Academic literature on the topic 'P53FL'

p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML.

Abstract Canonical p53-activated pathways can influence a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages (TAMs). We examined whether p53 activity in the tumor microenvironment (TME) influences antitumor immunity and show that p53 signaling induced pharmacologically with APR-246 (eprenetapopt) can augment the efficacy of immune checkpoint blockade (ICB) in preclinical models, a strategy that is also being tested in patients (NCT04383938). We first investigated the effects of combining APR-246 with ICB in wildtype C57BL6 (B6) mice bearing syngeneic p53 wildtype MC38 colon cancer and B16 melanoma tumors. The combination of an anti-PD-1 antibody (RMP1-14) with APR-246 in mice significantly delayed tumor growth (p < 0.001) and improved survival of tumor-bearing mice, compared to monotherapies (p < 0.01). To further dissect the effects of APR-246 on myeloid and T cells in the TME, we used a conditional knockout of p53 in CSF1R+myeloid cells (CSF1Rcre/p53fl mice), or T cells (CD8cre/p53fl mice). CSF1Rcre/p53fl had loss of tumor control and worse survival with APR-246+anti-PD-1. CD8cre/p53fl had intact tumor control. To study enhanced p53 activity in the TME, we performed flow cytometry, cytokine multiplex and global transcriptional profiling by RNA seq. We found enhanced p53-activity led to increased infiltration of T cells, increased MHC-II expression in TAMs and downregulation of M2-associated cytokines. This was associated with cellular senescence in TAMs and induction of canonical p53-induced senescence-associated secretory phenotype (SASP). Our preclinical findings informed the development of a phase I/II clinical trial using APR-246 with pembrolizumab for patients with advanced solid tumors (NCT04383938). We studied peripheral blood samples from two of the patients with tumor regression and two patients in whom tumors progressed on therapy. We analyzed peripheral blood mononuclear cells (PBMCs) and serum prior to therapy, and at the beginning of cycle 2 and 5 for the patients with tumor control, and at the end of therapy for patients who had progression. Single cell RNA-seq of PBMCs demonstrated a signature consistent with T cell activation and proliferation, and SASP-associated changes in the myeloid compartment as seen in mice. T cell profiling of PBMCs by flow cytometry demonstrated strong proliferation of T cells in patients with tumor control. Serum cytokine analysis demonstrated robust in IL-12, IFN-gamma and Eotaxin-1 in the two responders, which was not seen in the patients whose tumors progressed. Our study illustrates p53-induced SASP in TAMs as a mechanism to reprogram the TME and augment responses to ICB. Ongoing studies will help determine biomarkers that are predictive of response to APR-246+ICB therapy. Citation Format: Arnab Ghosh, Judith Michel, Divya Venkatesh, Riccardo Mezzadra, Lauren Dong, Fadi Samaan, Ricardo Gomez, Nathan Suek, Aliya Holland, Yu-Jui Ho, Mohsen Abu-Akeel, Luis Felipe Campesato, Levi Mark Bala Mangarin, Cailian Liu, Hong Zhong, Sadna Budhu, Andrew Chow, Roberta Zappasodi, Marcus Ruscetti, Scott W. Lowe, Taha Merghoub, Jedd D. Wolchok. Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 250.

Acute myeloid leukemia (AML) is a heterogeneous disease that develops within a complex microenvironment. Reciprocal interactions between the bone marrow mesenchymal stem/stromal cells (BM-MSCs) and AML cells can promote AML progression and resistance to chemotherapy (Jacamo et al., 2014). We have recently reported that BM-MSCs derived from AML patients (n=103) highly express p53 and p21 compared to their normal counterparts (n=73 p<0.0001) (Hematologica, 2018). To assess the function of p53 in BM-MSCs, we generated traceable lineage specific mouse models targeting Mdm2 or Trp53 alleles in MSCs (Osx-Cre;mTmG;p53fl/fl and Osx-Cre;mTmG;Mdm2fl/+) or hematopoietic cells (Vav-Cre;mTmG;p53fl/fl and Vav-Cre;mTmG;Mdm2fl/+). Homozygote deletion of Mdm2 (Osx-Cre;Mdm2fl/fl) resulted in death at birth and displayed skeletal defects as well as lack of intramedullary hematopoiesis. Heterozygote deletion of Mdm2 in MSCs was dispensable for normal hematopoiesis in adult mice, however, resulted in bone marrow failure and thrombocytopenia after irradiation. Homozygote deletion of Mdm2 in hematopoietic cells (Vav-Cre;Mdm2fl/fl) was embryonically lethal but the heterozygotes were radiosensitive. We next sought to examine if p53 levels in BM-MSCs change after cellular stress imposed by AML. We generated a traceable syngeneic AML model using AML-ETO leukemia cells transplanted into Osx-Cre;mTmG mice. We found that p53 was highly induced in BM-MSCs of AML mice, further confirming our findings in primary patient samples. The population of BM-MSCs was significantly increased in bone marrow Osx-Cre;mTmG transplanted with syngeneic AML cells. Tunnel staining of bone marrow samples in this traceable syngeneic AML model showed a block in apoptosis of BM-MSCs suggesting that the expansion of BM-MSCs in AML is partly due to inhibition of apoptosis. As the leukemia progressed the number of Td-Tomato positive cells which represents hematopoietic lineage and endothelial cells were significantly decreased indicating failure of normal hematopoiesis induced by leukemia. SA-β-gal activity was significantly induced in osteoblasts derived from leukemia mice in comparison to normal mice further supporting our observation in human leukemia samples that AML induces senescence of BM-MSCs. To examine the effect of p53 on the senescence associated secretory profile (SASP) of BM-MSCs, we measured fifteen SASP cytokines by qPCR and found significant decrease in Ccl4, Cxcl12, S100a8, Il6 and Il1b upon p53 deletion in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) compared to p53 wildtype mice. To functionally evaluate the effects of p53 in BM-MSCs on AML, we deleted p53 in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) and transplanted them with syngeneic AML-ETO-Turquoise AML cells. Deletion of p53 in BM-MSCs strongly inhibited the expansion of BM-MSCs in AML and resulted in osteoblast differentiation. This suggests that expansion of BM-MSCs in AML is dependent on p53 and that deletion of p53 results in osteoblast differentiation of BM-MSCs. Importantly, deletion of p53 in BM-MSCs significantly increased the survival of AML mice. We further evaluated the effect of a Mdm2 inhibitor, DS-5272, on BM-MSCs in our traceable mouse models. DS-5272 treatment of Osx-cre;Mdm2fl/+ mice resulted in complete loss of normal hematopoietic cells indicating a non-cell autonomous regulation of apoptosis of hematopoietic cells mediated by p53 in BM-MSCs. Loss of p53 in BM-MSCs (Osx-Cre;p53fl/fl) completely rescued hematopoietic failure following Mdm2 inhibitor treatment. In conclusion, we identified p53 activation as a novel mechanism by which BM-MSCs regulate proliferation and apoptosis of hematopoietic cells. This knowledge highlights a new mechanism of hematopoietic failure after AML therapy and informs new therapeutic strategies to eliminate AML. Disclosures Khoury: Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding. Bueso-Ramos:Incyte: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy. OffLabel Disclosure: Mdm2 inhibitor-DS 5272

Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by bone marrow infiltration of abnormally proliferating leukemic blasts which results in fatal anemia, bleeding and infectious complications due to compromised normal hematopoiesis. Patients with complete remission (CR) but incomplete blood cell count recovery (CRi) have significantly shorter survival compared to CR patients. Although there is a correlation between CRi and minimal residual disease (MRD), the two variables were shown to be independent risk factors for relapse development (1). The mechanism by which AML induces bone marrow failure in patients is largely unknown. Here, we demonstrate that AML derived MSCs highly express p53 and p21 proteins and are more senescent compared to their normal age-matched controls as demonstrated by high β-galactosidase staining (figure 1. A, B&C). Emerging evidence indicates that the aging of endosteal niche cells results in lower reconstitution potential of hematopoietic stem cells (2). To functionally evaluate the effects of AML on bone marrow MSCs, we utilized a murine leukemia model of the AML microenvironment. We transplanted Osx-Cre;mTmG mice with AML cells and compared the senescence of MSCs in normal bone marrow (Figure 1.D) with AML (Figure 1.E). Consistent with our initial findings in human, AML strongly induced senescence of osteoblasts. This suggests that AML suppresses normal hematopoiesis by inducing senescence in the hematopoietic niche. To address the role of p53 signaling in senescence of MSCs we generated a traceable conditional p53 gain/loss model specifically in bone marrow MSCs using Osx-Cre;mTmG; Mdm2fl/+ and Osx-Cre;mTmG;p53fl/fl mice respectively (Figure 1.F). Deletion of p53 in bone marrow MSCs resulted in an increased population of osteoblasts (GFP+) in Osx-Cre;mTmG;p53fl/fl mice in comparison to Osx-Cre;mTmG mice suggesting that p53 loss in osteoblasts inhibits senescence of osteoblasts. In order to evaluate p53 activity after recombination of p53fl alleles in the osteoblasts, we isolated MSCs from bone marrows and analyzed the expression of p21.P21 was significantly down regulated in osteoblasts (GFP+) derived from Osx-Cre;mTmG;p53fl/fl mice whereas its expression in the hematopoietic cells from same tissue (tdTomato+) remained comparable to p53 wild type suggesting that p21 as the master regulator of senescence is regulated by p53 in bone marrow mesenchymal cells. To evaluate the effect of p53 loss in osteoblasts and its impact on hematopoietic cells, we isolated the GFP+ cells (osteoblasts) and RFP + cells (hematopoietic) by FACS. Senescent cells, non-cell autonomously, modulate the bone marrow microenvironment through the senescence-associated secretory phenotype (SASP). We analyzed the expression of fifteen SASP cytokines by QPCR. Deletion of p53 in bone marrow mesenchymal cells strongly abrogated the expression of several SASP cytokines. Interestingly several Notch target genes such as Hey1 and Hey2 were highly induced in MSCs following p53 deletion suggesting a role for Notch signaling in hematopoietic failure following AML induced MSCs senescence. Our data suggest that AML induces senescence of endosteal niche resulting in hematopoietic failure. These findings contribute to our understanding of the role of p53 in leukemia MSCs and could have broad translational significance for the treatment of hematopoietic failure in patients with AML.Chen X, et al. (2015) Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia. J Clin Oncol 33(11):1258-1264.Li J, et al. (2018) Murine hematopoietic stem cell reconstitution potential is maintained by osteopontin during aging. Sci Rep 8(1):2833. Disclosures Andreeff: Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Jazz Pharma: Consultancy; Reata: Equity Ownership.

Abstract Bladder cancer (BCa) represents the fourth most common cancer in men with a poor patient prognosis for advanced disease. About 75% of patients present non-muscle invasive BCa (NMIBC) and 25% present muscle invasive BCa (MIBC) or metastatic disease. Although BCG immunotherapy is a common treatment of BCa, the dynamics of immune cell infiltration and its relevance for disease outcome are still not well understood. We took advantage of a genetic based mouse model of MIBC to obtain a profound understanding of the immune contexture over tumor development. P53Fl/Fl/PTENFl/Fl mice were injected with Cre recombinase-expressing adenoviral vectors directly into bladder by micro-surgery. We observed the presence of NMIBC, MIBC and the development of metastases at 5, 9 and 13 weeks after vector injection, respectively. As tumor evolves, we observed a transition from an anti-tumoral microenvironment (MHCIIhigh TAM, CD4 and CD8 TILs) to a pro-tumoral one (MHCIIlow TAM and MDSC), going along with an increased expression of PD-1 by T cells and accumulation of regulatory T cells. After testing various immunotherapy tretaments on mice with MIBC, we found out that combination of αCD40+αPD1 increases CD8 TILs infiltration and IFNg production, re-polarized TAM toward a M1-like phenotype and increased the survival of the mice.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and the leading cause of cancer-related deaths. The canonical NF-κB pathway displays critical functions in the development of PDAC. However, the relationship between the non-canonical NF-κB pathway and PDAC is still obscure. Here, we analyzed the role of the non-canonical NF-κB signaling in precancerous lesions and PDAC development by deleting NF-κB inducing kinase (NIK), an essential kinase for the activation of the non-canonical pathway. We used KC mice (Pdx1-cre/LSL-KrasG12D) that develop pancreatic intraepithelial neoplasias (PanINs) already at the age of 8 weeks. To evaluate the role of the non-canonical NF-κB signaling, KC mice were crossed with NIK-floxed mice (NiC) to generate the KNiC (Pdx1-cre/KRASG12D/NIKfl/fl) mouse model. Mice were injected with cerulein to promote the development of pancreatitis and support the development of PanINs. At the age of 8 weeks, mice were sacrificed and analyzed. Furthermore, to analyze the role of the non-canonical pathway in PDAC development, we crossed -the rapidly PDAC developing- KPC mice (Pdx1-cre/LSL-KrasG12D/p53fl/fl) to NiC mice and generated the KPNiC (Pdx1-cre/KRASG12D/NIKfl/fl/p53fl/fl) mouse model. Mice were analyzed at the age of 8 weeks or when they reached their humane endpoint. Analysis of the pancreata revealed that deletion of NIK supported the development of higher-grade precancerous lesions and an increased remodeling area. Importantly, NIK deletion accelerated the establishment of PDAC already by the age of 8 weeks. In addition, the pancreatic/body weight ratio was significantly higher in the KNiC group compared to the KC group (Mean: KC 0.018 vs KNiC 0.030, P<0.05). These results were further verified by Ki67 staining, where we detected more proliferating cells in KNiC pancreata (Mean: KC 66.53/mm2 vs KNiC 205.60/mm2, P<0.05). Moreover, FN1 expression level (Mean: KC 1.00 vs KNiC 2.14, P<0.05) and the number α-SMA+ cells (Mean: KC 6.73% vs KNiC 17.47%, P<0.05) were dramatically elevated in KNiC pancreata, indicating increased desmoplastic reaction after NIK deletion. Interestingly, KNiC pancreata displayed stronger activation of the STAT3 pathway which is associated with PanIN development and progression (Mean: KC 1.00 vs KNiC 1.73, P<0.05). With respect to the KPC and KPNiC mice, NIK deletion strongly accelerated the development of PDAC at the age of 8 weeks (83.3% of KPNiC mice, 25% of KPC mice). Finally, the median survival of KPNiC mice (71 days) was remarkably shorter than the survival of KPC mice (85 days). Our data demonstrate that deletion of NIK in KC mice promoted the progression of precancerous lesions, a stronger fibrotic reaction, and activation of the STAT3 pathway. Further, deletion of NIK in KPC mice accelerated cancer development and shortened the lifespan of the mice. These results indicate that inactivation of the non-canonical NF-κB pathway leads to PDAC deterioration. Analysis on later time points and further examination of the mechanism is ongoing. Citation Format: Ziwei Du, Miltiadis Tsesmelis, Thomas Wirth. NF-κB inducing kinase (NIK) deletion accelerates the progression of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A059.

Abstract Elevated levels of Interleukin 22 (IL-22) and its receptor (IL-22R) are associated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanism is currently unknown. Our aim is to investigate the role of the IL-22 axis in PDAC initiation and progression. Impact of IL-22 on tumor initiation was assayed by subcutaneous and intravenous inoculation of PDAC cells into wild-type and IL-22−/− mice. The PKCY (Pdx1-Cre; KrasG12D; p53fl/+; RosaYFP) model of pancreas cancer was used to study the in-vivo presence and significance of IL-22 in spontaneous tumors. Results were confirmed in human specimens of surgically resected pancreas cancer. IL-22R was present in all tested PDAC lines and IL-22 treatment led to STAT3 phosphorylation and subsequent increased expression of EMT (Epithelial to Mesenchymal Transition) transcription factors. Cells transitioned to a mesenchymal phenotype and robust tumor sphere formation was observed. While tumors readily formed in wild-type mice, initiation, establishment and growth were impaired in IL-22−/− mice and PKCY-IL-22−/− mice. Increased levels of IL-22 were found in both spontaneous murine tumors and surgical specimens compared to control tissue. IHC of tumors showed diffuse IL-22R staining with increasing intensity of pSTAT3 and EMT markers as tumors progressed to invasive cancer. FACS analysis identified type 3 innate lymphoid cells and TH22 cells as the source of IL-22 in both human and murine PDAC. Our data suggests that IL-22 is integral in the initiation, progression and establishment of pancreatic cancer, positioning it as an attractive target for cancer therapy.

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor that arises in the pons of children. Recent studies using single cell RNA-seq and enhancer analysis of DIPG tumor cells, together with analysis of the developing human pons, strongly suggest that an oligodendrocyte progenitor cell is the most likely cell-of-origin for DIPG. Here we describe a novel mouse model by expressing PDGF-B, with H3.3K27M or H3.3 wild-type in Olig2-expressing progenitors via injection into the 4th ventricle using Olig2-tva-cre;p53fl/fl mice. H3.3K27M tumors have high rate of Ki-67, Sox2, and Olig2 positivity and show a higher rate of leptomeningeal dissemination than H3.3 wild-type tumors (95.2％ vs 68.8%, p=0.0303) and mice harboring H3.3K27M tumors demonstrate a significantly shorter survival period than those harboring H3.3 wild-type tumors (31 days vs. 37 days, p=0.0473). While there is not any difference in survival between mice harboring PDGF-B; p53 wild-type; H3.3K27M tumors and those harboring PDGF-B; p53 wild-type; H3.3 wild-type tumors at the 6-month endpoint, we found that H3.3K27M tumors show a higher rate of high-grade glioma than H3.3 wild-type tumors (100% vs. 41.7%, p=0.0017). RNA-seq analysis identified 25 significantly differentially expressed genes with 23 upregulated and 2 downregulated genes in the PDGF-B; p53 null; H3.3K27M tumors compared with the H3.3 wild-type tumors. Phox2b, which is the most upregulated gene in the PDGF-B; p53 null; H3.3K27M tumors, was validated by qRT-PCR and expressed only in brainstem tumors and not expressed in tumors located in any other areas. IHC with Phox2b also revealed positivity in PDGF-B; p53 null; H3.3K27M tumors located only in the brainstem. Ongoing work includes validation of other significant differentially expressed genes as well as elucidation of their role in K27M-mediated gliomagenesis.

OBJECTIVEDrug clearance may be a limiting factor in the clinical application of convection-enhanced delivery (CED). Peptide-based nanofibers (NFPs) have a high aspect ratio, and NFPs loaded with drugs could potentially maintain effective drug concentrations for an extended period sufficient for cancer therapy. The objective of this study was to assess the volume of distribution (Vd) and clearance of variable lengths of NFPs when administered using CED.METHODSNFPs composed of multiple methoxypolyethylene glycol (mPEG)-conjugated constructs (mPEG2000-KLDLKLDLKLDL-K(FITC)-CONH2, for which FITC is fluorescein isothiocyanate) were assembled in an aqueous buffer. The NFPs were approximately 5 nm in width and were formulated into different lengths: 100 nm (NFP-100), 400 nm (NFP-400), and 1000 nm (NFP-1000). The NFP surface was covalently conjugated with multiple Cy5.5 fluorophores as the optical reporters to track the post-CED distribution. Forty-two 6- to 8-week-old Ntv-a;p53fl/fl mice underwent CED to the striatum. Animals were killed immediately, 24 hours or 72 hours after CED. The brains were extracted and sectioned for assessing NFP Vd to volume of infusion (Vi) ratio, and clearance using fluorescence microscopy.RESULTSCED of NFPs was well tolerated by all the animals. The average Vd/Vi ratios for NFP-100, NFP-400, NFP-1000, and unconjugated positive control (free Cy5.5) were 1.87, 2.47, 1.07, and 3.0, respectively, which were statistically different (p = 0.003). The percentages remaining of the original infusion volume at 24 hours for NFP-100, -400, and -1000 were 40%, 90%, and 74%, respectively. The percentages remaining at 72 hours for NFP-100, -400, and -1000 were 15%, 30%, and 46%, respectively. Unconjugated Cy5.5 was not detected at 24 or 72 hours after CED.CONCLUSIONSCED of NFPs is feasible with Vd/Vi ratios and clearance rates comparable to other nanocarriers. Of the 3 NFPs, NFP-400 appears to provide the best distribution and slowest clearance after 24 hours. NFP provides a dynamic theranostic platform, with the potential to deliver clinically efficacious drug payload to brain tumor after CED.

The TP53 tumour suppressor gene encodes for p53 protein which is the frequently altered gene in most of the cancers. p53 protein is activated in response to different stresses and plays major role in maintaining genome integrity by regulating cell cycle and cell survival. It is known that p53 has twelve isoforms generated using alternate splicing, alternate promoters and translation initiation sites. Out of these isoforms ∆40p53 (also known as ∆N-p53/p47) is the only translational isoform of p53, produced from the same mRNA by using an ‘internal ribosome entry site’ (IRES). From our laboratory, it has been shown earlier that p53 mRNA has two IRES elements, IRES1 and IRES2. IRES2 mediated translation of ∆40p53 is maximum during G1-S phase and that of full length p53 (p53FL) by IRES1 is maximum in G2-M phase. p53 gene expression levels are regulated at many levels, including transcription, splicing, mRNA transport, stability and protein translation. The focus of our laboratory is on the translational control of p53. Previous studies from our laboratory has demonstrated PTB (polypyrimidine binding protein), Annexin A2 (ANXA2), PTB-associated splicing factor (PSF) and Death-associated protein 5 (DAP5) to be the crucial ITAFs (IRES trans acting factors) for IRES mediated translation regulation of p53 and ∆40p53 under different stress conditions. PTB binds to the p53 mRNA IRESs and enhances the translation of p53 isoforms by translocating from nucleus to cytoplasm upon doxorubicin-induced DNA damage. ANXA2 and PSF proteins, the other p53 ITAFs, interact with p53 IRESs ex vivo in a stress-induced manner, showing greater association with the IRESs upon ER stress (thapsigargin treatment). DAP5 was demonstrated to bind to p53 IRESs and regulate the IRES2 mediated expression of Δ40p53.The tightly regulated p53 in turn regulates different target genes. p53 is also known to regulate miRNAs followed by their respective target genes and hence different cellular outcome. The present study focuses on translational regulation of p53 and its isoform ∆40p53 by binding of proteins and miRNAs at the untranslated regions (UTRs). Further, the effect of the differential expression of these two isoforms on cellular gene expression mediated by miRNA is also studied. Translation regulation of p53 involves interaction of proteins and microRNAs with the 5’and 3’ UTR of p53 mRNA. Earlier we have shown that PTB and ANXA2 interact with the 5’UTR of p53 mRNA to regulate its expression. Here we have studied the role of 3’UTR in regulating the expression of the two isoforms, that is, full-length p53 (p53FL) and ∆40p53. We have demonstrated that both PTB and ANXA2 bind to p53 3’UTR and delineated the specific binding regions within 3’UTR that contribute to these interactions. Knockdown of both PTB and ANXA2 led to significant decrease in translation of p53 isoforms., mediated by their interaction to the 5’UTR/3’UTR. Interestingly, we have observed that the addition of p53 3’UTR to the constructs led to a decrease in the expression of both reporter and p53 isoforms, indicating that microRNAs binding to 3’UTR might be playing a role in this regulation. Further, we have explored the role of possible interplay between protein and microRNAs in the 3’UTR mediated translational control of p53. Interestingly, PTB showed some overlapping binding regions in the p53 3′UTR with some miRNAs. In order to understand the interplay between PTB and miRNAs different approaches were taken. Firstly, after partial silencing of PTB, increase in the association of Ago-2 complex with p53 mRNA was observed. Secondly, this interplay was also observed under DNA damage (doxorubicin treatment), where PTB is known to be translocated to the cytoplasm from the nucleus. Under DNA damage there was decreased association of p53 mRNA with Ago-2 and vice versa association with PTB. So this increased binding of p53 mRNA with PTB under DNA damage suggests that there is interplay between miRNAs and PTB at the 3′UTR under normal and stress conditions like DNA damage. Interestingly, PTB showed some overlapping binding regions in the p53 3′UTR with miR-1285. In fact, knockdown of miR-1285 as well as expression of p53 3′UTR with mutated miR-1285 binding sites resulted in enhanced association of PTB with the 3′UTR and decreased association with Ago-2, which provides mechanistic insights of this interplay. Furthermore, to understand the physiological relevance, we curated single nucleotide variations (SNVs) in the p53 3′UTR, at the miRNA binding sites from literature. We investigated the effect of these SNVs on the 3′UTR mediated regulation of p53 expression by using reporter gene constructs containing wild type 3′UTR (Fluc-3′UTR WT) or 3′UTRs harbouring individual SNVs (Fluc-3′UTR SNV 93/287/737/806) in H1299 and A549 cells. SNV806 displayed highest reporter activity compared to the WT. Interestingly, in vitro experiments in the current study indicates that PTB binding to the 3′UTR with SNV 806 is higher compared to WT 3′UTR, thus suggesting a possibility of miR-1285 and PTB having common binding regions. Taken together, the results provide a plausible molecular basis of how the interplay between miRNAs and the PTB protein at the 3′UTR can play pivotal role in fine tuning the expression of the two p53 isoforms. Human cancer cells are often associated with a widespread decrease in miRNAs, which shows a crosstalk between p53 tumour suppressor pathway and miRNA regulation system. It is known that the p53 isoforms are differentially regulated and their differential expression leads to differential downstream target gene expressions, which can be a direct regulation or regulation mediated by miRNAs. The transcription of some pri-miRNAs is regulated by p53 through binding to consensus sites in their promoters. Also, p53 modulates miRNA processing through interaction with Drosha-p68 complex. miRNAs can target various transcripts, so they are involved in diverse processes such as cellular differentiation, metabolism and cell proliferation. Role of full length p53 in controlling expression of miRNAs is established but the role of ∆40p53 in regulating the p53 responsive miRNAs was not known. So here we have investigated the effect of differential expression of the two isoforms individually or both the isoforms together on the downstream miRNAs. Out of several differentially regulated miRNAs some miRNAs were found to be commonly regulated by both the isoforms individually and with their combination. However, out of these some were unique to the individual isoform. In order to understand the functions of ∆40p53 alone the miRNAs either uniquely regulated or the ones which showed the maximum fold change under ∆40p53 expression were short-listed for further investigation. Results from the microarray of the miRNA showed that there are different miRNAs whose expressions are regulated under ∆40p53 expression. The network analysis for ∆40p53 revealed its involvement in different cellular functions. ∆40p53 upregulated miRNAs which are involved in pathways viz. cell cycle regulation, apoptosis, cell proliferation, senescence etc. Interestingly ∆40p53 showed antagonistic regulation of miR-186-5p as compared to the dataset obtained with either p53 alone or the combination of both the isoforms. Also this miRNA is known to be involved in cell proliferation, senescence and cell cycle arrest pathways. Hence we pursued miR-186-5p for further characterization under ∆40p53 expression. In this study we focused on miR-186-5p mediated effect of ∆40p53 in cell proliferation. One of the established targets of miR-186-5p is YY1, which is also known to be involved in cell proliferation. Our results showed significant decrease in YY1 mRNA levels under the expression of ∆40p53. Further assays with anti-miR-186 established the interdependence of ∆40p53−miR-186-5p−YY1−cell proliferation. This unravels that ∆40p53 can also regulate cellular fate independent of p53FL. Furthermore, p53 and ∆40p53 are known to be deregulated in different stress conditions like DNA damage, endoplasmic reticulum stress, oncogene-induced senescence and cancer. We have explored the effect of nutrient-deprivation mediated translational regulation of p53 mRNA using glucose depletion as a model system. We found scaffold/matrix attachment region-binding protein 1 (SMAR1), a predominantly nuclear protein is abundant in the cytoplasm under glucose deprivation. SMAR1 knockdown decreased p53 IRES activity in normal conditions and under glucose deprivation. We also observed concomitant effect of SMAR1 knockdown on the p53 and Δ40p53 target genes involved in cell-cycle arrest, metabolism and apoptosis. In addition, rescue experiments ex vivo shows that the induction of p53 isoform levels on nutrient deprivation is reversible and also their targets show similar reversal in their mRNA levels. This study provides a physiological insight into the regulation of this critical tumour suppressor in nutrient starvation and also the downstream transcriptional targets. Interestingly, we also observed that SMAR1 can interact with 3’UTR with binding sites common to PTB, which results in interplay between the two proteins in vitro. Individual knockdown of these proteins decreased the p53 expression but silencing of both proteins together showed further decreases in the expression. These results suggest yet another interplay involving PTB with SMAR1 at the 3’UTR under different stress conditions. Taken together this study unfolds complex mechanisms by which p53 and Δ40p53 are regulated in different stress conditions; DNA damage and glucose starvation by proteins like PTB and SMAR1 respectively. This study also indicates that for the fine tuning of the regulation of p53 isoforms interplay between protein and miRNAs is required. Differential expression of miRNAs under p53 isoforms expression tells us the importance of these isoforms in regulation of miRNAs. It also contributes to the novel role of Δ40p53 in regulating miRNAs independent of p53 and hence consequent changes in cellular fate.