Restoration of p53 activity via intracellular protein delivery sensitizes triple negative breast cancer to anti-PD-1 immunotherapy

Background Although immune checkpoint inhibitors (ICIs) have been shown to yield promising therapeutic outcomes in a small subset of patients with triple negative breast cancer (TNBC), the majority of patients either do not respond or subsequently develop resistance. Recent studies have revealed the critical role of TP53 gene in cancer immunology. Loss or mutation of p53 in cancer cells has been found to promote their immune escape. Given the high mutation frequency of TP53 in TNBC cells, restoration of p53 function could be a potential strategy to overcome their resistance to anti-programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) therapy. Herein, we have assessed the use of Pos3Aa crystal-based platform to mediate the intracellular delivery of p53 protein to restore p53 activity in p53 null tumors and consequently augment anti-PD-1 activity.Methods The efficiency of Pos3Aa-p53 crystals in delivering p53 protein was evaluated using confocal imaging, immunofluorescence staining, flow cytometry and RNA-seq. The ability of Pos3Aa-p53 crystals to remodel tumor microenvironment was investigated by examining the markers of immunogenic cell death (ICD) and the expression of PD-L1, indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase 2 and type I interferon (IFN). Finally, both unilateral and bilateral 4T1 tumor mouse models were utilized to assess the efficacy of Pos3Aa-p53 crystal-mediated p53 restoration in enhancing the antitumor activity of ICIs. T cells in tumor tissues and spleens were analyzed, and the in vivo biosafety of the Pos3Aa-p53 crystal/anti-PD-1 antibody combination was also evaluated.Results Delivery of p53 protein into p53-null TNBC 4T1 cells via Pos3Aa-p53 crystals restored the p53 activity, and therefore led to the induction of ICD, activation of type I IFN signaling and upregulation of PD-L1 expression. Pos3Aa-p53 crystals significantly enhanced T cell infiltration and activation in 4T1 tumors, thereby sensitizing them to anti-PD-1 therapy. The combination of Pos3Aa-p53 crystals with anti-PD-1 antibody also induced a systemic antitumor immunity resulting in the inhibition of distal tumor growth with minimal toxicity.Conclusion This study validates that p53 restoration can be an effective approach to overcome ICI resistance and demonstrates that intracellular delivery of p53 protein can be an efficient, safe and potentially universal strategy to restore p53 activity in tumors carrying TP53 mutation

Overrepresented cell cycle genes in subclusters 3 and 5 and corresponding pathway enrichment analyses.

Overrepresented cell cycle genes in subclusters 3 and 5 and corresponding pathway enrichment analyses.</p

AD risk gene loci mapping to predicted CNV locations in subclusters 5, 3, and 0.

The yellow highlights indicate chromosomal locations of classic AD risk gene loci. The metadata underlying this figure can be found at https://zenodo.org/doi/10.5281/zenodo.10604562. (TIF)</p

Distribution of neuronal cells based on dataset source, sex, and disease status, as determined by cell cycle analysis in the integrated analysis setting.

(A) t-SNE plots of excitatory neuronal nuclei extracted from nondemented (ND) samples from different studies. (B) t-SNE plots of excitatory neuronal nuclei extracted from disease-affected (AD) samples from Mathys and colleagues and Lau and colleagues. (C) t-SNE plots of the excitatory neuronal nuclei distribution based on sex and disease status. (D) Violin plot illustrating the average feature counts of global transcriptomic profiles among excitatory neurons in different subclusters. (E) Violin plots presenting the cell cycle phase scores of all subclusters of excitatory neurons. Bolded violins highlighted in different phases indicate the subclusters that exhibit the most significant above-average cell cycle gene reexpression among all the subclusters. The corresponding significance values obtained for each subcluster compared to the rest of the others are shown in (F). The metadata underlying this figure can be found at https://zenodo.org/doi/10.5281/zenodo.10604562. (TIF)</p

Original cell cycle gene lists extracted from Whitfield and colleagues and Tirosh and colleagues studies and the final refined gene list.

Original cell cycle gene lists extracted from Whitfield and colleagues and Tirosh and colleagues studies and the final refined gene list.</p

Common marker genes shared between human and mouse late senescent (LS) neurons and pathway enrichment analysis.

Common marker genes shared between human and mouse late senescent (LS) neurons and pathway enrichment analysis.</p

Postmitotic cells other than excitatory neurons revealed limited signs of predicted CNV events.

Estimation of copy number variants by the InferCNV algorithm in (A) inhibitory neurons, (B) oligodendrocytes, and (C) astrocytes. The heatmap located at the top of each panel indicates the copy number alteration regions identified by the hidden Markov model, i.e., regions of gain (red) and loss (blue) in expression along each chromosome at various regions from the p-arm (left side of each box) to the q-arm (right side of each box), in all subclusters. The heatmap located at the bottom of each panel is an outcome of the Bayesian latent mixture model implemented to identify the posterior probabilities of alteration status in each cell and whole CNA region. Red: gain of copy number. Blue: loss of copy number. (JPG)</p

Pathway enrichment analysis of up-regulated DEGs in ESAD with reference to DisGeNET.

Pathway enrichment analysis of up-regulated DEGs in ESAD with reference to DisGeNET.</p

Validation of cortical layer markers by spatial transcriptomic analysis.

(A) Visualization of cortical layers in sample #151707 from the jhpce#HumanPilot10x dataset using spatialLIBD. (B) Visualization of the distribution and counts of cortical layer-specific markers per spot. (C) Boxplots showing the expression levels of various layer-specific markers across different spatial locations defined in (A) to validate their layer specificities. (D) t-SNE and (E) UMAP plots illustrating the differential enrichment of cortical layer-specific markers in different neuronal subclusters in the integrated cohort analyses. (TIF)</p