Potential role of p53 deregulation in modulating immune responses in human malignancies: a paradigm to develop immunotherapy

Abstract

The crucial role played by the oncogenic expression of TP53, stemming from mutation or amyloid formation, in various human malignancies has been extensively studied over the past two decades. Interestingly, the potential role of TP53 as a crucial player in modulating immune responses has provided new insight into the field of cancer biology. The loss of p53’s transcriptional functions and/or the acquisition of tumorigenic properties can efficiently modulate the recruitment and functions of myeloid and lymphoid cells, ultimately leading to the evasion of immune responses in human tumors. Consequently, the oncogenic nature of the tumor suppressor p53 can dynamically alter the function of immune cells, providing support for tumor progression and metastasis. This review comprehensively explores the dual role of p53 as both the guardian of the genome and an oncogenic driver, especially in the context of regulation of autophagy, apoptosis, the tumor microenvironment, immune cells, innate immunity, and adaptive immune responses. Additionally, the focus of this review centers on how p53 status in the immune response can be harnessed for the development of tailored therapeutic strategies and their potential application in immunotherapy against human malignancies.

1. Introduction

One of the most common genetic aberrations in human malignancies is the loss of function of the TP53 gene (tumor suppressor; localized on chromosome 17) either due to loss of heterozygosity or somatic, and germline mutations[1–4]. These genetic dysregulations are linked with an increased risk of cancer[5]. The very first evidence came from the Li-Fraumeni Syndrome where these patients represent the predominance of TP53 germ-line mutation leading to the development and progression of different types of cancers[6, 7]. The frequent mutations in the TP53 gene are observed in a variety of solid tumors and leukemia[8–13]. Several TP53 mutations are well studied and characterized which display loss of DNA binding ability as well as gain of oncogenic potential especially when these mutations are present in hotspots[14, 15]. The p53 is an essential transcription factor with defined functions in regulating the expression of a variety of genes involved in the cell cycle, senescence, apoptosis, DNA damage, genome instability, and cellular metabolism[16, 17] (Fig. 1).

Fig. 1. Timeline figure showing the key milestone in the fields of p53 research and immunotherapy.

p53 research advancement from the discovery to targeting mutant p53 is presented below the arrow. All the key advancements in the field of immunology from 1883 are represented in the upper panel of the figure. The year 2003 marked the merge of both fields when clinical trials related to gene therapy with adenovirus expressing p53 were tested in Head and Neck cancer. Onwards several clinicals were conducted using p53 to elicit the immunological responses.

In normal cells, p53 levels are suppressed by its negative regulator murine double minute 2 (MDM2) by inducing ubiquitination and degradation of the p53 protein. Under stress conditions, p53 is phosphorylated thereby inhibiting the interaction of p53 with MDM2, resulting in its stabilization and activation of p53[16]. Also, p53 was reported to reverse the cell cycle arrest under stress conditions, thereby allowing the cells to proliferate[18, 19]. In contrast, the mutant p53 can drive the expression of several anti-apoptotic genes thereby driving the cell toward tumorigenesis. p53 mutants can participate in EMT and stemness leading the cell toward metastasis[20]. Interestingly, it was discovered that the loss of p53 functions in several cases was caused by the wildtype p53’s inability to enter the nucleus and its subsequent accumulation in the cytoplasm[21]. Therefore, the usual protective function of wildtype p53 in the cells was eliminated by another mechanism which to date is a weakly understood process that underlies this unique cytoplasmic preservation of wildtype p53. Additionally, it has been shown that mutant p53 can accumulate both cytoplasmic and nucleus in a subset of primary human tumors, including inflammatory breast carcinoma, undifferentiated neuroblastoma, and colon carcinoma, among other tumor types[20, 22–24]. Immunocytochemistry detected mutant p53 in each of these instances as distinct punctate structures that were resistant to Mdm-2-mediated destruction[25].

To understand p53 aggregation, and characteristics in tumor biopsies, a thorough screening of 163 biopsy tissues, with mutant or wildtype p53, was recently carried out. Additionally, patients with tumors containing p53 aggregates across six different cancer types demonstrated poor clinical outcomes, which were comparable to those in patients with p53 expression loss[26]. These findings demonstrate that p53’s functional inactivation by mutation and/or aggregate formation plays a vital role in the tumor’s malignant state[27]. Since p53 aggregates were demonstrated in a variety of cancer tissues and cells, it is logical to assume that p53 can form amyloid fibrils, which could result in a loss of function[28–31]. Natively structured and unstructured proteins and peptides undergo a structural change during the amyloid formation, resulting in partially folded intermediates that can self-associate to form oligomers followed by the cross β-sheet-rich fibrils[32, 33]. In this context, several studies have shown that p53 has the propensity for amyloid formation which resulted in the loss and gain of function phenotypes in cells and tissues[28–31, 34]. p53 amyloids have been observed to drive the expression of anti-apoptotic and proliferative genes leading the cell toward tumorigenesis[28]. These characteristics are demonstrated to be heritable, due to the transmissibility properties of the p53 prions[28]. Several of the hotspot p53 mutants were reported to aggravate these amyloid kinetics by destabilizing the protein fold[28, 35, 36]. The cells containing p53 amyloids also resulted in the establishment of tumors in mouse xenograft models[31]. The emergence of tumor-like lesions in immunosuppressed SCID mice after p53 core fibrils injection supports the link between p53 amyloid and cancer initiation. Further, the role of the immune system was assessed by using immunocompetent (C57BL/6) mice which showed delayed tumor induction and a lower rate of tumorigenesis[37]. However, any direct mechanism of immune modulation by p53 amyloids is still unexplored. Altogether, several roles have been noticed for p53 that are suggestive of the fact that p53 can regulate diverse molecular mechanisms and biological pathways in human malignancies[38].

In 1909, Paul Ehrlich was the first scientist to propose that the immune system can regulate the growth of tumor cells[39]. Burnet and Thomas have demonstrated this fact and coined the term ‘immune surveillance[40, 41]. This is an independent line of defense by the immune system called immunosurveillance- where immune cells can identify and destroy the tumor cells thereby helping in the eradication of tumor cells from the body[42, 43] (Fig. 2). This has been observed that the components of innate and adaptive immune responses play a crucial role in immune surveillance including T helper (Th; CD4⁺) cells, T cytotoxic (Tc; CD8⁺) cells, and natural killer (NK) cells with very few cases where neutrophils are also involved[44–46]. On the other immune suppressive cells like regulatory T cells (Tregs), myeloid-derived suppressive cells (MDSC), and macrophages have been reported to promote tumor progression. Treg and MDSCs allow the tumor cells to survive and grow aggressively by inhibiting the functions of both CD4⁺ and CD8⁺ T cells while macrophages and polymorphonuclear cells (PMNs) can support and favor the process of angiogenesis, metastasis, and suppressive immune environment within the tumor via modulation of suppressive cytokines, immune checkpoints, and other surface ligands[47–49]. Several key oncogenic events like gain of function mutations, amplification, and activation of oncogenes (like HRAS, KRAS, MYC) as well as loss of function, and deletion, have been displayed to be associated with the evasion or inhibition of anti-tumor immune responses (PTEN, TP53)[50–52]. The emerging pieces of evidence have shown that p53 is one of the key players in regulating crosstalk between tumor and immune cells within the tumor microenvironment[52–54].

Fig. 2. Schematic showing the major steps involved in immune editing.

The first step in anticancer immunity is elimination where immune cells survey and destroy the malignant cells by producing inflammatory cytokines, and activation of anticancer immune responses. The next step in the process of carcinogenesis is the equilibrium where the activation of T cells, IL-12, and interferon-gamma keeps check on the tumor cells. Also, the tumor cells modulate the expression of negative immune regulators to maintain them in a dormant state. The last state is an escape which is marked by the ability of tumor cells to grow and expand quickly inside the body thereby escaping from immune cells by following mechanisms like shedding tumor recognition antigens, accumulation of immunosuppressive cells like Tregs, MDSC, TAMs, expression of immune checkpoint like PD-L1, PD-L2, CTLA4 leading to tumor progression.

In the present review, we have discussed the potential role of p53 in autophagy, apoptosis, modulation of the immune responses during cancer progression within the tumor microenvironment, and a particular focus on enhancing the power of p53-mediated immune responses for the development of immunotherapeutics for human cancers.

2. p53 mediated crosstalk between autophagy and apoptosis

Autophagy and apoptosis are vital catabolic pathways for maintaining organismal homeostasis[55]. Autophagy serves as a cell-survival mechanism by recycling obsolete cellular components under stress conditions[56]. However, excessive autophagy can contribute to type II cell death[57]. Both autophagy and apoptosis are interconnected through a variety of molecular crosstalk to influence tumor suppressor pathways for the prevention of human cancers[57]. However, autophagy has been reported to perform dual functions in cancers either by promoting survival or inducing cell death. Interestingly, autophagic modulation impacts apoptosis, which is regulated by factors like AMPK, MAPK, PI3K-AKT, BECN1, ATG proteins, and non-coding RNAs[58].

p53 is a well-known master regulator for both the extrinsic and intrinsic apoptotic pathways by influencing death receptors, caspases, and mitochondrial dynamics[59]. Also, p53 can enhance apoptosis through transcriptional regulation of pro-apoptotic and anti-apoptotic genes[59]. The translocation of p53 within the mitochondria resulted in the engagement with the members of the Bcl-2 family, a group of proteins comprising both anti-apoptotic mediators (Bcl-2, Bcl-x, Bcl-XL, Bcl-XS, Bcl-w, BAG, Mcl-1) and pro-apoptotic mediators (Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, Blk)[16]. The delicate balance between these mediators dictates the cellular choice between survival and apoptosis by regulating mitochondrial outer membrane permeabilization and the subsequent release of cytochrome c into the cytoplasm[60]. Moreover, p53 also intricately regulates autophagy in a manner contingent on its cellular location and the specific context. The cytoplasmic p53 generally inhibits autophagy by interacting with FIP200, preventing ulk1-fip200-Atg13-Atg101 complex activation and autophagosome formation[61]. Conversely, nuclear p53 promotes autophagy by suppressing the PI3K/AKT/mTOR signaling pathway through direct interaction with PTEN and enhancing the expression of autophagy-related genes[62]. This increased autophagy significantly contributes to p53-dependent apoptosis resulting in cancer suppression but can paradoxically promote cancer in cells with impaired p53.

The mutant p53 proteins play a significant role in various autophagic pathways involved in cell migration, proliferation, invasion, and anti-apoptotic functions[63]. The mutant p53 proteins influence autophagy by suppressing vesicle formation and lysosome fusion through transcriptional repression of key downstream autophagy-associated genes[63]. This can indirectly block autophagy by interacting with transcription factors and triggering growth factor receptors, contributing to sustained PI3K/Akt/mTOR signaling in cancer cells[64]. For example, in colorectal carcinoma, p53 is frequently mutated and loss of p53 is linked to the overproduction of LC3, a marker for autophagosomes, under prolonged nutrient deprivation[65]. Mutant p53 expression in AML cells is associated with increased autophagy, while pancreatic and breast cancer cell lines with mutant p53 exhibit reduced autophagy[66]. This discrepancy may be attributed to p53 localization in the subcellular compartment of the cells; cytosolic p53 mutants suppress autophagy, whereas nuclear counterparts do not. These variations in p53 mutant impact on autophagy can influence responses to autophagy inhibitors offering potential avenues for cancer treatment through autophagy modulation[62]. The upregulation of mutant TP53 in AML cells reduces sensitivity to hydroxychloroquine (HCQ) treatment whereas the TP53 wild-type AML cells were sensitive to the HCQ treatment suggesting the importance of autophagy inhibitors in AML with wild-type TP53[67]. Similarly, the mutated p53 glioblastoma cells were less responsive to chloroquine (CQ) treatment[68].

Recently, the involvement of p53-mediated autophagy has been noticed in inflammation and immune responses as an immune effector[69]. Autophagy plays an essential role during thymic selection, lymphocyte homeostasis, antigen presentation, cytokine regulation, and in both innate and adaptive immunity. For instance, pro-inflammatory cytokines such as IL-1α and IL-1β can induce autophagy[70]. In the case of innate immunity, autophagy works downstream of pattern recognition receptors while activating receptors like TLRs and NLRs[71]. This is important because it promotes a variety of effector responses, including phagocytosis, cytokine production, and NKT cell activation[71]. In the case of adaptive immunity, autophagy contributes as an important source of antigens to be presented onto histocompatibility complex-II (MHC-II) and may be crucial for cross-priming CD8+ T-cells[72]. The dynamic relationship between p53, apoptosis, autophagy, and its relation to immune responses underscores p53 pivotal role in cancer progression.

3. p53 mediated immune responses

The extended role of p53 is found in the process called inflammation[73, 74]. The wild-type p53 is an important key regulator in autoimmune diseases[75] However, the deregulation of p53 can lead to autoimmune diseases like arthritis, diabetes, and cancer by suppressing the expression of many pro-inflammatory factors[76, 77]. p53 directly takes part in the activation of various immune signaling pathways including pathogen sensing and the production of cytokines[52]. p53 can upregulate the expression of MHC class I molecules thereby enhancing the role of CD8⁺ T cells which successfully target endogenous pathogens, especially the viruses and tumor antigens (intracellular pathogens). p53 is also reported as a transcriptional target of type 1 interferon (IFN) signaling [78, 79]. The positive feedback loop was discovered between p53 and the expression of IFN as p53 was directly associated with the transactivation of Interferon regulatory factor 9, which is a key component of the IFN signaling pathway. This transactivation leads to the activation of innate immune signaling and the production of many cytokines and chemokines including IFNα, IFNβ, and CCL2. Therefore, it is evident that p53 has a vital role in the production of cytokines and chemokines during viral infections and other cellular stress like ROS. Further, p53 regulates antigen cell-signaling pathways of T and B lymphocytes. Therefore, p53 is involved in various adaptive and innate immune pathways like pathogen recognition (TLRs). p53 protein plays a vital role by activating a variety of immune cells like T-cells, B-cells, natural killer cells, and reprogramming of macrophages which is discussed shortly. Recently, programmed cell death protein-1 (PD-1) was discovered as one of the direct targets of p53. This study revealed that acetylation of p53 at K120/164 residues was essential for the transcription of PD-1. Moreover, acetylated p53 recruited cofactors required for acetyltransferase activity at the PD-1 promoter that increased PD-1 transcription. Interestingly, HDAC inhibitors demonstrated PD-1 activation through p53 acetylation, suggesting that p53-dependent PD-1 activation is important for anti-tumor immunity[80].

4. Potential role of p53 in modulating the tumor immune environment

Immune cells play an essential role in the tumor microenvironment (TME) and have a prominent impact on cancer progression, metastasis, drug resistance, and prognosis. Several studies have displayed that p53 regulates the variety of functions[81] of immune cells and thereby keeps neoplastic growth in check[76]. Mutant p53 or loss of p53 can alter the function of the immune system, and secretion of cytokines/chemokines leads to evasion of the anti-tumor immune responses, tumor progression, and metastasis[52]. Interestingly, this has been observed that p53 knockout murine models were highly vulnerable to inflammation and autoimmunity to favor the initiation and progression of several diseases including cancers[82]. Further, the involvement of p53 was noticed as an important event in modulating the immune microenvironment through the alteration of the immune cell population (Fig. 3). For instance, depletion of p53 in cells of myeloid origin enhances the formation of intestinal tumors in the Apc (Min/+) murine model whereas the restoration of p53 in the cells of myeloid lineage impaired the inflammatory response and tumor progression[83]. This study reported that the restoration of p53 inhibited the polarization of tumor-associated macrophages (TAMs; M2) and Myc expression. A marked increase in the TAMs population is associated with the loss of p53 in pancreatic carcinoma, melanoma, pancreatic cancer, and ovarian carcinoma[84, 85]. A positive correlation between loss of p53, macrophage infiltration, and colony-stimulating factor 1 (CSF1) signaling has been observed. The blocking of CSF1R using AZD7507 and PLX3397 was found to decrease tumor growth through suppression of TAMs in mutant-p53 pancreatic tumors[86]. The CSF1R blockade enhanced the sensitivity of these p53 mutant pancreatic cancer cells toward immune checkpoint inhibitors[86, 87]. Guo and colleagues have reported that the melanoma cells (B16F1) accelerated the tumor growth in the p53^null murine model along with a significant increase in the population of T-regulatory (FoxP3⁺) cells, CD11b⁺Gr-1⁺ myeloid-derived suppressor (CD11b⁺Gr-1⁺) cells, with the loss of effector function than p53-WT murine[88]. Further, nutlin-3a treatment was shown to upregulate p53 in TME through the infiltration of leukocytes and induction of antitumor immunity. This study suggested that targeting the p53 specifically TME may be utilized to break the immunosuppressive environment with limited toxicity[89]. Apart from this, it has also been observed that the loss of function of p53 due to mutations in breast cancer cells leads to dysregulation of the WNT signaling by this means increasing the number of neutrophils in the tumor microenvironment, which helps in the progression, metastasis, and growth of the oncogenic cells. Several groups have further confirmed that the loss of function of p53 in human malignancies such as prostate, breast, and ovarian leads to the recruitment of tumor-supporting myeloid cells[52]. Blagih and colleagues have demonstrated that the reprogramming of myeloid cells into tumor-promoting macrophages within the p53 null tumors was due to the secretome of p53-deficient malignant cells and upregulation of CXCR3/CCR2 and M-CSF[90]. Moreover, R248W p53 mutants have been found to gain the capability to modify and alter the behavior of the macrophages, thereby accelerating tumor growth. Interestingly, the knockout murine model of pancreatic cancer harboring Kras^G12D/+; Trp53^R172H/+ revealed the enrichment neutrophils (CD11b⁺Ly6G⁺) and decreases in T-cell populations[85]. Inhibition of neutrophil-associated chemokine (CXCL2) resulted in the suppression of neutrophil phenotype. Depletion of neutrophils in this murine model sensitizes the CD40 immunotherapy and gemcitabine[85].

Fig. 3. Role of wildtype p53 and mutant p53 in tumor microenvironment.

The tumors with mutant p53 can reprogram macrophages into tumor-promoting macrophages or M2 macrophages for tumor progression, and metastasis (Right). On the contrary, the expression wild wild-type p53 repressed polarization of macrophages and maintained an antitumor immune environment (Left). In the context of cancer-associated fibroblasts (CAFs), mutant p53 has been observed to negatively impact the proliferation and immune responses of CD8+ and CD4+ T-cells. Interaction between cancer-associated fibroblasts (CAFs) and cancer cells triggers the Interferon-β pathway within CAFs, which, in conjunction with wild-type p53 present in fibroblasts, hinders the tumor growth, and migration of cancer cells. Conversely, the presence of mutated p53 modulates the functions of CAFs to promote cancer cell proliferation, metastasis, and drug resistance. Importantly, p53 facilitates the activation of programmed death-ligand 1 and L2 (PD-L1/L2) and its corresponding receptor programmed death-1 (PD-1) in both cancer cells and normal T-cells in response to stress, thereby suppressing the activity of CD8+ T cells.

T-cells are a crucial component of the immune system for generating effective anti-tumor immunity for eliminating malignant growth[91]. T-regulatory cells (Tregs) are highly immune-suppressive cells that help in the maintenance of immune homeostasis and self-tolerance through the expression of FoxP3[91]. Treg cells prevent the anti-tumor activity of the T-cells by suppressing the proliferation of T-cells and the production of cytokines. The mutant p53 has been noticed to increase the number of Treg cells leading to inhibition of the T-cells mediated immune responses (Fig. 3). The loss of p53 in human cancers was correlated with the enrichment of Treg populations and repressed effector T cells in the periphery and tumor mass[92]. In ovarian, pancreatic, and prostate cancers, the loss of p53 is displayed as increased intratumoral Treg cells. The de novo differentiation of Tregs was induced by p53-null tumor-guided PMNs in prostate cancer[93]. The p53-null tumor was positively linked with highly suppressive Treg cells which in turn suppressed CD4⁺ and CD8⁺ T-cell responses in the murine model as compared to p53-WT tumors and this was independent of Kras mutation[90]. CSF1R signaling inhibition reactivates the anti-cancer response of the T-cells by reducing the number of Treg cells[87, 90].

Also, the recognition of the antigenic peptide through T-cell receptors is an important step for T-cell activation. Once T-cells are fully activated, the effector T-cells switch on the expression of co-inhibitory receptors like PD-1 to keep protective immunity in check[94]. This has been reported that cancer cells overexpress inhibitory ligands such as PD-L1, and PDL-2 to suppress T-cell activity[95]. The p53 mutations have been involved in regulating the expression of PD-1 in cancers like Diffuse large B-cell lymphoma, and TNBC (Fig. 3). The miR-34a is one of the transcriptional targets of p53 and is displayed to regulate PD-L1 expression[96]. Moreover, the loss of p53 activity increases PD-L1 surface expression, which can suppress T-cell function[96]. Altogether, these data indicated that the expression of mutant p53 in cancers is correlated with increased PD-L1 expression, which may help to identify patients responsive to checkpoint inhibitors targeting PDL1 (Fig. 3). Recently, several clinical trials have been initiated as first for the patients with p53 mutations and PD-L1 expression using PD-1 antibody and R-CHOP (ClinicalTrials.gov, Identifier: NCT05280626).

Future research in this direction could focus on exploring how mutant p53 influences the functioning of the different cell types within the TME, expression of immune checkpoint ligands, like PD-L1/2, and CTLA4, and their subsequent effects on T-cell function. These studies can inform the development of strategies to manipulate the TME for enhanced cancer immunotherapy and improved patient outcomes.

5. Effect of mutant p53 on immune responses

5.1. Mutant p53 and extracellular matrix remodeling

Extracellular matrix (ECM) is majorly responsible for providing structural support, aiding in cell-cell adhesion, communication, and cell movement as they form a complex network of proteins and macromolecules[97]. The tumor cells can easily reprogram the extracellular matrix to form a positive tumorigenic environment. p53 has been shown to have a prominent role in regulating ECM, especially in hypoxic conditions[53]. Recent studies have demonstrated that p53 is the key regulator of rearrangement of the extracellular matrix as they upregulate the expression of certain transcription factors like the hypoxia-inducible factor (HIF) and vascular endothelial growth factors (VEGF) which have pro-angiogenic activity[98]. The loss of p53 results in cytokine production such as the macrophage-colony stimulating factor in the TME which leads to the infiltration of immunological suppressor cells, such as regulatory T-cells and myeloid cells. p53 mutants have established a well-studied role in the alteration of cellular secretome involving ECM remodeling, autocrine signaling, stromal cell recruitment, and activation. The mutant p53 is reported to promote an immune suppressive TME that is enriched with M2 macrophages. Such tumors failed to respond to the immune checkpoint inhibitor therapies in oral cavity squamous cell carcinoma. Additionally, as mutant p53 may be packed within extracellular vehicles (EVs), its gain-of-function activities are transmitted to nearby cells and distant regions via EV transfer and circulating vesicles, both of which aid in the growth and metastasis of tumors. Several of the mutant p53 such as R273H, R175H, and R248W have been shown to influence the TME and support TME-driven chemoresistance in different cancer types[99]. Future research in cancer biology should delve into the intricate interplay between p53 and the ECM, particularly in hypoxic conditions.

5.2. Role of mutant p53 in NK cells activation in human malignancies

NK cells play a crucial role in the immune system through the elimination of foreign, transformed, and senescent cells. NK cells can efficiently eliminate the foreign, transformed, and senescent cells via specialized cell surface receptors like natural-killer group 2, member D (NKG2D) which interacts with a variety of stress or viral-induced ligands known as ULBP1 and ULBP2 (expressed on surfaces of the target cells)[100, 101]. The expression of both ULBP1 and ULBP2 is dependent on the status of the p53[102]. The induction of wild-type p53 under stressed conditions has been reported to upregulate the expression of both ULBP1 and ULBP2. This resulted in the elimination of transformed cells by NK cells. Therefore, loss of p53 can help the cancer cells not only to proliferate and survive but also to escape from NK cells. Further, coculturing of NK cells with cancer cells that express p53-WT displayed the NKG2D-dependent generation of IFN whereas p53 mutants were not recognized by NKG2D[53]. This suggested that lack of p53 activity may potentially affect the NK cell-dependent priming of T cells, as NK cell-derived IFN- is crucial for the activation of Th1 and Tc1 effector responses. As a result, the loss of p53 activity in tumor cells is anticipated to have an impact on immune cell-mediated pathways as well as intrinsic mechanisms within the cancer cells. In contrast, the mutant p53 cells lead to the repression of ULBP1 and ULBP2 translation, allowing the neoplastic cells to escape from cytotoxic destruction. Evidence has shown that murine p53 mutations G242A reduce host natural killer (NK) cell activation thereby protecting breast cancer cells from immunological attack[53]. Serial injection of EMT6 breast cancer cells, harboring p53-WT increased NK activity in mice, while serial injection of SVTneg2 cells, harboring G242A+/+ mutation, decreased the number of NK cells and increased the number of CD8+ T lymphocytes in the spleen. The level of innate immunity based on NK cells and CD8 T cells was also decreased in transgenic mice expressing the p53 mutation R172H/+. Additionally, in co-culture with isolated NK cells, EMT6 cells significantly increased NK cell proliferation and interferon-gamma (IFN- γ) production. In contrast, SVTneg2 cells significantly decreased NK cell activation. Another study has reported that miR-34a and miR-34c can regulate the expression of ULBP2 by targeting the 3’-untranslated region of ULBP2. Further, miR-34 mimics were found to repress the ULBP2 expression and decreased the cancer cell recognition by NK cells. Nutlin-3a treatment was reported to downregulate the levels of ULBP2 in a p53-dependent fashion[103]. This was because of a p53-mediated increase in cellular miR-34 levels[104]. These findings indicated the role of p53 in ULBP2 regulation, underlining the functional role of NKG2DL in tumor immune surveillance. These data indicated that restoration of p53 after the adoptive transfer of NK cells can be used for NK cell-based immunochemotherapy against human cancers.

5.3. Mutant p53 and MHC-I endogenous pathway

The major MHC-I antigen processing pathway plays a crucial role in the initiation of an appropriate immune response through peptide fragment presentation derived from intracellular proteins[105]. Every nucleated cell possesses MHC class I molecules that bear peptide fragments derived from intracellular proteins. In the case of a virally infected cell, peptides from viral proteins may also be detected. The cytotoxic T-cells recognize the presented peptide fragments and eliminate the infected cells. These fragments are generated by proteasome digestion of the antigenic protein and are then translocated into the endoplasmic reticulum (ER) via the transporters associated with antigen processing 1 and 2 (TAP1 and TAP2). Once the fragments have reached the ER, they then form the peptide-loading complex with numerous components, including that of the MHC-I subunits. The length of the peptides generated by the proteasome has to be extremely specific for it to be presented by MHC-I, hence requiring the enzyme endoplasmic reticulum aminopeptidase 1 (ERAP1) to fit them to the required length which is around 8-10 amino acids. Autophagy plays a crucial role in antigen presentation by APCs like dendritic cells (DCs) and macrophages, influencing the surface expression of major histocompatibility complex class I (MHC-I) molecules[106]. Inhibition of autophagy increases MHC-I surface levels, affecting T-cell responses, particularly in cross-presentation by DCs. Autophagy is essential for generating diverse antigenic peptides, and its inhibition can reduce the presentation of immunodominant epitopes in MHC, ultimately negatively impacting T-cell immunity against tumor antigens[106]. In pancreatic cancer, immune evasion, often linked to low MHC-I expression, poses a significant hurdle in immune checkpoint blockade therapy[107]. Autophagy intensifies immune evasion in PC by promoting tumorigenesis, even with low MHC-I surface expression. Inhibiting autophagy enhances MHC-I levels, facilitating antigen presentation and boosting T-cell responses, suppressing tumorigenesis[107].

p53 has been shown to regulate the endogenous pathway of antigen processing and presentation by MHC-I, along with transcriptional repression of human leukocyte antigen B7 (HLA-B7) – one of the polymorphic proteins of MHC-I. The p53 triggers the expression of ERAP1 and TAP1, in response to stress caused due to DNA damage, which eventually causes enhanced surface MHC-I and peptide complexes in cancer cells. The number of peptides accessible for MHC-I loading would likewise increase if p53-dependent stimulation of ERAP1 expression were to occur TAP-1 protein localized on the surface of rough endoplasmic reticulum (RER)[108]. TAP-1 acts as a transport channel through which small fragments of endogenous antigens are transported to RER. The expression and function of TAP1 is directly influenced by p53 because of the presence of a p53-responsive element downstream to the transcription start site of gene TAP1[103]. This study showed that forced expression of p53 along with IFNγ treatment in H1299 (p53-null) cells resulted in the upregulation of TAP1. However, the p53 mutants such as V13A, R273H, R175H, and 249S failed to increase the expression of TAP1. This suggested that mutant p53 can contribute to the evasion of tumor surveillance in human malignancies. A similar effect was also observed by the family members of p53 such as p73, that have the ability to induce p53-mediated TAP1 activation. The mechanism used by tumor cells is that by acquiring mutations, they downregulate the MHC class I pathway thereby evading recognition by the complex immune system. Furthermore, with no stable MHC-peptide complex on the surface of cells, the tumor cells also evade CTL recognition. Therefore, it was concluded that induction of TAP1 by p53 will lead to increased transport of MHC class I peptides[103] In p53-mutant and p53-null cell lines, both components of antigen presentation are downregulated. Intriguingly, ablation of important MHC-I pathway components, TAP1 or β2M decreases p53 function, indicating a probable interaction between the MHC-I presentation pathway and p53 activity in cancer cells. In mutated p53 cells, both expressions of ERAP1 and TAP1 are altered, having a direct effect on the endogenous pathway, causing tumor cells to evade immune surveillance. Exploring the impact of p53 mutations on MHC-I pathway components could unveil novel approaches to counter immune evasion by tumor cells.

5.4. Mutant p53 and Toll-like Receptors (TLRs)

TLRs are widely expressed on several types of immune cells to recognize a variety of pathogen-associated molecular patterns derived from viruses, bacteria, protozoans, and fungi. This recognition activates the innate immune responses[109]. The TLRs initiate the immune responses by activating the NF-κB and interferon regulatory factors. Different TLRs are crucial to alert the immune system about any foreign pathogen through their signaling pathway. The TLRs can have the dual functionality of either suppressing or contributing to the inflammation-induced proliferation of tumors by altering their signaling pathways. TLRs expression can have a detrimental impact on the growth and viability of cancer cells, as well as can directly destroy tumor cells by inducing TNF-α. In response to p53 activation and genotoxic stress, the majority of the TLR gene family members can express themselves in human cancer cells. This is anticipated to boost the potential for TLR-based cancer therapy, especially considering that some treatments may also involve higher amounts of p53 and DNA-damaging chemicals/agents. For all TLRs, except for TLR7, many putative p53 binding sites were found close to the transcription start sites. Interestingly, it was noticed that most of the functioning sites for TLR2 and TLR10 were predicted to be noncanonical and have only a partial p53RE. Previously, it was demonstrated that p53 directly controls the genes FLT1 and RAP80 through half-site REs, and several additional target genes have been found that are predicted to be regulated by noncanonical p53 RE. In-depth, studies have shown that the p53 gene can target TLR3 and to a lesser extent TLR9, both of which are responsible for the induction of the apoptotic pathway in abnormal and malignant cells. TLR3 expression was induced by all genotoxic agents tested in the p53-positive cells and overexpression of p53 protein in p53-null cells induces apoptosis of cancer cells.

In MCF7 cells simultaneous activation of the p53 and TLR5 pathways was able to boost the expression of more than 200 genes, largely linked to immunity and inflammation[110]. The enhanced phosphorylation of p38 MAPK, PI3K, and STAT3 were some of the molecular pathways by which the transcriptional response to TLR5 activation was amplified in a p53-dependent manner[110, 111]. A wide link between p53 and these signaling pathways is further suggested by the fact that p53 activation boosted cytokine expression in response to TNF, another activator of the NF-B and MAP kinase pathways. On the other hand, if there is the presence of mutant p53 instead of the WT-p53, these TLRs are not able to function properly, thereby allowing the cancer cells to evade cell death through apoptosis. R175H and G245S, two well-known loss-of-function mutants, failed to activate any TLR gene[112]. The most significant modifications were caused by the R337H mutation, which included a loss or gain of transactivation for various TLRs[112]. These findings provide credence to the idea that when developing adjuvant cancer medicines that involve TLR pathways, the precise state of p53 should be considered [112].

5.5. Role of the mutant p53 and cytokine signaling during pro-inflammatory responses

Pro-inflammatory cytokines have a major role in initiating defense against foreign infections. Cytokines can lead to both the activation and the inhibition of p53-mediated functions in human cancers. Nowak and colleagues have shown that the codeletion of Trp53/Pten leads to the development of metastatic prostate cancer in murine models by increasing the transcription and marked secretion of IL-The secretion of IL-6 activates the STAT3 signaling cascade and stabilizes the Myc protein[113]. Wormann et al have noticed that the deletion of p53 in the pancreatic cancer murine model stimulated the JAK2/STAT3 pathway and enhanced tumor proliferation and gemcitabine resistance[114]. The p53 loss resulted in increased levels of reactive oxygen species which therefore inhibits the SHP2 phosphatases and promotes STAT3 activity. Interestingly, the phosphorylation of STAT3 was positively linked with the mutated p53 in human pancreatic tissue specimens. Moreover, the STAT3 pathway inhibition displayed retarded tumor growth and stroma, increased immune infiltration, and increased survival of the mice[114]. Many studies have established the antagonistic relationship between p53 and NF-κB, an essential protein complex responsible for the transcription of DNA, regulation of cytokines, and cell survival. This has been observed that constitutive activation of NF-κB leads to a reduction in the tumor suppressor activity of p53 that supports a pro-inflammatory environment to increase the risk of tumor formation[82]. R175H mutant was reported to enhance the p65 nuclear localization that constitutively activates NF-κB signaling[115, 116]; Other studies displayed that mutant p53 proteins can bind with p65 which in turn upregulates NF-κB transcription[117]. R273H mutant was found to support IL-1β signaling through suppression of IL-1 receptor antagonist (IL-1RA) transcription[118].

Importantly, the mutant p53 has been reported to modify the cancer cell secretome and subsequently regulate the features of the tumor microenvironment[119, 120]. Among its other effects, mutant p53 has been shown to activate the unfolded protein response (UPR), particularly the activating transcription factor 6 (ATF6) branch can positively regulate the production of pro-inflammatory cytokines and activate the pro-inflammatory/pro-oncogenic transcription factors NF-B, MAPK, STAT3, and mTOR[119–122]. Also, mutant p53 has the potential to activate all of the aforementioned pathways, which results in increased stability, a characteristic feature of its oncogenic action[20, 123, 124]. Therefore, these pathways may inhibit the degradation of mutant p53 by macroautophagy, a procedure that is known to contribute to the degradation of mutant p53. Given that mutp53 is frequently misfolded, the presence of chaperoning molecules like HSP90, whose expression can be controlled by pathways including PI3K/AKT/mTOR and STAT3, may also be necessary for mutant p53’s stability.

During chromosomal instability, DNA is prone to rupturing and is discharged into the cytoplasm[125] The DNA-binding protein cyclic GMP-AMP synthase (cGAS) can detect these cytoplasmic DNAs, thereby cascading innate immune signaling and type I interferon production (IFNs)[126]. This results in cGAS homodimerization and the production of the second messenger cyclic-GMP-AMP (cGAMP)[127] The endoplasmic resident Stimulator of Interferon Genes (STING) then recognizes this cGAMP molecule and translocates to the ER-Golgi intermediate compartment (ERGIC), where it recruits TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3)[128] IRF3 joins STING and TBK1 to create a trimeric complex, which is phosphorylated by TBK1 to enable IRF3 to homodimerize and go to the nucleus to control gene expression[128]. IRF3 can translocate to the mitochondria and induce apoptosis by interacting with Bax[129] Thus, this innate immune signaling pathway is essential for the inhibition of immune cell-mediated tumor suppression. The cytoplasmic DNA induces IRF3 transcriptional activity and death in cells lacking p53[130]. In contrast, mutant p53 binds to TBK1, disrupts the downstream signaling from cGAS/STING to TBK1, and henceforth prevents the phosphorylation of its substrates[131] Additionally, mutant p53 reduces the TBK1-dependent activation of IRF3, which encourages cytoplasmic DNA tolerance. Importantly, mutant p53 inhibits IRF3 activation in vivo, which enhances immune evasion[131]. The gain-of-function activity of mutant p53 can prevent both cell-autonomous and non-cell-autonomous surveillance systems, thereby accelerating the development of cancer. Future research directions should explore the mechanisms through which mutant p53 modifies the cancer cell secretome and activates pathways like UPR, NF-κB, MAPK, STAT3, and mTOR is crucial for developing targeted therapies. Additionally, further exploration of the cGAS/STING pathway and its inhibition by mutant p53 could provide insights into immune evasion mechanisms, contributing to the development and progression of cancer.

5.6. Mutant p53 and leucocyte recruitment through SASP

The p53 plays a crucial role in inducing senescence phenotype, with the production of certain angiogenic and growth-promoting cytokines termed senescence-associated secretory phenotype (SASP)[132]. These SASP molecules can recruit different types of immune cell populations which can either lead to the suppression or promote the growth of the tumor[133] For example, the forced expression of p53 in the established transplantable lung adenocarcinomas and hepatocellular carcinomas led to the induction of senescence along with the production of SASPs that recruited immune cells such as NK cells, T-cells and myeloid cells leading to the clearance of the cancer cells[134]. However, persistent secretion of SASP components can have pro-tumorigenic effects, especially when normal p53 function is lost[135] In contrast, it was also observed that the production of IL-1 by senescent cells, attracting the PMNs caused the production of IL-1RA, eventually suppressing the IL-1 and p53 expression and helping the malignant cells to bypass proliferation arrest. Future studies explore the intricate interplay between p53-induced senescence and the SASP. Investigating how SASP components recruit immune cell populations and impact tumor growth is crucial.

5.7. Potential role of mutant p53 in cancer-associated fibroblasts (CAFs)

CAFs play a crucial role within the TME by modulating cancer progression, development, metastasis, and therapeutic resistance. Recent research demonstrates that p53 inhibits tumor growth in CAFs by acting as a cell non-autonomous tumor suppressor. TP53 mutations have been displayed to be prevalent in the CAFs of highly inflamed cancers[136]. CAFs with altered p53 have displayed increased tumor metastasis, invasion, and poor prognosis via increased expression of various cytokines and chemokines such as IL-6, SDF-1, and CXCL12[137–139]. The mutant p53^N236S was reported to activate CAFs resulting in the enhanced expression of α-SMA due to the regulation by the STAT3 pathway. Mutant p53^N236S was able to modulate the functions of CAFs through Stat3. Notably, even in the absence of WT p53, low-level Stat3 activation already takes place. The upregulation of the Stat3 pathway is thus a result of the GOF property by the mutant p53. Altogether, these findings suggested that restoring the p53 in CAFs could be essential for the development of anti-cancer therapies. Therefore, future studies are needed for the development of anti-cancer therapies by restoring p53 in CAFs to inhibit tumor growth, and metastasis along with improving prognosis.

6. Therapeutic aspects to activate the immune response in cancer cells via p53 functions

Therapeutic targeting of p53 can play a vital role in modulating the immune responses within the tumor microenvironment and can efficiently eliminate cancer cells[140]. This is possible because either mutant p53 or inactivated p53 controls the immune responses[141]. Several studies have shown that restoration of p53 function in cancer cells makes the tumor cells more susceptible to chemotherapy or radiotherapy as compared to those cancer cells having mutant p53[142–144]. However, developing activators of p53 for tumor therapy is still a concern as they might also cause harmful or deadly effects on normal cells[145]. In this regard, several approaches have been made to restore p53 function to target the tumor cells by modulating the immune responses and are discussed in detail in this section.

6.1. Vaccination strategies for p53 in human malignancies

The potential p53 in tumor development has attracted the scientific community towards the generation of vaccines against p53[140]. Initially, this was thought that generating a T-cell response against wild-type p53 can lead to autoimmunity. Interestingly, several groups have displayed that p53-dependent T-cell immune response did not cause autoimmunity in murine models. These data suggested that p53 could be safe for the development of a vaccine[146, 147]. Different vaccine approaches that have been used to induce T-cell response against p53 are under development (Fig. 4). Based on the preclinical studies, many groups have initiated clinical trials including phase-I and II by employing a variety of approaches against p53. Two independent studies led by Leffers et al and Vermeji et have used synthetic long peptides of p53 (SLP-p53) for vaccination in clinical phase I/II trials in patients with ovarian carcinoma after chemotherapy. In these studies, p53-specific CD4+ T cell immune activation has been observed and was sustained after chemotherapy[148, 149] (Fig. 4, Table 1). However, there was no significant difference in overall survival in both trials. In a phase 1/2 trial for platinum-resistant p53-positive epithelial ovarian cancer, a combination of Pegintron (IFN-α), gemcitabine, and p53 synthetic long peptide (SLP) vaccine was studied. The treatment was well-tolerated, with reduced myeloid-derived suppressor cells and increased immune-supportive M1 macrophages. Patients displayed strong p53-specific T-cell responses, suggesting the feasibility and immunogenicity of this chemo-immunotherapeutic approach for cancer treatment[150–152](Table 1). Roth and colleagues have used recombinant canarypox virus vaccine containing human p53 (ALVAC) in preclinical tumor models. This vaccine has generated promising p53-specific antitumor immune responses in preclinical models[153]. However, no significant response was noticed against this vaccine in patients with lung cancer or advanced colorectal carcinoma, even though the p53-specific response was high[154, 155]. Modified vaccine Ankara expressing murine p53 (MVAp53) vaccine has been shown to stimulate the immune response in cancer mouse models[156, 157]. Moreover, the use of MVAp53 gene therapy resulted in the increased survival of platinum-resistant patients with ovarian carcinoma. In a heavily pretreated patient with triple-negative breast cancer and cutaneous metastases, the combination of p53MVA vaccine and pembrolizumab led to a significant regression of cutaneous metastases, confirmed by pathological response, activation of p53-specific T cell responses, and elevated immune response genes, sustaining a rapid and lasting clinical response for 6 months[158] (Table 1). Interestingly, more promising results have been shown by combining MVAp53 with TLR9 agonists CPG-ODN (CpG deoxynucleotides) or CTLA-4 blockade can mediate tumor rejection[159]. The trials using SLPp53 in combination with other agents like Pegnitron (IFNα) and gemcitabine are ongoing.

Fig. 4. Therapeutic aspects of activating immune response via p53 functions.

a, CXCR4 targeted p53 mRNA nanoparticle when combined with anti PD1 therapy has shown to downregulate M2 TAM (Tumor-associated macrophages) and upregulate CD+8 t cell, CD+4 T cell, M1 TAM, and NK cells. This results in immune system reprogramming and tumor death. b, p53 Vaccines like p53MVA work by activation of CD+8 T cells which results in recognition of neoantigens on MHC molecules and immune attack on tumor cells. c, Biseptic antibodies work by creating a link between T-cells and cancer cells, which will lead to the release of cytotoxic proteins that will help T-cells kill the tumor cells.

Table 1. Summary of the preclinical and clinical trials which are associated with the effect of p53 modulation to influence anti-tumour immune responses against human malignancies.

Name of the compound/drug/pharmacological inhibitor (s)	Detail of the clinical trial (s)	Molecular mechanism (s)	Type of human malignancy (s)	Clinical benefit (s)	Adverse effect (s)	Reference (s)
Alrizomadlin (APG-115) and pembrolizumab	NCT03611868, Phase II	Combination restores p53 functions and acts as an immunomodulator and may restore antitumor activity in pts with cancers that progressed on PD-1/PD-L1 inhibitors	Solid tumors including adults and children.	The combination was well tolerated with antitumor activity in solid tumors.	Thrombocytopenia, neutropenia, peripheral edema, anemia, nausea, vomiting, fatigue.	(140, 178)
SLP-p53 + cyclophosphamide	NCT00844506; Phase II	The combination of SLP-p53 and cyclophosphamide resulted in a marked increase in the p53-specific Th1/Th2 immune response leading to disease stabilization.	Ovarian carcinoma	The disease was stable, and treatment was well tolerated. This study provides evidence that low doses of cyclophosphamide can be combined with p53-SLP vaccine or other antitumor vaccines.	Anemia, muscle spasm, diarrhea, dizziness, fatigue, neutropenia, weight loss, rashes, gastroesophageal reflux, constipation, cardiac murmur, monocytopenia, lymphopenia, leukopenia,	[148, 149]
Synthetic long peptides-p53 vaccine and PegIntron (Pegylated IFN-α2b)	NCT01639885, Phase I/II	Enhanced the T-cell activation by increasing activated T-cell/T-regulatory cell ratios. The p53-specific immune responses were markedly improved for SLP-p53 vaccination with the combination of PegIntron.	Ovarian cancer resistant to platinum drugs	The patients under this regimen displayed strong p53-specific T-cell immune responses.	Local skin allergic reactions, fatigue, Pegintron-related flu-like symptoms, nausea, vomiting, dyspnea, and hypokalemia, Anorexia	[150, 151, 152]
p53MVA	NCT01191684, Phase I	CD4+ and CD8+ T cells recognized p53 overlapping peptides after the first immunization. However, most of the patients failed to expand their T-cells during subsequent immunizations. Moreover, the blockade of PD-1 boosted p53 immune responses during the second or third immunizations.	Pancreatic and colorectal cancers	Immunization with p53MVA was well tolerated.	No adverse effect above grade 2.	(156, 157)
Heterologous p53 Immunization combined with synthetic double-strand RNA {poly (I:C)} and unmethylated CpG-oligodeoxynucleotide (CpG-ODN)	Preclinical evaluation	Heterologous p53 immunization with the activation of toll-like receptors by poly (I:C)/CpG-ODN leads to marked and significant regression of tumors	p53 null murine mammary adenocarcinoma (4T) cells	Treatment displayed safety, immunogenicity, and antitumor immune response.	None	[159]
p53MVA + pembrolizumab	NCT03113487 Phase II trial	Based on the observation from the previous trial Immune checkpoint inhibition in combination with p53MVA will be more beneficial.	28 patients having resistant ovarian cancer	Not completed	Not completed	[156, 157]
p53MVA + pembrolizumab	NCT02432963	immune checkpoint blockade enhanced antitumor immune responses of the p53MVA vaccine for better clinical benefit. Patients with cutaneous metastases showed complete regression after 2 cycles.	cutaneous metastases, advanced solid tumors including pancreatic, breast, liver, head, and neck cancer	Well tolerated, safe, and displayed clinical benefit in selected patient groups.	Dizziness, nausea, diarrhea, fatigue, manageable cardiac toxicity.	[156, 157, 158, 159, 160]
Ad.p53-DC combined with nivolumab and ipilimumab	NCT03406715, Phase II	The combination of dendritic cell-based p53 vaccine involving the immune checkpoint inhibition using nivolumab and ipilimumab was decided to improve efficacy and clinical outcomes of the patients with recurrent lung cancer.	Recurrent lung cancer			[155, 162, 163]
Eprenetapopt (APR-246) and pembrolizumab	NCT04383938, Phase Ib	The first clinical trial assessed the combination of a p53 reactivation with immune checkpoint inhibition which modulates the tumor microenvironment, favors infiltration of T-cells, and restores antitumor T-cell responses.	Advanced or metastatic urothelial bladder cancer and NSCC	The combination displayed promising anticancer clinical efficacy with acceptable tolerability and safety profile.	Nausea, dizziness, fatigue, diarrhea, vomiting, loss of appetite.	(176, 177)

Active clinical trials are also ongoing using the MVAp53 vaccination with pembrolizumab, an anti-PD1 antibody (NCT03113487, NCT02432963)[160, 161] pembrolizumab. As an alternative strategy, trials involving modified autologous DCs expressing p53 peptides on MHC class I and II are being carried out[140]. Out of the 28 patients who received treatment, 16 individuals with small-cell lung cancer (SCLC) responded to the DC-p53 vaccination by developing an immune response specific to p53[155, 162] (Table 1). It’s significant to note that 13 of the 21 patients who underwent additional chemotherapy after receiving the p53 vaccine demonstrated an objective clinical response although, during the phase II trial, there were no changes in the overall response rate between the groups receiving paclitaxel followed by the DC-p53 vaccine to that of a control group. The priming DCs with mutant p53 peptides and adoptively transferring them into tumor-bearing hosts. This approach was shown to successfully promote T-cell activation and tumor regression[163]. In a separate phase I trial involving patients with advanced solid tumors, combining p53MVA vaccine with pembrolizumab (anti-PD-1) resulted in clinical responses in 3 out of 11 patients, with stable disease lasting up to 49 weeks. Enhanced p53-reactive CD8+ T cells and increased immune response gene expression were observed in responsive patients, indicating the potential clinical benefit of this combination therapy, although further studies are needed to identify specific patient groups. Safety concerns, including a fatal myocarditis case, led to enhanced cardiac monitoring in subsequent patients[160] (Table 1). Thus, more studies on the optimization of different vaccine strategies, and safety concerns in p53-targeted cancer immunotherapy are needed.

6.2. Bispecific antibody targeting mutant p53

The basic principle for developing the bispecific antibody is to create a link between the T-cell and the tumor cell so that the T-cell can get activated and eliminate the tumor cells. Interestingly, bispecific antibodies have two arms where one arm targets the T-cell receptor (CD3), and the second arm targets the mutant peptide HLA complex (neoantigens). The bispecific antibodies that target the mutant p53 and Ras were found to be effective in generating specific immune responses against tumors having these mutations. Initially, polymorphic HLA class I and II receptors were used to bind R175H (p53) or G12V mutant peptides of KRas and reported poor affinity, limiting the efficacy of these bispecific antibodies[164, 165]. Later, bispecific antibodies were designed based on the HLA-A1 or HLA-A3 receptors and enhanced the affinity of the antibody to recognize the antigens at lower levels. Hsiue and colleagues have used phage display antibody libraries against HLA-A*02:01 peptide–HLA monomers containing R175H (mutant p53) peptide, along with a negative selection against peptide–HLA monomers with wild-type peptide against p53[166]. These selected phage clones were used to generate bispecific antibodies, joining in a single-chain diabody (scDb) to an anti-CD3e scFv to recognize CD3 and stimulate the T-cell mediated immune response. Moreover, the specificity of the phage peptide binder scDbs (H2-scDb) for R175H (mutant p53) was checked in several functional assays against the R175H expressing cells. Also, X-ray crystallography experiment data supported that scDb has a specific binding with mutant R175H/HLA-A*02:01. H2-scDb has been reported to eliminate tumor cells with R175H (mutant p53) in both in-vitro and murine models while activating T-cell responses[166]. Therefore, improving the design of bispecific antibodies targeting specifically the diverse p53 mutants, including optimizing their affinity and targeting mechanisms, will be crucial for enhancing their specificity and efficacy in inducing immune responses against tumors.

6.3. Activation of p53 using gene therapy approach

Gendicine also known as rAd-p53, is the first commercial gene therapy that has received clinical approval in China. This is an adenovirus that has been genetically altered to carry the human Tp53 gene. At Beijing Hospital, between 1998 and 2003, the first Gendicine clinical trial was conducted. In October 2003, the China Food and Drug Administration (CFDA) gave its approval[167]. A year later, Gendicine launched its commercial and clinic operations[168]. It was initially used in conjunction with radiotherapy to treat head and neck squamous cell cancer (HNSCC)[167, 169]. Later, gendicine was used to treat a range of malignancies, such as malignant gynecological tumors and cancers of the ovary, bladder, liver, lung, and soft tissue[167]. Gendicine is administered through intratumoral injection, intracavity or intravascular infusion. It is particularly successful when paired with chemotherapy for the treatment of liver metastases and uterine sarcomas[170]. Additionally, the use of gendicine in conjunction with radiation, chemotherapy, and other types of therapy can enhance complete response (CR) and patient survival[171]. According to preliminary findings, Ad-p53 can elicit immune responses against co-stimulatory molecules, tumor antigens, and numerous cytokine genes, among other processes that strengthen anticancer immunity (Fig. 4). In earlier adenoviral vector clinical trials, the vast majority of patients had neutralizing antibodies and nearly all displayed a notable rise in viral titer following the initial Ad vector injection[172]. The focus should be the development of less toxic vehicles and better delivery mechanisms for p53-mediated gene therapy in diverse cancer types.

6.4. Activation of p53 using immune checkpoint inhibitors

p53 works by promoting T-cell infiltration and the checkpoint inhibitors affect tumor cells[173]. rAD p53 when combined with pembrolizumab shows effective results against tumor cells[174]. ALRN-6924 is a first-in-class, clinical-stage, alpha-helical peptide-based inhibitor that dismisses the interaction of the p53 with MDMX and MDM2 to induce transient cell cycle arrest in p53-wild-type tissues[175] ALRN-6924 was reported to be markedly active in wild-type TP53 cancer cells. However, no anticancer activity was noticed in mutant TP53 cancer cells. ALRN-6924 in combination with paclitaxel displayed increased apoptosis and decreased tumor growth of ER-positive breast cancer. Stimulating peripheral blood mononuclear cells (PBMCs) with ALRN-6924 ex vivo induces the transcriptional activation of genes related to both innate and adaptive immunity. This stimulation also leads to the production of immune-stimulating cytokines such as INF-γ, IL-6, and IL-12. Analysis of mRNA from tumor biopsies taken before and after treatment with ALRN-6924 in patients reveals a distinctive gene expression pattern indicative of a shift towards an inflamed tumor phenotype. In syngeneic mouse models, ALRN-6924 facilitates the infiltration of CD8+ T cells, the polarization of M1 macrophages within mouse tumors, and the establishment of immunological memory. Additionally, ALRN-6924 demonstrates synergy with anti-PD-1 and anti-PD-L1 treatments, fostering anti-tumor immunity. This synergy results in an increased proportion of mice achieving complete regressions (CR) when compared to the outcomes observed with single-agent treatments, regardless of the p53 status in tumors (wild-type or mutant). (Fig. 3). ALRN-6924 works by activating the interferons which as a result boosts the immune response via p53 functions. This has been observed that p53 signaling can be induced either with APR-246 or overexpression of p53 in transgenic mice which can enhance anti-tumor T-cell immune responses and extend the activity of immune checkpoint blockade. This study displayed enhanced p53 expression in TAMs and activated p53-associated functions like senescence and secretory phenotype[176]. This study has noticed the decreased expression of proteins associated with M2 polarization in TAM. These data led to the foundation for clinical trials in combination with APR-246 and pembrolizumab against solid tumors[176, 177] (Table 1).

The combination of alrizomadlin (APG-115) and pembrolizumab restores p53 functions which in turn acts as an immunomodulator. This resulted in the restoration of antitumor activity in patients with cancers that progressed on PD-1/PD-L1 inhibitors[178]. The clinical trial data displayed inhibition of M2-polarized myeloid cells and an increase in T cell proliferation in the responder group. Studies towards the identification of reliable biomarkers that can predict patient responses against immune checkpoint combination therapies will be developed for patient selection and the development of more targeted and effective treatment strategies.

6.5. p53 mRNA nano-therapy with immune checkpoint blockade

Immune checkpoint blockade, which uses anti-PD-1, anti-CTLA-4, and anti-PD-L1 antibodies to boost anti-tumor immunity, has shown promise in transforming the treatment strategies of several malignancies, including HCC[179, 180]. The transcriptional control of important cytokines, chemokines, and pathogen recognition receptors such as TLRs, and p53 has been demonstrated to generate an anticancer immune response by modulating the TME[181]. Restoration of p53 was reported to induce the activation of myeloid cells for promoting tumor antigen-specific adaptive immunity and by suppressing the pro-tumorigenic M2-type macrophage (TAM) polarization. Henceforth attempts were made recently to target p53-based mRNA nanoparticles (NPs) to activate p53 and rewire the TME[182] (Fig. 1). Using HCC models, p53 expression was induced by altering the nanoparticles with the chemokine receptor CXCR4-specific targeting peptide CTCE-9908 (KGVSLSYRCRYSLSVGK) and as a combination therapy along with anti-PD1. They concluded that the CXCR4 targeted p53 mRNA nanoparticle has antitumor activity in ectopic and intrahepatic models of p53 deficient HCC (Fig. 4). p53 mRNA NPs effectively restored p53 expression in vivo in subcutaneously grafted immunocompetent C57Bl/6 mice thereby significantly enhancing the anti-tumor effects of anti-PD1 therapy in HCC growing outside the liver. The combination therapy of CXCR4 p53 mRNA nanoparticle and anti-PD1 works by reprogramming the tumor microenvironment by promoting anti-tumor immunity, and MHC1 expression, and additionally by lowering the levels of immunosuppressive cytokines in HCC[183]. Further, the exploration of TP53 mRNA-based nano-therapeutics should include investigations into its efficacy across diverse cancer types, and optimization of delivery systems, as well as address the challenges associated with the restoration of p53 expression in the in vivo model system.

7. Conclusion and future perspective

The tumor-suppressive functions of p53 have been proved in past decades through extensive research in several human malignancies[1, 3, 9, 17, 145]. Interestingly, emerging studies have established the potential of p53 on the immune system, especially in human cancers[74, 184]. The p53 is now recognized to promote the immune and inflammatory hallmarks of cancer through its involvement in altering the activity of infiltrating immune cells, fueling the inflammatory cascades, shaping the TME, and repressing the innate tumor immunity[52, 184]. Dysregulation of p53 in tumor cells is one of the factors for the evasion of immune surveillance leading to an immunosuppressive microenvironment. Mutant p53 in the tumors has been found to modulate immune responses through several mechanisms like repression of the antigen presentation by MHC-I as well as recruitment of MDSCs and Tregs. Also, p53 is essential for the stromal compartment and plays diverse roles either by inhibiting or favoring tumor initiation and progression. Interestingly, p53 activity in T-cells is important to prevent inappropriate proliferation in the absence of TCR signaling, allowing tight regulation of antigen-specific T-cell expansion[185]. Several efforts have been demonstrated in both in vitro and in vivo models for generating an anti-tumor immune response against mutant p53 for immunotherapy and vaccines in human malignancies[181]. For instance, p53 can enhance PD-L1 expression to improve the efficacy of anti-PD-L1 and anti-PD-1 immune checkpoint inhibitors, with varying effects across cancer types. Investigating context-specificity across cancer types, mutation types, and cell types is essential for optimal clinical translation of p53 restoration in immune-suppressed TME. Evidence also suggests that treatment with DNA-damaging agents may sensitize cancer cells to immunotherapy either through upregulation of the PD-L1 or induction of immunogenic cell death[186]. However, the specific contribution of p53 to these mechanisms requires further clarification, and significant work is needed to identify optimal dosing and treatment timing when combining DNA-damaging agents with immunotherapy to induce maximal anti-tumor immune responses. While immune checkpoint inhibitors and p53 vaccines have shown efficacy, other immunotherapies like adoptive cell therapy, monoclonal antibodies, oncolytic viruses, and immune system modulators need to be explored in combination with p53-reactivating therapies. Testing the efficacy of these approaches in combination with p53-reactivating compounds, especially in cancers with mutant p53, is more relevant. During the past decade, our knowledge has been greatly increased to understand how p53 modulates several immune responses to cancer[76, 187]. More comprehensive studies are required to delineate the molecular mechanisms and signaling cascades associated with p53 expression and the immune landscape within the TME. We hope this will certainly fasten and benefit the process of development of new targeted therapies including vaccines, immunotherapy, and pharmacological small molecules for prolonged anti-tumor responses in patients.

Highlights.

• TP53 alterations is crucial for initiation, progression, and metastasis of human malignancies.

• p53 deregulation modulate innate and adaptive immunity, aiding immune evasion in human cancers.

• Highlighted is the potential of p53 in the development of immunotherapy and vaccines for anticancer therapies.

• p53 governs intricate crosstalk between autophagy and apoptosis in cancer by modulating autophagy through cytoplasmic inhibition and nuclear promotion, impacting cellular fate.