P53 Target Research Articles

Syntaxin-6, a Reliable Biomarker for Predicting the Prognosis of Patients with Cancer and the Effectiveness of Immunotherapy

Syntaxin-6 (STX6), a vesicular transport protein, is a direct target of the tumor suppressor gene P53, supporting cancer growth dependent on P53. However, STX6's function in the tumor microenvironment has yet to be reported. In this research, we comprehensively explored the role of the oncogene STX6 in pan-cancer by combining data from several databases, including the Cancer Genome Atlas, CPTAC, cBioPortal, and TIMER. Then, we verified the carcinogenic effect of STX6 in hepatocellular carcinoma (HCC) and colorectal cancer (CRC) through a series of experiments in vitro and in vivo. Bioinformatics analysis demonstrated that STX6 is an oncogene for several cancers and is mainly involved in the cell cycle, epithelial-mesenchymal transition, oxidative phosphorylation, and tumor immune modulation, especially for tumor-associated fibroblasts (CAFs) and NKT cells. Additionally, a high level of STX6 could indicate patients' resistance to immunotherapy. Our own data indicated that the STX6 level was upregulated in HCC and CRC. Knockdown of the STX6 levels could arrest the cell cycle and restrain cell proliferation, migration, and invasion. RNA-seq indicated that STX6 was significantly involved in pathways for cancer, such as the MAPK signal pathway. In a mouse model, knockdown of STX6 inhibited tumor growth and potentiated anti-PD-1 efficacy. In light of the essential roles STX6 plays in carcinogenesis and cancer immunology, it has the potential to be a predictive biomarker and a target for cancer immunotherapy.

Interaction of C-terminal p53 isoforms depends strongly upon DNA sequence and topology

The p53 protein is a key tumor suppressor and the most commonly mutated and down-regulated protein in human tumors. It functions mainly through interaction with DNA, and p53 acts as a transcription factor that recognizes the so-called p53 target sites on the promoters of various genes. P53 has been shown to exist as many isoforms, including three C-terminal isoforms that are produced by alternative splicing. Because the C-terminal domain is responsible for sequence-nonspecific binding and regulation of p53 binding, we have analyzed DNA recognition by these C-terminal isoforms. Using atomic force microscopy, we show for the first time that all C-terminal isoforms recognize superhelical DNA. It is particularly noteworthy that a sequence-specific p53 consensus binding site is bound by p53α and β isoforms with similar affinities, whilst p53α shows higher binding to a quadruplex sequence than both p53β and p53γ, and p53γ loses preferential binding to both the consensus binding sequence and the quadruplex-forming sequence. These results show the important role of the variable p53 C-terminal amino acid sequences for DNA recognition.

Inhibition of p21 activates Akt kinase to trigger ROS-induced autophagy and impacts on tumor growth rate

Owing to its ability to induce cellular senescence, inhibit PCNA, and arrest cell division cycle by negatively regulating CDKs as well as being a primary target of p53, p21 is traditionally considered a tumor suppressor. Nonetheless, several reports in recent years demonstrated its pro-oncogenic activities such as apoptosis inhibition by cytosolic p21, stimulation of cell motility, and promoting assembly of cyclin D-CDK4/6 complex. These opposing effects of p21 on cell proliferation, supported by the observations of its inconsistent expression in human cancers, led to the emergence of the concept of “antagonistic duality” of p21 in cancer progression. Here we demonstrate that p21 negatively regulates basal autophagy at physiological concentration. Akt activation, upon p21 attenuation, driven ROS accumulation appears to be the major underlying mechanism in p21-mediated modulation of autophagy. We also find p21, as a physiological inhibitor of autophagy, to have oncogenic activity during early events of tumor development while its inhibition favors survival and growth of cancer cells in the established tumor. Our data, thereby, reveal the potential role of autophagy in antagonistic functional duality of p21 in cancer.

BI-907828, a novel potent MDM2 inhibitor, inhibits glioblastoma brain tumor stem cells in vitro and prolongs survival in orthotopic xenograft mouse models.

The tumor suppressor TP53 (p53) is frequently mutated, and its downstream effectors inactivated in many cancers, including glioblastoma (GBM). In tumors with wild-type status, p53 function is frequently attenuated by alternate mechanisms including amplification and overexpression of its key negative regulator, MDM2. We investigated the efficacy of the MDM2 inhibitor, BI-907828, in GBM patient-derived brain tumor stem cells (BTSCs) with different amplification statuses of MDM2, in vitro and in orthotopic xenograft models. In vitro growth inhibition and on-target efficacy of BI-907828 were assessed by cell viability, co-immunoprecipitation assays, and western blotting. In vivo efficacy of BI-907828 treatments was assessed with qPCR, immunohistochemistry, and in intracranial xenograft models. BI-907828 decreases viability and induces cell death at picomolar concentrations in both MDM2 amplified and normal copy number TP53 wild-type BTSC lines. Restoration of p53 activity, including robust p21 expression and apoptosis induction, was observed in TP53 wild-type but not in TP53 mutant BTSCs. shRNA-mediated knock-down of TP53 in wild-type BTSCs abrogated the effect of BI-907828, confirming the specificity of the inhibitor. Pharmacokinetic-pharmacodynamic studies in orthotopic tumor-bearing severe combined immunodeficiency (SCID) mice demonstrated that a single 50mg/kg p.o. dose of BI-907828 resulted in strong activation of p53 target genes p21 and MIC1. Long-term weekly or bi-weekly treatment with BI-907828 in orthotopic BTSC xenograft models was well-tolerated and improved survival both as a single-agent and in combination with temozolomide, with dose-dependent efficacy observed in the MDM2 amplified model. BI-907828 provides a promising new therapeutic option for patients with TP53 wild-type primary brain tumors.

Selective ablation of P53 in pancreatic beta cells fails to ameliorate glucose metabolism in genetic, dietary and pharmacological models of diabetes mellitus

ObjectiveBeta cell dysfunction and death are critical steps in the development of both type 1 and type 2 diabetes (T1D and T2D), but the underlying mechanisms are incompletely understood. Activation of the essential tumor suppressor and transcription factor P53 (also known as TP53 and Trp53 in mice) was linked to beta cell death in vitro and has been reported in several diabetes mouse models and beta cells of humans with T2D. In this article, we set out to determine the beta cell specific role of P53 in beta cell dysfunction, cell death and development of diabetes in vivo. MethodsWe generated beta cell specific P53 knockout (P53BKO) mice and used complementary genetic, dietary and pharmacological models of glucose intolerance, beta cell dysfunction and diabetes development to evaluate the functional role of P53 selectively in beta cells. We further analyzed the effect of P53 ablation on beta cell survival in isolated pancreatic islets exposed to diabetogenic stress inducers ex vivo by flow cytometry. ResultsBeta cell specific ablation of P53/Trp53 failed to ameliorate glucose tolerance, insulin secretion or to increase beta cell numbers in genetic, dietary and pharmacological models of diabetes. Additionally, loss of P53 in beta cells did not protect against streptozotocin (STZ) induced hyperglycemia and beta cell death, although STZ-induced activation of classical pro-apoptotic P53 target genes was significantly reduced in P53BKO mice. In contrast, Olaparib mediated PARP1 inhibition protected against acute ex vivo STZ-induced beta cell death and islet destruction. ConclusionsOur study reveals that ablation of P53 specifically in beta cells is unexpectedly unable to attenuate beta cell failure and death in vivo and ex vivo. While during development and progression of diabetes, P53 and P53-regulated pathways are activated, our study suggests that P53 signaling is not essential for loss of beta cells or beta cell dysfunction. P53 in other cell types and organs may predominantly regulate systemic glucose homeostasis.

Distinct p53 phosphorylation patterns in chronic lymphocytic leukemia patients are reflected in the activation of circumjacent pathways upon DNA damage.

TP53 gene abnormalities represent the most important biomarker in chronic lymphocytic leukemia (CLL). Altered protein modifications could also influence p53 function, even in the wild-type protein. We assessed the impact of p53 protein phosphorylations on p53 functions as an alternative inactivation mechanism. We studied p53 phospho-profiles induced by DNA-damaging agents (fludarabine, doxorubicin) in 71 TP53-intact primary CLL samples. Doxorubicin induced two distinct phospho-profiles: profile I (heavily phosphorylated) and profile II (hypophosphorylated). Profile II samples were less capable of activating p53 target genes upon doxorubicin exposure, resembling TP53-mutant samples at the transcriptomic level, whereas standard p53 signaling was triggered in profile I. ATM locus defects were more common in profile II. The samples also differed in the basal activity of the hypoxia pathway: the highest level was detected in TP53-mutant samples, followed by profile II and profile I. Our study suggests that wild-type TP53 CLL cells with less phosphorylated p53 show TP53-mutant-like behavior after DNA damage. p53 hypophosphorylation and the related lower ability to respond to DNA damage are linked to ATM locus defects and the higher basal activity of the hypoxia pathway.

P53 maintains gallid alpha herpesvirus 1 replication by direct regulation of nucleotide metabolism and ATP synthesis through its target genes.

P53, a well-known tumor suppressor, has been confirmed to regulate the infection of various viruses, including chicken viruses. Our previous study observed antiviral effect of p53 inhibitor Pifithrin-α (PFT-α) on the infection of avian infectious laryngotracheitis virus (ILTV), one of the major avian viruses economically significant to the poultry industry globally. However, the potential link between this antiviral effect of PFT-α and p53 remains unclear. Using chicken LMH cell line which is permissive for ILTV infection as model, we explore the effects of p53 on ILTV replication and its underlying molecular mechanism based on genome-wide transcriptome analysis of genes with p53 binding sites. The putative p53 target genes were validated by ChIP-qPCR and RT-qPCR. Results demonstrated that, consistent with the effects of PFT-α on ILTV replication we previously reported, knockdown of p53 repressed viral gene transcription and the genome replication of ILTV effectively. The production of infectious virions was also suppressed significantly by p53 knockdown. Further bioinformatic analysis of genes with p53 binding sites revealed extensive repression of these putative p53 target genes enriched in the metabolic processes, especially nucleotide metabolism and ATP synthesis, upon p53 repression by PFT-α in ILTV infected LMH cells. Among these genes, eighteen were involved in nucleotide metabolism and ATP synthesis. Then eight of the 18 genes were selected randomly for validations, all of which were successfully identified as p53 target genes. Our findings shed light on the mechanisms through which p53 controls ILTV infection, meanwhile expand our knowledge of chicken p53 target genes.

Dual IKZF2 and CK1α Degrader Targets Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematologic disease for which several epigenetic regulators have been identified as therapeutic targets. We previously found that a transcription factor and Ikaros family member, IKZF2 (HELIOS) is required for myeloid leukemic stem cell activity (Park et al. Cell Stem Cell 2019). Additionally, IKZF2 enhances self-renewal by increasing HOXA9 gene expression and in parallel blocks myeloid differentiation by reducing C/EBP expression. Thus, our studies suggested that targeting IKZF2 could be a new therapeutic strategy in AML. Immunomodulatory drugs (IMiDs; lenalidomide and others) have been used clinically to specifically target and degrade other Ikaros family members. However, these have been unable to target IKZF2. Utilizing a structure-guided approach, we developed a small molecule with low nanomolar degrader activity against IKZF2 (DEG-35). DEG-35 treatment in different AML cell lines induce apoptosis and differentiation with IC50 values of 5 ±0.4 nM in MOLM-13 cells to 27± 2.5 uM in NB4 cells. To understand if DEG35 has activity against other substrates, we performed global quantitative proteomics and found casein kinase 1 alpha (CK1α), a serine/threonine kinase previously identified to be critical for myeloid leukemogenesis and a known IMiD substrate, to be the most depleted protein (Jaras et al. JEM). Also, we performed a PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) screen assay which utilizes 770 barcoded cell lines from 20 lineages and identified multiple correlations with the p53 pathway. Previous studies found that CK1α suppresses the activity of p53 phosphorylation or binding MDM proteins. To further probe the mechanism behind DEG35 activity, we performed RNA sequencing at 24hrs in MOLM-13 cells. Gene set enrichment analysis (GSEA) using the rank list of differentially expressed genes from the MOLM-13 cells treated with DMSO and DEG-35 revealed enrichment for genes upregulated in CK1α KO keratinocytes and p53 targets. Additionally, we found enrichment for the MLL-AF9 IKZF2 KO LSC gene sets, myeloid differentiation, loss of MEIS1-HOXA9 and MYC targets. Immunoblotting validated the reduction of MYC and HOXA9 protein in DEG-35 treated MOLM-13 cells. To determine the contribution of CK1α and IKZF2 to the cellular phenotypes in MOLM13 cells, we developed CK1α and IKZF2 nondegradable mutants. CK1α, G40N was able to significantly reduce the cell killing activity of DEG-35 by 94% compared to vector cells (**p<0.01). Moreover, p53 deletion was also able to block DEG-35 induced apoptosis by 98% and differentiation by 88% compared to control. Additionally, expression of IKZF2 H141Q rescued DEG-35 mediated differentiation by 55% (***p<0.001), but not apoptosis. Thus, we demonstrate that the degradation of IKZF2 and CK1α by DEG-35 blocks cell growth and induces myeloid differentiation in human AML cell lines through CK1α-p53- and IKZF2-dependent pathways. To further probe the efficacy and therapeutic index of targeting both IKZF2 and CK1α. We found that AML cells were more sensitive to DEG-35 compared to normal bone marrow cells (murine MLL-AF9 Crbn I1391V cells has 10 fold and AML PDX cells have 2-5 fold higher sensitivity to normal mouse or human bone marrow cells).Treatment of DEG-35 at 50 mg/kg in AML PDX-transplanted mice led to significant prolonged survival compared to vehicle treated mice (median survival of DMSO mice: 45 days vs DEG-35 mice: 61 days; ***p< 0.001) and reduced disease burden. To improve pharmacological properties, we developed analog DEG-77, which has 10-fold increased solubility than DEG-35 in DMSO. DEG77 exhibited better in vivo efficacy in the MLL-AF9 Crbn I1391V model (Increase in median survival by DEG-35: 2 days *p<0.05 vs DEG-77: 31 days ****p<0.0001). Importantly, the increase of median survival of 30 days (****p<0.0001) was also observed in the secondarily transplanted DEG-77 mice compared to DMSO mice, suggesting that DEG-35 can reduce LSC activity. In summary, we report the development of nanomolar monofunctional cereblon-dependent degraders of IKZF2 and casein kinase 1 alpha (CK1α). DEG-35 or the analog DEG-77 delays leukemia progression in murine and human AML mice models. Thus, we provide a novel strategy for multi-targeted degradation of IKZF2/CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Pharmacological Disruption of the Interplay between HDM2-p53 and NFκB Depletes Myelofibrosis Progenitor/Stem Cells and Dampens the Accompanying Inflammatory Milieu

We and others have observed that myelofibrosis (MF) hematopoietic stem/progenitor cells (HSC/HPCs) are characterized by upregulation of HDM2 which promotes the degradation of wild type p53 (1-4). NFκB which serves as a transcription factor (TF) for many inflammatory cytokines and promotes resistance to programmed cell death, and HIF1α, the master TF for cellular and developmental responses to hypoxia, with implied roles in angiogenesis and MF progression (5) was also upregulated. Using RT-qPCR, we determined that a correlation existed between increased transcripts of HDM2, HIF1α and NFkB and decreased transcripts of p53. Furthermore, transcripts of cytokines (LCN2, IL8 and VEGF) that are known to be regulated by HIF1α and NFkB were elevated in MF CD34+ cells as compared to normal donor (ND) CD34+ cells (p-value <0.05 for each gene). These findings indicate that the interplay between HDM2/p53, HIF1α and NFκB pathways play a role in the resistance of MF CD34+ cells to apoptosis and the generation of MF pro-inflammatory cytokines. The p53 and NFκB target genes play crucial roles in cell proliferation and survival and cancer progression. BET (Bromodomain and Extra-Terminal domain) protein family inhibitor (BETi) studies have implicated the BET protein member BRD4 in NFκB-dependent promoter and super-enhancer modulation. In order to develop a therapeutic regimen that would deplete MF CD34+ and dampen the MF pro-inflammatory milieu, we evaluated the effects of a combination of an HDM2 antagonist (HDM2a), which results in increased p53 activity, with a BETi, which repressed NFκB activity, on depletion of MF HSPCs. Treatment with either an HDM2a or BETi alone increased apoptosis of MF CD34+ cells. Combination of these the two drugs (HDM2a+BETi) led to an even greater degree of apoptosis (p=0.01 and p=0.02, respectively). RT-qPCR data showed that treatment with HDM2a increased the transcripts of downstream p53 targets including p21 and PUMA in a dose dependent fashion while HDM2a+BETi increased both genes to an even greater extent (p<0.01). The ability of these drugs to eliminate MF HSPCs was evaluated with 10 MF and 5 ND samples using colony formation assays. Treatment with either drug alone or in combination displayed limited effects on colony formation by ND CD34+ cells. By contrast, treatment with HDM2a or BETi alone decreased MF colony formation by 35% and 50% respectively, while the HDM2a+BETi combination led to an 80% reduction in MF colony numbers. JAK2V617F genotyping revealed that treatment with either drug alone decreased JAK2V617F+ colony number by 21.3% and 47%, respectively, while the combination decreased JAK2V617F+ colonies by 64% as compared to control cells. To evaluate the ability of these drugs to eliminate MF HSCs, an immunocompromised NSGS mouse (expressing human IL3, GM-CSF and SCF) xenograft transplantation model was used. MF CD34+ cells were pretreated with vehicle, HDM2a or BETi alone or HDM2a+BETi for 3 days, followed by transplantation into NSGS mice. After 12 weeks, mice were sacrificed and the degree of human cells chimerism in the bone marrows (BM) and spleens of recipient mice evaluated. As compared to vehicle treatment, the mice receiving HDM2a treated grafts had on average 40% less hCD45+ cell chimerism in both organs. BETi treated grafts led to on average a 20% decrease in hCD45+ cells chimerism in the BM but surprisingly not the spleen (Figure 1A). Mice receiving grafts treated with HDM2a+BETi exhibited on average a 60% reduction in hCD45+ cell chimerism in the BM and spleen. Moreover, the grafts treated with HDM2a+BETi generated reduced numbers of cells belonging to multiple hematopoietic lineages (CD34+, CD33+ and CD41+ cells) in both organs of NSGS as compared to mice receiving grafts pretreated with either drug alone indicating that the HDM2a+BETi combination is effective in depleting MF HSCs (Figure 1). Furthermore, treatment of MF CD34+ cells with WT p53 with an HDM2a or BETi decreased activation of the NFkB pathway, and IL8 and VEGF transcript levels. In addition, treatment of MF CD34+ cells co-cultured with ND mesenchymal stem cells and endothelial cells with the HDM2a+BETi combination led to a 50% reduction in protein levels of IL8, VEGF in the culture supernatants. These results indicate that combinations of pharmacological agents that disrupt the interplay between HDM2/p53, HIF1α and N-κB pathways could serve as effective therapeutic strategies to treat MF patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TP53 CRISPR/Cas9 Reveals Strict BAX Dependence in the Response to BH3 Mimetic Targeting MCL1

Background In multiple myeloma (MM), 1q gain/amplification (1q+) and deletion of the short arm of chromosome 17 (del17p), encompassing MCL1 and TP53, respectively, as well as the t(4;14) translocation are adverse events that reduce both event-free survival and overall survival of patients. The genome gatekeeper p53 is well known for its multiple roles in the regulation of mitochondrial apoptosis: p53 induces early direct transactivation of the BH3-only protein PUMA (BBC3) and the death effector BAX (BAX) and late direct-to-indirect transactivation of NOXA (PMAIP1) and BAK (BAK1). To investigate the direct impact of loss of p53 function in myeloma cells, we generated TP53 CRISPR/Cas9 isogenic cells from cell lines harboring t(4;14) and 1q+. Materials and Methods To knockout the expression of all p53 isoforms in two TP53wt t(4;14)+ 1q+ myeloma cell lines, NCI-H929 and XG7 cells were sequentially transduced with 2 lentiviruses expressing Cas9 and a doxycycline-inducible TP53-specific sgRNA targeting exon 5. TP53-/-clones (n=3) and control clones (n=3) were derived for each cell line by limiting dilution. Results RNAseq analysis was performed in triplicate in the 12 clones of the two MM cell lines: analysis of the 6 TP53-/-clones versus the 6 control clones showed significant changes (log2FC>0.4, p>0.05) in the expression of 92 genes under steady-state conditions (and of more than 3000 upon nutlin3a-induced p53 accumulation) : 27% of the genes were known to be direct p53 target genes, 39% were known to be indirectly regulated, and 35% of the genes were not identified as p53 targets in solid cancer cell lines (Andrysik Z Genome Res 2017). Among the 51 downregulated genes, only 2 belonged to apoptosis pathways, namely BAX and TNFRSF10B, both direct early targets of p53. Of note, the expression of BAK1, PMAIP1 or BBC3 was not significantly modified. To directly assess the impact of BAX decrease in the 6 TP53-/- clones, we used 3 BH3 mimetics specific for BCL2, Venetoclax, MCL1, S63845, and BCLXL, A1155463. The XG7 and NCI-H929 cell lines were predominantly resistant to Venetoclax and to A1155463, and TP53 knockout did not alter responses. In contrast, as compared to control clones, the response to S63845 was decreased 5.7 to 7.4-fold in NCI-H929 TP53-/- (LD50 median values increased from 7 to 40nM, p<0.0001) and XG7 TP53-/- clones (LD50 median values increased from 25 to 186nM, p=0.0007), respectively. This result suggested a major role of BAX in the response to S63845. Indeed, BAX silencing significantly inhibited S63845-induced cell death while BAK silencing had no protective effect, demonstrating the BAX dependence of the S63845 response. Of note, this strong dependence on p53-dependent BAX expression was not limited to XG7 or NCI-H929 : in 21 TP53abnormal myeloma cell lines, BAX expression (GEP) was 1.7-fold lower than in 11 TP53wt cell lines (LD50 median values 34 vs. 59, p=0.0004), and sensitivity to S63845 was also significantly lower in TP53abn MM cell lines compared to MM TP53wt (LD50 median values 75 nM vs. 12 nM, p=0.0296). The resistance of TP53-/- cells to S63845 was overcome by the addition of Venetoclax, although the death remained lower in TP53-/- clones compared to control clones (p=0.001). BAX and BAK activation and MCL1 co-immunoprecipitation assays further demonstrated the central role of MCL1/BAX expression and complexes in the response to S63845. In 63 consecutive independent samples from patients with MM (23 at diagnosis, 40 at relapse), 33 1q+ and 30 1q-, we show that 1q gain/amplification favored response to 25 nM S63845 alone (death median 16% in 29 1q- vs 30% in 34 1q+, p=0.0132) and had a trend in combination with 300 nM Venetoclax (80% vs 89%, p=0.152). Finally, we show that 1q was sufficient to induce responses to S63845 plus Venetoclax regardless of del17p status (death median 30% in 26 1q+ del17p- vs. 40% in 6 1q+ del17p+ samples). To analyze the response to the combination of S63845 and Venetoclax at the single cell level, we performed scRNASeq analysis in t(4;14)+ 1q+ del17p+/- patients and determined the transcriptomic profile of cells surviving the combination: we show that BH3 mimetics combination heterogeneously induced death in and between patient's samples and we identified a resistance signature that is under evaluation. All these results support the S63845 / Venetoclax combination for patients with double (1q+, del17p) to triple (t(4;14), 1q+, del17p) hit. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Histone Deacetylase Inhibition Restores Therapy Response in TP53mutated Acute Lymphoblastic Leukemia to Chemotherapy

Background: Although TP53 is the most frequently mutated gene in cancer, in Acute Lymphoblastic leukemia (ALL), mutations or deletions affecting TP53 are rare, with an incidence of only 3% at diagnosis. However, at relapse TP53 aberrations are found in about 12% of the patients and predict a very poor outcome. As a consequence, relapsed TP53 deleted ALL is now classified as 'very high risk, together with t(1;19) and t(17;19) ALL. Since p53-mediated apoptosis is an endpoint for many (cytotoxic) drugs, loss of p53 function induces therapy resistance not only to classical chemotherapeutics, but also newly introduced immunotherapies such as bi-specific T cell engagers such as Blinatumomab or CAR-T cell therapies. Therefore, there is an urgent clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Methods: Using CRISPR/Cas9, we introduced frameshift mutations in the TP53 gene to generate isogenic wildtype and knockout models in the TP53WT B-cell precursor ALL (BCP-ALL) cell lines Nalm6 and RCH-ACV. After confirming the presence of TP53 deletions, we treated these isogenic models with a panel of treatment protocol drugs, demonstrating that TP53 loss negatively affects response to therapy to most contemporary drugs used in the treatment of relapsed ALL. To identify drug combinations that may reverse TP53 mediated therapy resistance, we assessed the viability of these isogenic models in a drug screen containing 198 (pre)clinical drugs. The most interesting targets were subsequently combined with the previously tested treatment protocol drugs to determine potential synergies. Effects on cell viability were determined by amine exposure and MTT assay and confirmed by measuring PARP cleavage. Additionally, positive drug interactions were validated in vivo using immunocompromised mice engrafted with RCH-ACV p53KO cells expressing a luciferase reporter gene. Results: We observed that loss of p53 protects BCP-ALL cell lines Nalm6 and RCHACV from drug-induced apoptosis, leading to increased resistance to all drugs used currently in the treatment of relapsed ALL, except for Asparaginase. In a subsequent drug screen, we found that the histone deacetylase (HDAC) 1/2 inhibitor romidepsin shows strong single agent activity at single nanomolar concentrations. Moreover, romidepsin was found to strongly synergize with contemporary ALL treatment drugs in TP53KO and TP53WT cells (Figure 1A). This effect was strongest in TP53KO cells, effectively reversing therapy resistance induced by TP53 loss. Gene expression profiling suggest that romidepsin may lead to reactivation of TP53 target genes in response to cellular stress, even in the absence of functional p53. Finally, preliminary mouse experiments using TP53 deleted RCH-ACV cells showed that romidepsin improves the response to cytarabine in TP53 aberrant leukemia in vivo (Figure 1B) without signs of added toxicity. A mouse trial testing a potential synergy between romidepsin and cytarabine using TP53 deleted patient derived xenografts (PDX) is currently underway. Conclusions: Loss of p53 protects BCP-ALL cells from currently used (chemo)therapy strategies. Romidepsin can restore response to therapy in TP53 deleted ALL, presumably by reactivation of p53 target genes in response to cellular stress. Therefore, romidepsin may be used to improve therapy response in relapsed TP53 deleted ALL. Since romidepsin already has FDA approval for use in other hematological malignancies, our findings may be rapidly translated into clinical practice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Idasanutlin and Navitoclax Induce Synergistic Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

We report here investigation of the combination of idasanutlin and navitoclax in the context of T-cell acute lymphoblastic leukemia (T-ALL), including detailed mechanistic characterization and in vivo patient derived xenograft results. T-ALL is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. An unmet clinical need remains for the development of better therapies for T-ALL. Notch pathway activation mutations are present in the majority of T-ALL and are thought to induce transformation, in part, through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, and critical to our approach, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these genetic data, we hypothesized that treatment with MDM2 inhibitors, by bypassing the loss of ARF, would increase p53 expression and lead to T-ALL cell apoptosis. To test this hypothesis, we initially treated T-ALL patient derived xenograft (PDX) lines in vitro with single-agent idasanutlin (a second generation MDM2 inhibitor) and observed upregulation of pro-apoptotic p53 target genes Bax and Puma1. However, only modest T-ALL cell apoptosis was observed in response to MDM2 inhibition alone. Based on these observations, particularly the induction of pro-apoptotic p53 targets, we hypothesized that inhibition of pro-survival BH3 domain proteins would synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with navitoclax (ABT-263), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient and dose-limiting thrombocytopenia was observed. Combination therapy with idasanutlin could permit on-target inhibition with tolerable dosing. In vitro treatment of a panel of 5 different PDX lines derived from pediatric and adult T-ALL with dual idasanutlin and navitoclax led to robust cell death in all lines. In addition, treatment of a T-ALL PDX line with a matrix of dose combinations showed strong evidence of synergic activity. Synergy was assessed by ZIP (zero interaction potency) score, a statistical method developed by B Yadav et al. (Comput. Struct. Biotechnol. J. 2015) that compares the change in potency of individual dose response curves in the presence of a second agent. Generally, a ZIP score greater than 5 is considered significant; Idasanutlin and navitoclax combination therapy had a global ZIP score=16-18, with the most synergic dose area ZIP score=40-52. Combined treatment resulted in marked apoptosis, with strong induction of pro-apoptotic p53 targets, including PUMA and BAX. Finally, we assessed the in vivo response of four different T-ALL xenografts to dual treatment. In each case, human T-ALL cells were injected into immunodeficient NSG mice and randomized into treatment groups. Monotherapy with idasanutlin or navitoclax resulted in a modest but significant decrease in T-ALL burden in all cases, with the exception of an ETP T-ALL, which was resistant to idasanutlin monotherapy. A marked response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts. Specifically, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change in tumor burden for synergy interaction. Furthermore, overall survival was significantly increased in the combination treatment group. These data suggest that the rational combination of idasanutlin and navitoclax is highly active, with induction of an apoptotic cell death in T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of 3-Phenylquinoline Derivative PQ1 as an Antagonist of p53 Transcriptional Activity.

Transcription factor p53 regulates cellular responses to environmental perturbations via the transcriptional activation of downstream target genes. Inappropriate p53 activation can trigger abnormal cellular responses, therefore leading to acute or chronic tissue damage, human developmental syndromes, and neurodegenerative diseases. Antagonists of p53 transcriptional activity provide prospective therapeutic applications and molecular probes. In this article, we identified five 3-phenylquinoline derivatives as potential p53 inhibitors through screening a chemical library consisting of 120 compounds, in which PQ1 was the most active compound. PQ1 had no effect on p53 protein levels and decreased the expression of p53 target gene p21. PQ1 thermally stabilizes the wild-type p53 protein. Further, transcriptomics confirmed that PQ1 exposure generated a similar regulatory effect to transcription profiles with a reported p53 transcriptional inhibitor pifithrin-α. However, compared to pifithrin-α, PQ1 increased the sensitivity of SW480 cells to 5FU. Taken together, PQ1 was a novel antagonist of p53 transcriptional activity. We propose that PQ1 could be developed as a chemical tool to pinpoint the physiological functions of p53 and a novel lead compound for targeting dysfunctional p53 activation.

ΔNp63α transcriptionally represses p53 target genes involved in the radiation-induced DNA damage response

BackgroundThe DNA damage response (DDR) is a mechanism that protects cells against radiation-induced oxidative DNA damage by causing cell cycle arrest and apoptosis. TP63 is a member of the tumour suppressor TP53 gene family, and ΔNp63α, a TP63 splicing variant, is constitutively expressed in the stem cell-containing basal layer of stratified epithelial tissues, including the mammary gland, where it plays a critical role in stemness and tissue development. ΔNp63α has been reported to transcriptionally inhibit the tumour suppression protein p53. This p53-repressive activity may cause genomic instability in epithelial stem cells exposed to radiation. In this study, we analysed the inhibitory effect of ΔNp63α on radiation-induced DDR.MethodsTo elucidate the role of the p53-repressive effect of ΔNp63α in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing ΔNp63α and then performed ectopic and entopic expression experiments using human induced pluripotent stem cells (hiPSCs). After irradiation, the expression of DDR-related genes and proteins in ΔNp63α-expressing and control cells was analysed by RT–qPCR, Western blotting, and flow cytometry.ResultsThe mRNA/protein expression levels of BAX and p21 were significantly increased in p63-siRNA-treated HMECs (sip63) after X-ray irradiation (4 Gy, 0.7 Gy/min) but not in scramble-siRNA treated HMECs (scr). Transcriptomic analysis showed decreased RNA expression of cell cycle-related genes and increased expression of programmed cell death-related genes in sip63 cells compared to scr cells. Furthermore, flow cytometric analysis revealed an increase in apoptotic cells and a decrease in 5-ethynyl-2´-deoxyuridine uptake in sip63 cells compared to scr cells. On the other hand, both the ectopic and entopic expression of ΔNp63α in apoptosis-sensitive hiPSCs reduced the expression levels of BAX after irradiation and significantly decreased the number of apoptotic cells induced by radiation.ConclusionTaken together, these results indicate that ΔNp63α represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells.

EXTH-19. OPTIMIZING MDM2 INHIBITION FOR THE TREATMENT OF GLIOBLASTOMA

Abstract Over 60% of glioblastomas retain wild-type p53, resulting in potential susceptibility to MDM2 inhibitors. These molecules disrupt the interaction between p53 and its negative regulator MDM2, allowing downstream pathway signaling and cell fate decisions including growth inhibition and death resulting from cellular stress. We have analyzed clinical samples from 10 patients newly diagnosed with glioblastoma multiforme (GBM) and treated with the MDM2 inhibitor KRT-232. We detected upregulation of CDKN1A transcript (a target of p53) in all p53 wild-type tumors (8 of 10), but not in p53-mutant tumors (2 of 10). In patient-derived cell lines, treatment with KRT-232 at the clinically detected concentration was only sufficient to stall tumor growth. Cell death via apoptosis can be achieved when MDM2 inhibition is combined with the chemotherapeutic agent temozolomide. As the effects and resistance mechanisms of MDM2 inhibition remain poorly understood in GBM, we have additionally performed genomic and transcriptomic analyses in patient-derived cell lines to better characterize sensitive tumors and identify putative biomarkers of drug response. Dose response curves and growth assays showed that tumors with inactivating p53 mutations are highly resistant to treatment, but those that retain wild-type p53 exhibit various degrees of drug sensitivity. This suggests that other factors, in addition to p53 mutational status, mediate response to MDM2 inhibition in gliomas. Transcriptional analyses of patient samples following drug treatment suggest that cell division, chromatin reorganization, cell differentiation state and immune response programs become deregulated under MDM2 inhibition and in the absence of p53-inactivating mutations.

TRIM11, a new target of p53, facilitates the migration and invasion of nasopharyngeal carcinoma cells

BackgroundAlthough tripartite motif-containing protein 11 (TRIM11) is known to be associated with a variety of cancers, its role in nasopharyngeal carcinoma (NPC) is unclear.Methods and resultsTo investigate the role of TRIM11 in NPC, TRIM11 was stably overexpressed in 6–10B and CNE2 cells with lentiviral vectors and knocked down in S18 and 5–8F cells using the CRISPR/Cas9 system. Transwell assays and wound-healing assays revealed that TRIM11 facilitated the migration and invasion of NPC cells. Mechanistically, we found that p53 inhibits TRIM11 expression by binding to its promoter.ConclusionsTRIM11 may serve as a potential diagnostic marker for NPC and has a certain therapeutic value.

CSIG-24. EPIGENETIC SUPPRESSION ALLOWS GBM TO MAINTAIN P53 WILD-TYPE STATUS

Abstract Introduction Glioblastoma (GBM) is the most common primary brain malignancy in adults. The vast majority of GBM cases maintain wild-type status of p53, the protein considered to be the most critical tumor suppressor. How GBM displays such a malignant phenotype despite retaining normal p53 protein is unknown. We also aim to translate these molecular findings into therapeutics via a randomized clinical trial. METHODS A variety of molecular biological techniques were employed. These include CRISPR-CAS, next generation sequencing, stereotactic mouse brain injections, and histological analyses of human GBM tumors. Approval for a small, single site, blinded, randomized clinical trial for newly diagnosed GBM is underway. Overall survival, recurrence-free survival, immunohistochemical analyses will be assessed. RESULTS Human p53 wild-type GBM cell lines express Brd at higher levels compared to p53 mutant cell (~10 fold, p &amp;lt; 0.05). Cell viability assays show that deletion of Brd leads to a ~50% decrease (p &amp;lt; 0.05) in cell survival. Critically, this effect is abrogated upon deletion of p53 itself, showing that Brd functions through the inhibition of p53’s tumor suppressive effects. Furthermore, immunoprecipitation of Brd shows that it is a member of a complex of proteins that bind to p53 target genes. The importance of these findings are corroborated in vivo, as mice stereotactically injected with human GBM cells that lack Brd survive ~2-fold longer (p &amp;lt; 0.05) than mice that retain Brd. We have begun the process to start clinical trials with an already FDA-approved drug (currently used for the treatment of lymphoma and sarcoma) that blocks the Brd pathway. CONCLUSION Brd is the key suppressor of p53 target genes in GBM. Brd binds to a multimeric protein complex, which inhibits p53 target genes. Thus, the tumor suppressive effects of p53 are prevented in GBM. Inhibition of this pathway in GBM patients is underway.

An MDM2 degrader for treatment of acute leukemias.

In acute myeloid leukemia (AML), p53 tumor suppressor activity can be reduced due to enhanced expression of MDM2 which promotes the degradation of p53. In TP53 wild-type malignancies, therapy with small molecule antagonists of MDM2 results in antileukemic activity. Current treatment strategies, however, have been limited by poor tolerability and incomplete clinical activity. We have developed a proteolysis-targeting chimera (PROTAC) MS3227 that targets MDM2 by recruiting the E3 ligase Von Hippel-Lindau, resulting in proteasome-dependent degradation of MDM2. In WT TP53 leukemia cell lines, MS3227 led to activation of p53 targets p21, PUMA, and MDM2 and resulted in cell-cycle arrest, apoptosis, and decreased viability. The catalytic PROTAC MS3227 led to more potent activation when compared to a stoichiometric inhibitor, in part by dampening the negative feedback mechanism in the p53 - MDM2 circuit. The effectiveness of MS3227 was also observed in primary patient specimens with selectivity towards leukemic blasts. The addition of MS3227 enhanced the activity of other anti-leukemic agents including azacytidine, cytarabine, and venetoclax. In particular, MS3227 treatment was shown to downregulate MCL-1, a known mediator of resistance to venetoclax. A PROTAC-based approach may provide a means of improving MDM2 inhibition to gain greater therapeutic potential in AML.

PLTP is a p53 target gene with roles in cancer growth suppression and ferroptosis

The tumor suppressor protein p53 suppresses cancer by regulating processes such as apoptosis, cell cycle arrest, senescence, and ferroptosis, which is an iron-mediated and lipid peroxide-induced cell death pathway. Whereas numerous p53 target genes have been identified, only a few appear to be critical for the suppression of tumor growth. Additionally, while ferroptosis is clearly implicated in tumor suppression by p53, few p53 target genes with roles in ferroptosis have been identified. We have previously studied germline missense p53 variants that are hypomorphic or display reduced activity. These hypomorphic variants are associated with increased risk for cancer, but they retain the majority of p53 transcriptional function; as such, study of the transcriptional targets of these hypomorphs has the potential to reveal the identity of other genes important for p53-mediated tumor suppression. Here, using RNA-seq in lymphoblastoid cell lines, we identify PLTP (phospholipid transfer protein) as a p53 target gene that shows impaired transactivation by three different cancer-associated p53 hypomorphs: P47S (Pro47Ser, rs1800371), Y107H (Tyr107His, rs368771578), and G334R (Gly334Arg, rs78378222). We show that enforced expression of PLTP potently suppresses colony formation in human tumor cell lines. We also demonstrate that PLTP regulates the sensitivity of cells to ferroptosis. Taken together, our findings reveal PLTP to be a p53 target gene that is extremely sensitive to p53 transcriptional function and which has roles in growth suppression and ferroptosis.

ZFP14 Regulates Cancer Cell Growth and Migration by Modulating p53 Protein Stability as Part of the MDM2 E3 Ubiquitin Ligase Complex.

Multi-zinc finger proteins that contain a KRAB domain are part of the biggest family of transcription factors in mammals. However, the physiological or pathological functions for the majority of them are unknown. Here, we showed that ZFP14 (also known as ZNF531) is a p53 target gene that can be induced upon genotoxic stress in a p53-dependent manner. To determine the function of ZFP14 in mouse and human cancer cell lines, we generated multiple cell lines where ZFP14 was knocked out. We showed that ZFP14-KO inhibits cancer cell growth and migration. We also showed that, as a target of p53, ZFP14, in turn, represses p53 expression and that the knockdown of p53 restores the potential of ZFP14-KO cells to proliferate and migrate. Mechanistically, we found that ZFP14 modulates p53 protein stability by increasing its ubiquitination via associating with and possibly enhancing MDM2/p53 complex integrity through its zinc finger domains. Our findings suggest that the reciprocal regulation of p53 and ZFP14 represents a novel p53-ZFP14 regulatory loop and that ZFP14 plays a role in p53-dependent tumor suppression.