists of differentially expressed genes between HER2hi and HER2lo cells in the HCC1954 and 21PT models growing in monoculture or in co-culture.