P53 Target Research Articles

Abstract 515: Single-cell transcriptomic analysis of HR+/HER2- breast cancer identifies gene signatures that predict outcomes of luminal A and B subtypes

Abstract Introduction: Patients with luminal A and B early-stage ER+/HER2- breast cancer (BrCa) are uniformly treated with adjuvant endocrine therapy (ET) (±chemotherapy). A better understanding of drivers of ET resistance is required as subsequent lethal metastatic disease remains a major clinical problem. Therefore, there is an unmet clinical need to identify biomarkers that select low- and high-risk patients who may benefit from de-escalation of current treatments or alternative therapeutic interventions. In the present study, we hypothesized that while luminal A and B tumors both arise from normal hormone-responsive cells, they are transcriptionally distinct, hence allowing the identification of unique gene signatures that can predict outcomes in each subtype. Methods: Tumors from 10 early-stage ER+/HER2- BrCa patients were subjected to single-cell RNA-sequencing (scRNA-seq) analysis (10X Genomics); 6/10 tumors were classified as luminal A and 4/10 as luminal B based on combined PAM50 and immunohistochemical classification (Ki67 cut-off=20%). We performed a direct transcriptional comparison between luminal A and B tumors, using well-established signatures and unbiased differential gene expression analysis. To identify unique luminal A and B tumor-specific genes, we compared the gene expression profile of each luminal subtype with 10 non-neoplastic breast tissues. A predictive model (LASSO) was applied to select genes with the highest frequency using a training dataset. This resulted in 5- and 4-gene signatures for luminal A and B, respectively, which were used to calculate risk scores that divided each subtype into low- and high-risk groups. The prognostic value of the above signatures was validated in an independent dataset. Results: The integrated scRNA-seq analysis of luminal A and B tumors revealed transcriptionally distinct tumor cell clusters while tumor microenvironment (TME) clusters were well intermixed. Luminal B tumors had higher cell cycle and BrCa-specific scores, low ER pathway-gene expression scores, and increased 8q amplifications. IFNγ, OXPHOS, p53, hypoxia and MYC targets were the most upregulated pathways in the luminal B subtype. The TME of luminal B tumors was comprised of lower CD4+ and CD8+ T cell but higher Treg levels. Comparison with normal breast tissues revealed that early-stage ER+ BrCa arises from hormone-responsive epithelial cells and provided a number of tumor-specific genes that were used to generate prognostic signatures. These signatures were capable of differentiating high- from low-risk patients within each subtype and predicting survival outcomes in two large-scale training and validation cohorts. Conclusions: We developed a novel prognostic tool that can be used to determine duration of adjuvant ET and/or new therapeutic strategies for high-risk luminal A and B patients in the early-stage ER+/HER2- setting. Citation Format: Sofia Mastoraki, Jerome Lin, Xiayu Rao, Sophie R. Liu, Harsh Batra, Maria G. Raso, Edwin R. Parra Cuentas, Akshara S. Raghavendra, Komal S. Rasaputra, Min Yi, Jing Wang, Aysegul Sahin, Debasish Tripathy, Kelly K. Hunt, Nicholas E. Navin, Khandan Keyomarsi. Single-cell transcriptomic analysis of HR+/HER2- breast cancer identifies gene signatures that predict outcomes of luminal A and B subtypes [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 515.

Abstract 5854: Therapeutic targeting of p73-activated c-FLIP switches cell fate from growth arrest and survival to apoptosis in p53-deficient cancer cells

Abstract The tumor suppressor p73, a member of the p53 family, transcriptionally activates numerous p53-target genes in cell cycle regulation and apoptosis. Therapeutic activation of p73 can restore p53-signaling in mutant p53-expressing cancer cells effectively bypassing the p53 deficiency. In addition to pro-apoptotic signaling, outcomes of p73 activation contain survival signals and this may provide insights in cell fate outcomes between cell survival and apoptosis after cellular stress. We report that cellular FLICE inhibitory protein (c-FLIP), a master antiapoptotic factor, is a novel transcriptional target of p73. To test if p73 regulates c-FLIP expression in p53-deficient cancer cells, we overexpressed p73 in p53-deficient cancer cells using p73-adenovirus. Overexpression of p73 dramatically increased c-FLIP at the protein and RNA levels. The effect of p73 on c-FLIP upregulation was blocked by knock-down of p73. ChIP assay showed that p73 binds to the c-FLIP promoter in a p73-level dependent manner. Furthermore, a luciferase reporter assay showed that p73 increased the c-FLIP-L-promoter-mediated luciferase reporter expression in p53-deficient cancer cells. To test the effect of the outcomes of p73 signaling on cell survival or death, we performed cell viability assay and found that p73 reduces cell viability in a p73-level dependent manner. We found an increase of both p21 and c-FLIP at the protein levels in response to overexpression of p73 in p53-deficient cancer cells, but no cleaved-PARP was detected and flow cytometric analysis showed the cells were arrested in G1 phase. These results suggest that p73 arrests the cell cycle and prevents apoptosis at the tested levels of p73. We knocked down c-FLIP using siRNA in p53-deficient cancer cells, and found that knockdown of c-FLIP enhanced cleaved-PARP, cleaved caspase- 3 and -8 correlated to high level of apoptotic signaling with Noxa and puma upregulation in response to the overexpression of p73. Our results suggest that the depletion of c-FLIP primes cellular apoptosis switching from p73-induced cell cycle arrest (cell survival) and sensitizes cells to p73-induced apoptotic signals in p53-deficient cells. The cell fate decision following p73 activation is determined by the adjustment of the balance of outcomes of p73 activation between p73-induced pro-apoptotic signaling and c-FLIP expression in p53-deficent cancer cells. Our discovery of the new function of p73 to upregulate c-FLIP provides a rationale for depleting c-FLIP to improve antitumor efficacy of p73-targeting cancer therapy. Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Therapeutic targeting of p73-activated c-FLIP switches cell fate from growth arrest and survival to apoptosis in p53-deficient cancer cells [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 5854.

Abstract 3964: Co-targeting MDM2-p53 and BCL-2 apoptosis pathways overcomes resistance conferred by acquired BCL-2 gene mutations in preclinical models

Abstract The BCL-2 inhibitor venetoclax has shown impressive efficacy in patients with chronic lymphocytic leukemia, but its clinical benefits are limited by acquired BCL-2 gene mutations that confer drug resistance. Among acquired mutations, those proximal to BH3 binding motifs (e.g., G101V, D103E, and V156D) have the most significant impact on BCL-2 binding to BH3-only prodeath proteins and BH3 mimetics (e.g., venetoclax). Hence, it is important to identify novel therapeutics that address this emerging unmet need. To this end, we investigated the effect of co-targeting BCL-2 and MDM2-p53 apoptosis pathways in preclinical models of acute lymphoblastic leukemia (ALL) with BCL-2 mutations. We first genetically engineered cell lines that express clinically relevant BCL-2 gene mutations in ALL cell line RS4;11, which shows greater expression of mutants, compared to endogenous wild-type BCL-2 protein. Relative to parental cells, sensitivity to venetoclax and investigational, clinical-stage BCL-2 inhibitor lisaftoclax (APG-2575) in mutant cell lines was reduced 160-fold. Using these BCL-2-inhibitor-resistant cell lines, we assessed the effect of combining lisaftoclax with investigational, clinical-stage MDM2-p53 inhibitor alrizomadlin (APG-115). Alrizomadlin inhibits the MDM2-p53 interaction and activates the prodeath p53 tumor suppressor signaling pathways (Aguilar A et al. J Med Chem 2017). Also potentially consequential was the observation that combining alrizomadlin with lisaftoclax synergistically inhibited proliferation and induced apoptosis in RS4:11 cells carrying G101V, V156D, or D103E mutations. Treatment with this combination also synergistically enhanced antitumor activity in mouse xenograft tumor models derived from RS4:11 cells carrying G101V or V156D mutations. In these animal models, lisaftoclax alone exhibited no or limited activity, with T/C (%; i.e., relative tumor volume of treatment group vs. control) values ranging from 86.40% to 99.15%. In contrast, single-agent alrizomadlin exerted slightly more potent activity, with T/C values ranging from 57.25% to 62.35%. Importantly, the combination of lisaftoclax and alrizomadlin demonstrated synergy, with T/C values ranging from 12.30% to 32.16%. Mechanistically, alrizomadlin likely induced expression of prodeath BCL-2 family proteins BAX, PUMA, and Noxa, which are p53 target genes. Further, lisaftoclax plus alrizomadlin more effectively blocked cell cycle entry into the G2/M phase, resulting in accumulation of sub-G1 apoptotic cells. In summary, our study demonstrates, for the first time, that co-targeting BCL-2 and MDM2-p53 apoptosis pathways overcame resistance to BCL-2 inhibitors conferred by acquired BCL-2 gene mutations, potentially offering a viable strategy to overcome drug resistance and thus providing a rationale for clinical investigation. Citation Format: Jing Deng, Kaixiang Zhang, Qiuqiong Tang, Xiaojing Huang, Qixin Wang, Na Li, Douglas D. Fang, Dajun Yang, Yifan Zhai. Co-targeting MDM2-p53 and BCL-2 apoptosis pathways overcomes resistance conferred by acquired BCL-2 gene mutations in preclinical models [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 3964.

Abstract 361: MicroRNA expression in murine spontaneous non-small cell lung cancer and human cell lines post-treatment of PRIMA-1

Abstract A variety of cancers have been correlated with having a mutation on the TP53 gene, including lung cancer. Lung cancer has been one of the deadliest cancers in the United States. TP53-dependent reactivation and induction of massive apoptosis (PRIMA-1) changes mutant TP53’s conformation, resulting in the restoration of DNA binding and activation of p53 target genes. PRIMA-1 methylated analog, PRIMA-1Met (APR-246, Eprenetapopt) have been used in clinical trials to target cancers containing mutant TP53. It has been reported that PRIMA-1 induced apoptosis through the regulation of microRNAs. Our research group has previously developed a transgenic mice model with a mutant form of TP53 being expressed lung specifically, to closely model human lung cancer development. The aim of the current study is to investigate the effects of PRIMA-1 on miRNA expressions in a murine spontaneous lung cancer mice model, human lung cancer cell lines H211 and H1155. The transgenic FVB/N mice lung tumor model was developed using the lung specific human surfactant protein C (SPC) promoter to control the expression of mutant TP53(273H) following the standard injection method. Tumor bearing mice were identified with a micro-CT and were treated with the PRIMA-1 at a dose of 100mg/kg. Additionally, we treated two human cell lines H211 (containing mutant TP53 R248Q) and H1155 (containing mutant TP53 R273H) with the PRIMA-1. The miRNA expression profile was evaluated with the NanoString nCounter array analysis and qt-PCR array analysis. We found that three miRNAs were up-regulated (>1.5-fold change) in the human lung cancer cells H211, H1155, and murine spontaneous non-small cell lung cancers model post treatment of PRIMA-1. The result of the study showed that miR-200c, miR-181c, and miR-183 was up-regulated in the two human cell lines and the transgenic mice lung tumors after treatment of PRIMA-1. While there was no significant downregulation in miRNA expression in these human lung cancer cells and the murine spontaneous lung cancer model. However, the overall miRNA expression is lower in murine spontaneous lung tumors when compared to the overall miRNA expression of the cell lines post PRIMA-1 treatment. This difference is due to model specific expression. Further investigation is needed to better understand the effects of PRIMA-1 in regulation of micro-RNA expression in the treatment of lung cancers containing mutant p53. Citation Format: Melanie Perez, Shaylyn Grier, Li Gao, Wenrui Duan. MicroRNA expression in murine spontaneous non-small cell lung cancer and human cell lines post-treatment of PRIMA-1 [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 361.

Abstract 829: p53 dimers elicit tumor suppressive activities through an altered metabolic program

Abstract p53 is the most frequently mutated gene in human cancer. As a tetrameric transcription factor, mutation of the p53 Tetramerization Domain (TD) is a mechanism by which cancers abrogate wild-type (WT) p53 function. p53 TD mutations result in a protein that preferentially forms monomers or dimers. Although it is accepted that tetrameric p53 is required for full tumor suppressive activities, the physiological relevance of monomeric and dimeric states of p53 is not well understood. We have established the first in vivo models for hot-spot p53 TD mutants, p53R339P and p53A344D (murine), that result in monomeric and dimeric p53, respectively. These models closely resemble Li-Fraumeni Syndrome (LFS) patients with germline p53 TD alterations and will be used to characterize their physiological activities. p53R339P and p53A344D proteins failed to form tetramers. These mutants were deficient in their transactivation ability for canonical p53 target genes, and unable to induce apoptosis following IR. Both p53 TD mutant mice rescued the p53-dependent embryo lethality caused by genetic deletion of Mdm2, suggesting these are loss-of-function alleles. Monomeric p53 bearing mice succumbed mainly to thymic lymphomas (~60%), with a median survival similar to p53-/- mice (~130 days). Dimeric p53 bearing mice displayed lower incidence of thymic lymphomas (~25%) and longer survival (~180 days). Analyses of the transcriptomes elicited after DNA damage in normal thymus revealed that the p53 signaling pathway is dampened in dimeric p53 thymi. However, under basal conditions, p53 dimers upregulate a lipid metabolic program that is associated with PPAR activation. Further RNA sequencing of common metabolic tissues of the mouse (pancreas, liver, and intestine) revealed that they upregulate lipid-related metabolic programs in p53 dimer samples. This study describes, for the first time, the consequences of altering p53 tetramerization in vivo. Monomer and dimer forms of p53 do not retain most WT p53 features. p53 dimers may exert tumor suppression as seen by extended survival and tumor spectra shift. p53 dimer-specific activities on PPAR signaling and lipid metabolism may explain these phenotypes. These activities are possible therapeutic targets for LFS patients. Citation Format: Jovanka Gencel-Augusto, Xiaoping Su, Guillermina Lozano. p53 dimers elicit tumor suppressive activities through an altered metabolic program [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 829.

Abstract 5288: Development of lead mutant-p53 reactivating peptides towards clinical application

Abstract p53 is the most extensively studied tumor suppressor. It serves as a major barrier against malignant transformation and the p53 pathway is inactivated in almost all human tumors. A large percentage of tumors inactivate p53 by point mutations in the DNA binding domain (DBD) of the protein. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, improperly folded form. Previously, we reported the use of phage display technology to select for peptides that reactivate mutant p53 by stabilizing a wt protein conformation, taking advantage of a unique combination of selection steps (Tal P, at al Oncotarget. 2017 Jan 3;8(1):164-178). We now describe further progress in the development of one lead peptide, pCAP-250, which contains 9 amino acids and a myristic fatty acid at its N-terminus. The pCAP-250 sequence bears 100% homology to RAD9, a known p53 interacting protein. We show physical interaction between pCAP-250 and the p53DBD. Characterization of this interaction by NMR reveals a significant chemical shift of amino acids located in the p53DBD L1 loop and Helix-2, two regions critical for p53 transcriptional activity and interaction with DNA. We also report the optimization of our lead sequence by rational design to yield more potent derivatives with higher efficacy in cancer cell elimination and longer in vivo half-life. RNA-seq analysis shows that our lead peptides induce robust activation of p53 target genes. Furthermore, we report that in addition to p53 activation, pCAP-250 also has a p53-independent anti-cancer activity. Overall, our lead peptides cause specific cell death in a wide spectrum of mutant p53 expressing cell lines, with an IC50 of 0.1-12µM, while having no effect on non-transformed cells at these concentrations. The pharmacokinetic profile of pCAP-250 and its derivatives shows that they are completely stable in human serum and have plasma half-life of 1.5-10h in vivo when administered IV or subcutaneously, presumably due to interaction with albumin through the fatty acid moiety. Remarkably, our lead peptides elicit remarkable regression of very aggressive tumors in several preclinical mouse models of renal, colon, pancreatic and ovarian cancer. In vivo efficacy can be achieved through several routes of administration, including intra-tumor injection as well as systemic subcutaneous continuous slow release and subcutaneous bi-daily injections. Thus, such peptides might serve as novel therapeutic agents for p53-mutated human cancer. Citation Format: Perry Tal, Moshe Oren, Orna Palgi, Varda Rotter, KWANGIL JEONG. Development of lead mutant-p53 reactivating peptides towards clinical application [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 5288.

Abstract 6228: APR-246, which restores p53 function, is highly active against alternative lengthening of telomere (ALT) cell lines and PDXs

Abstract Introduction: Most cancers proliferate by activating telomerase (TA+) while 10% of cancers utilize alternate lengthening of telomeres (ALT). ALT has been associated with resistance to DNA damaging agents, p53 loss-of-function (p53LOF), ATRX mutations, and very poor survival. ATM kinase, which activates functional p53, is constitutively activated in ALT cancers (Science Translational Medicine 18:eabd5750, 2021). We hypothesized that the constitutive activation of ATM kinase in ALT cancers would confer high sensitivity to pharmacological reactivation of p53 function. Methods: We used patient-derived ALT (telomeric DNA C-circle positive) and TA+ neuroblastoma, sarcoma, colorectal, and breast cancer cell lines and xenografts treated with the clinical-stage p53 reactivator eprenetapopt (APR-246) and irinotecan. In vitro cytotoxicity was assayed by DIMSCAN digital imaging microscopy, ATM activation by immunofluorescence microscopy, and protein expression by western blotting. Results: ALT p53LOF cell lines were significantly (p<0.05) less sensitive to DNA-damaging agents relative to TA+ p53LOF comparators. Constitutive phosphorylation (activation) of ATM kinase and the DNA damage marker 53BP1 were observed at telomeres in ALT but not TA+ cell lines. APR-246 induced p53 targets p21 and NOXA and was significantly more cytotoxic (p<0.001) for ALT relative to TA+ cell lines, regardless of TP53 status. Overexpression of p53 in a TP53-null ALT cell line increased sensitivity (p<0.0001) to APR-246. Knockdown of p53 or ATM kinase in ALT TP53 mutated and wild-type cell lines antagonized (p<0.0001) APR-246 activity. Induction of telomere dysfunction in a TA+ p53LOF neuroblastoma cell line using dominant-negative TRF2 (a shelterin protein that blocks ATM activation at telomeres) activated ATM and sensitized cells to APR-246 (p<0.01). APR-246 enhanced the cytotoxicity of irinotecan (as SN38) in ALT cell lines in vitro to a higher degree than in TA+ p53LOF cell lines (p<0.05). Single-agent APR-246 significantly (p<0.0001) increased event-free survival (EFS) of mice with ALT xenografts relative to controls. APR-246 enhanced (p<0.0001) the activity of irinotecan in 3 neuroblastoma, 2 rhabdomyosarcoma, 1 colorectal, and 1 triple-negative breast ALT xenograft models with most mice (47/56) achieving complete responses and an EFS of >100 days (45/56) compared to no complete responses (median EFS ~ 34 days) in mice treated with only irinotecan (p<0.0001). APR-246 + irinotecan had no significant effect relative to irinotecan alone (p=0.08) on EFS of mice with TA+ p53LOF xenografts (median EFS ~ 40 days). Conclusion: ALT cancers of a variety of histologies are highly resistant to DNA damaging agents, have p53LOF, and constitutive activation of ATM kinase, which confers high sensitivity to p53 reactivation by APR-246. APR-246 warrants clinical testing in patients with ALT cancers. Citation Format: Shawn J. Macha, Balakrishna Koneru, Trevor Burrow, Charles Zhu, Dzmitry Savitski, Jonas Nance, Kristyn McCoy, Cody Eslinger, C Patrick Reynolds. APR-246, which restores p53 function, is highly active against alternative lengthening of telomere (ALT) cell lines and PDXs [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 6228.

Abstract 3934: KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI)

Abstract Background: The murine double minute 2 (MDM2) oncoprotein is a key E3 ubiquitin ligase that degrades the tumor-suppressor p53. Reversible SMIs of the MDM2/p53 interaction have been developed to stabilize p53 and to induce apoptosis in wildtype p53 tumors. However, MDM2 SMIs induce a p53/MDM2 feedback loop, resulting in upregulation of MDM2 protein levels and p53 pathway inhibition, thus drastically limiting their biological activity and clinical application. MDM2 targeted protein degradation suppresses p53-dependent MDM2 protein feedback upregulation and is therefore expected to lead to a superior response compared to SMIs. Objectives: We discovered KT-253, a novel, highly potent heterobifunctional MDM2 degrader. We characterized the activity and molecular mechanism of KT-253 in wildtype p53 cell lines and xenograft models and demonstrated that KT-253 has superior activity compared to SMIs in these models. Methods: We characterized KT-253 in MDM2-dependent cell lines in vitro, as well as in in vivo mouse AML and ALL xenograft models. Methods included in vitro cell proliferation and apoptosis assays, gene expression profiling, and in vivo pharmacological studies. Results: KT-253 selectively depletes MDM2 protein levels with sub-nanomolar cellular potency. We demonstrated in the Acute Lymphoblastic Leukemia (ALL) cell line RS4;11 that KT-253 can overcome the p53-dependent upregulation of MDM2 protein levels as seen for reversible SMIs. In addition, even brief exposures to KT-253 in these cells could more potently stabilize p53 compared to SMIs, leading to apoptosis mediated through p53 target genes. The superior MDM2/p53 pathway inhibition and induction of apoptosis by KT-253 translates into a >200-fold stronger cell growth inhibition, compared to SMIs, across a panel of solid and hematological tumor cell lines. Using mouse xenograft models, we established an intermittent dosing schedule that drives anti-tumor efficacy. Importantly, a single dose of KT-253 at 1 mg/kg resulted in sustained tumor regression in the RS4;11 mouse xenograft model. In this model, KT-253 exposures correlated with induction of apoptotic p53 target genes and tumor growth inhibition. In addition, weekly administration of KT-253 significantly prolonged the survival in a disseminated Acute Myeloid Leukemia (AML) model when compared to vehicle treated animals. Conclusions: In summary, KT-253, our development candidate, has significantly improved potency relative to reversible SMIs leading to potent in vitro and in vivo efficacy that is superior to all clinically active agents. In addition, an intermittent dosing schedule of the highly potent KT-253 degrader can induce rapid apoptosis in MDM2-dependent cancer cells potentially leading to improved efficacy and safety profile. Citation Format: Yogesh Chutake, Michele Mayo, Dapeng Chen, Bradley Enerson, Patricia Cho, Jessica Filiatrault, Crystal Brown, Michael Placke, Madison Adams, Rahul Karnik, James Shaw, Yatao Shi, Dirk Walther, Alice McDonald, Frank Qi, Phillip Liu, Joseph D. Growney, Kirti Sharma, Duncan Walker, Stefanie Schalm, Juliet Williams, Matthew Weiss. KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI) [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 3934.

Abstract 1837: Low dose actinomycin D preferentially activates p53-p21 pathway in aerodigestive tract cancers: Implication for cyclotherapy

Abstract Actinomycin D (ActD) was the first anti-cancer antibiotic approved for the management of human cancers. However, the notorious toxicity profile limits its widespread application in cancers including cancers of the aerodigestive tract. Recent studies show that combining low-dose ActD with existing chemotherapies could potentially reduce the toxicity of chemotherapy drugs through p53 mediated cyclotherapy (protection of normal cells while eliminating cancer cells), a recently coined concept. An understanding of ActD’s effect on p53 signaling is critical for meaningful application of ActD in cyclotherapy-based combinations. This study evaluated the antitumor efficacy and mechanism of action of ActD in aerodigestive tract cancers. We found that ActD strongly inhibited the growth of a panel of aerodigestive tract cancer cell lines and induced efficient apoptosis although the sensitivity varies among cell lines. The IC50 values of ActD spanned between 0.021-2.96nM. Mechanistic studies revealed that ActD time- and dose-dependently increased the expression of total and phosphorylated p53 (ser15). Furthermore, ActD-induced apoptosis is dependent on p53 in cells expressing wild-type p53. Further studies demonstrated that ActD induced context-dependent differential expression of downstream targets p21 and PUMA without significant effects on p27. Interestingly, our study revealed that p53-p21 is the predominant pathway activated by low-dose ActD in cells with wild type p53. Therefore, our findings further rationalize the premise for future studies to explore the application of low-dose ActD as a chemoprotectant for cyclotherapy combinations in aerodigestive tract cancers. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence) Citation Format: Adeoluwa A. Adeluola, A. R. M Ruhul Amin. Low dose actinomycin D preferentially activates p53-p21 pathway in aerodigestive tract cancers: Implication for cyclotherapy [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 1837.

Abstract 5733: Targeting nonsense-mediated decay restores p53 function in HPV-associated head and neck cancers

Abstract HPV-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) tumors typically have p53 loss due to the activity of the human papillomavirus (HPV)-encoded E6 protein and the E6-associated protein (HPVE6-AP) which mediate the degradation of wild-type (WT) p53 (p53α). The loss of p53 is thought to be a major contributor to the pathogenesis of HPV+ HNSCC, which comprise approximately 35% of all HNSCC. Currently, standard care for HPV+HNSCC includes radiation and chemotherapy. However long-term toxicity related to these treatments is a concern, and there is a need for newer therapeutic strategies. Previously, we reported that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising of exons 1 to 9β or 9γ, respectively) are degraded by nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. Here, using HPV+HNSCC cell line models, we show that NMD inhibition rescues p53β/γ isoforms and activates p53 pathway. Furthermore, we show that p53β/γ isoforms are more stable compared to p53α in these cells, with reduced vulnerabililty to HPVE6-AP- mediated degradation, and that p53β/γ isoforms contribute to increased expression of p53 transcriptional targets p21 and PUMA following NMD inhibition. Consistent with p53 pathway activation, NMD inhibition enhanced radiosensitivity of HNSCC cells. NMD inhibition attenuated colony forming ability and disrupted cell cycle progression. To evaluate the therapeutic implications of NMD inhibition, we assessed the in vivo growth of HPV+ UMSCC47 tumors. Nude mice were injected with UMSCC47 cells either subcutaneously or orthotopically in the tongue and randomized to receive vehicle or with an NMD inhibitor. In both tumor models, we observed a significant reduction in tumor volume with NMD inhibition as compared to the vehicle-treated animals. To investigate whether NMD inhibition induced the expression of p53β/γ isoforms and activated the p53 pathway in vivo, we collected tumor tissues from animals and evaluated expression of p53 isoforms and transcriptional targets by RT-PCR. We observed increased expression of p53γ, p21, GADD45A and PUMA mRNAs in NMD inhibitor treated UMSCC47 tumors, compared to their respective vehicle treated controls. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in major subgroups of p53-deficient HPV+ HNSCC tumors. Citation Format: Jayanthi Gudikote, Tina Cascone, Alissa Poteete, Piyada Sitthideatphaiboon, Sonia Patel, Yan Yang, Fahao Zhang, Lerong Li, Li Shen, Monique Nilsson, Phillip Jones, Jing Wang, Jean-Christophe Bourdon, Faye M. Johnson, John V. Heymach. Targeting nonsense-mediated decay restores p53 function in HPV-associated head and neck cancers [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 5733.

Enhanced expression of p21 promotes sensitivity of melanoma cells towards targeted therapies.

Metastatic melanoma patients benefit from the approved targeted BRAF inhibitor (BRAFi) therapy. Despite the great progress in the therapeutic approach to combat metastatic melanoma, fast emerging drug resistance in patients limits its long-term efficacy. In this study, we aimed to unravel the role of the p53 target gene CDKN1A/p21 in the response of melanoma cells towards BRAFi. We show that p53 activation increases BRAFi sensitivity in a synergistic manner exclusively in cells with a high expression of CDKN1A/p21. In a similar way, high expression of p21 was associated with a better response towards the mouse double minute 2 inhibitor (MDM2i) compared to those with low p21 expression. Indeed, p21 knockdown decreased the sensitivity towards both targeted therapies. The results indicate that the sensitivity of melanoma cells towards targeted therapies (BRAFi and MDM2i) is dependent on the p21 protein level in the cells. In addition to that, we found that p53 negatively regulates p73 expression; however, p73 seems not to have an influence on p53 expression. These findings offer new potential strategies for the treatment improvement of melanoma patients with high basal p21 levels with BRAFi by increasing treatment efficacy using combination therapies with p53 activating substances, which are able to further increase p21 expression levels. Furthermore, the data suggest that the expression and induction level of p21 could be used as a predictive biomarker in melanoma patients to forecast the outcome of a treatment with p53 activating substances and BRAFi. All in all, this manuscript shows the distinct role of p53 family members and its impact on melanoma therapy. In future, individualized treatment regimens based on p21 basal and induction levels could help melanoma patients with limited treatment options.

Genomic and immunologic profiles of concurrent RB1 and CDKN1A/p21(WAF1) truncating mutations (RW+) in bladder cancer.

4571 Background: p53 target and cell cycle inhibitor CDKN1A/p21(WAF1) was initially not found to be mutated in cancer. TCGA analysis identified CDKN1A mutations are present but rare with frequencies of < 1%, but enrich in bladder cancer (̃8%). Truncating WAF1 mutations are associated with sensitivity to cisplatin and are associated with truncating Rb mutations in bladder cancer (RW+). We hypothesized RW+ bladder cancers may represent a unique subgroup with sensitivity to therapeutics. Methods: A total of 1104 urothelial tumors underwent molecular profiling at Caris Life Sciences (Phoenix, AZ) utilizing NGS of DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS). Wilcoxon, Fisher’s exact were used for statistical significance (p value without and q value with multi comparison correction). Immune cell fraction (QuanTIseq) and pathway analysis (ssGSEA) were assessed by mRNA analysis. Immune epitope prediction was performed using the NetMHCpan v4.0 method in the Immune Epitope Database. Results: Concurrent truncating mutation (frameshift, nonsense) for RB1 and WAF1 were detected in 47 tumors (RW+, 4.25%) and tumors with wild-type status for both RB1 and WAF1 genes were classified as RW- group (54.08%). Tumors harboring only one RB1 or WAF1 mutation were excluded for further analysis. When compared to RW- group, RW+ tumors showed lower mutation rate of TP53 (54.5% vs 80.9%, q < 0.05), ARID1A (23.5% vs 38.3%, p < 0.05), and PIK3CA (18.4% vs 31.9%, p < 0.05). Interestingly, RW+ was mutually exclusive with FGFR3 mutation (18.0% vs 0%, p < 0.05). We further evaluated RNA expression of DNA repair and checkpoint arrest pathways. Notably, E2F pathway (Normalized Enrichment Scores, NES: 0.89 vs 0.86, q < 0.01) and DNA G2M checkpoint (NES: 0.89 vs 0.86, q < 0.01) were found to be the most enriched in RW+ with respect to RW- group. In addition, mRNA levels of FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 were found to be overexpressed in RW+ group (q < 0.05). RW+ tumors also displayed a distinct immunological profile: They were associated with higher PD-L1 status (63.8% vs 37.3%, q < 0.01), higher median TMB (11 mut/Mb vs 8 mut/Mb, q < 0.01) and with less frequent loss of heterozygosity for HLA-DPA1 (51.1% vs 66.7%, p < 0.05), with more high-binding-affinity neoantigen load (4.78 vs 3.89, p < 0.05) to MHC proteins, consistent with the significantly more myeloid dendritic cells in in RW+ group (0.3 vs 0.04, q < 0.001). Conclusions: Concurrent truncating mutation in RB1 and WAF1 (RW+) bladder carcinomas have fewer p53, ARID1A, and PIK3CA mutations but are enriched for E2F targets, G2/M checkpoint genes, FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 and have a distinct immunological profile. The findings suggest therapeutic strategies for RW+ bladder cancers including Chk1/Wee1, PARP inhibitors, -/+ immunotherapy that may impact on clinical outcomes.

Dichotomous role of autophagy in cancer.

Autophagy is an evolutionary conserved catabolic process that plays physiological and pathological roles in a cell. Its effect on cellular metabolism, the proteome, and the number and quality of organelles, diversely holds the potential to alter cellular functions. It acts paradoxically in cancer as a tumor inhibitor as well as a tumor promoter. In the early stage of tumorigenesis, it prevents tumor initiation by the so-called "quality control mechanism" and suppresses cancer progression. For late-staged tumors that are exposed to stress, it acts as a vibrant process of degradation and recycling that promotes cancer by facilitating metastasis. Despite this dichotomy, the crucial role of autophagy is evident in cancer, and associated with mammalian targets of rapamycin (mTOR), p53, and Ras-derived major cancer networks. Irrespective of the controversy regarding autophagic manipulation, promotion and suppression of autophagy act as potential therapeutic targets in cancer treatment and may provide various anticancer therapies.

The mitochondrial‐derived lncRNA MDL1 mediates a mitochondria‐to‐nucleus retrograde regulation by inhibiting the nuclear translocation of p53

AbstractIt has been clear that mitochondria are not just nucleus‐relying entities; instead, they also modulate nuclear events reversely. Long noncoding RNAs (lncRNAs) are classified into nuclear‐encoded and mitochondrial‐derived lncRNAs (nulncRNAs and mtlncRNAs, respectively). Relative to nulncRNAs, the mtlncRNAs are far from being well characterized. Here we report a mtlncRNA, MDL1, that interacts with both p53 and Tid1 proteins and acts as retrograde signaling. MDL1 can regulate a network of nuclear genes, including p53 targets, and functions in cells in a p53‐dependent manner. Mechanistically, MDL1 is necessary for maintenance of the reciprocal interaction between p53 and Tid1, thereby facilitating formation of a ternary MDL1/p53/Tid1 complex that inhibits the nuclear translocation of p53. Since p53 is a canonical transcription factor, the observed inhibition of p53 nuclear translocation would contribute to the MDL1‐mediated mitochondrial retrograde regulation on nuclear genes expression. This study advances our understanding of lncRNA biology, and also sheds new light on the role of mtlncRNAs in mitochondrial‐nuclear functional network and in diverse biological processes.

Differential cofactor dependencies define distinct types of human enhancers.

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors1,2. Emerging evidence suggests that not all cofactors are required at all enhancers3-5, yet whether these observations reflect more general principles or distinct types of enhancers remained unknown. Here we categorized human enhancers by their cofactor dependencies and show that these categories provide a framework to understand the sequence and chromatin diversity of enhancers and their roles in different gene-regulatory programmes. We quantified enhancer activities along the entire human genome using STARR-seq6 in HCT116 cells, following the rapid degradation of eight cofactors. This analysis identified different types of enhancers with distinct cofactor requirements, sequences and chromatin properties. Some enhancers were insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4 and regulated distinct transcriptional programmes. In particular, canonical Mediator7 seemed dispensable for P53-responsive enhancers, and MED14-depleted cells induced endogenous P53 target genes. Similarly, BRD4 was not required for the transcription of genes that bear CCAAT boxes and a TATA box (including histone genes and LTR12 retrotransposons) or for the induction of heat-shock genes. This categorization of enhancers through cofactor dependencies reveals distinct enhancer types that can bypass broadly utilized cofactors, which illustrates how alternative ways to activate transcription separate gene expression programmes and provide a conceptual framework to understand enhancer function and regulatory specificity.

Snrpb is required in murine neural crest cells for proper splicing and craniofacial morphogenesis.

ABSTRACTHeterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for cerebrocostomandibular syndrome (CCMS). We show that Snrpb heterozygous mouse embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon skipping and intron retention in association with increased 5′ splice site strength. We found increased exon skipping in negative regulators of the P53 pathway, along with increased levels of nuclear P53 and P53 target genes. However, removing Trp53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53 activity and craniofacial-specific genes contributes to craniofacial malformations. This article has an associated First Person interview with the first author of the paper.

Complementary omics strategies to dissect p53 signaling networks under nutrient stress

Signaling trough p53is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Biophysical characterisation of human LincRNA-p21 sense and antisense Alu inverted repeats.

Human Long Intergenic Noncoding RNA-p21 (LincRNA-p21) is a regulatory noncoding RNA that plays an important role in promoting apoptosis. LincRNA-p21 is also critical in down-regulating many p53 target genes through its interaction with a p53 repressive complex. The interaction between LincRNA-p21 and the repressive complex is likely dependent on the RNA tertiary structure. Previous studies have determined the two-dimensional secondary structures of the sense and antisense human LincRNA-p21 AluSx1 IRs using SHAPE. However, there were no insights into its three-dimensional structure. Therefore, we in vitro transcribed the sense and antisense regions of LincRNA-p21 AluSx1 Inverted Repeats (IRs) and performed analytical ultracentrifugation, size exclusion chromatography, light scattering, and small angle X-ray scattering (SAXS) studies. Based on these studies, we determined low-resolution, three-dimensional structures of sense and antisense LincRNA-p21. By adapting previously known two-dimensional information, we calculated their sense and antisense high-resolution models and determined that they agree with the low-resolution structures determined using SAXS. Thus, our integrated approach provides insights into the structure of LincRNA-p21 Alu IRs. Our study also offers a viable pipeline for combining the secondary structure information with biophysical and computational studies to obtain high-resolution atomistic models for long noncoding RNAs.

A Critical Role for p53 during the HPV16 Life Cycle.

ABSTRACTHuman papillomaviruses (HPV) are causative agents in ano-genital and oral cancers; HPV16 is the most prevalent type detected in human cancers. The HPV16 E6 protein targets p53 for proteasomal degradation to facilitate proliferation of the HPV16 infected cell. However, in HPV16 immortalized cells E6 is predominantly spliced (E6*) and unable to degrade p53. Here, we demonstrate that human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), and HPV16 positive oropharyngeal cancers, retain significant expression of p53. In addition, p53 levels increase in HPV16+ head and neck cancer cell lines following treatment with cisplatin. Introduction of full-length E6 into HFK+HPV16 resulted in attenuation of cellular growth (in hTERT immortalized HFK, E6 expression promoted enhanced proliferation). An understudied interaction is that between E2 and p53 and we investigated whether this was important for the viral life cycle. We generated mutant genomes with E2 unable to interact with p53 resulting in profound phenotypes in primary HFK. The mutant induced hyper-proliferation, but an ultimate arrest of cell growth; β-galactosidase staining demonstrated increased senescence, and COMET assays showed increased DNA damage compared with HFK+HPV16 wild-type cells. There was failure of the viral life cycle in organotypic rafts with the mutant HFK resulting in premature differentiation and reduced proliferation. The results demonstrate that p53 expression is critical during the HPV16 life cycle, and that this may be due to a functional interaction between E2 and p53. Disruption of this interaction has antiviral potential.IMPORTANCE Human papillomaviruses are causative agents in around 5% of all cancers. There are currently no antivirals available to combat these infections and cancers, therefore it remains a priority to enhance our understanding of the HPV life cycle. Here, we demonstrate that an interaction between the viral replication/transcription/segregation factor E2 and the tumor suppressor p53 is critical for the HPV16 life cycle. HPV16 immortalized cells retain significant expression of p53, and the critical role for the E2-p53 interaction demonstrates why this is the case. If the E2-p53 interaction is disrupted then HPV16 immortalized cells fail to proliferate, have enhanced DNA damage and senescence, and there is premature differentiation during the viral life cycle. Results suggest that targeting the E2-p53 interaction would have therapeutic benefits, potentially attenuating the spread of HPV16.

Identification of mutant p53-specific proteins interaction network using TurboID-based proximity labeling

BackgroundsAlthough several studies on mutant p53 reported cancer-promoting activities via “gain-of-function”, the mechanism underlying these differences in function between p53 R175H, R175P, and p53 wild-type (WT) remains unclear. MethodsLinking miniTurbo with p53 WT, R175H, and R175P, the expression of fusion and biotinylated proteins were assessed by Western blotting. The function and subcellular localization of fusion proteins were detected by apoptosis assay and immunofluorescence, respectively. Biotinylated proteins were analyzed by liquid chromatography-tandem mass spectrometry, followed by bioinformatics analysis. Small-scale pull-downs and Co-Immunoprecipitation were performed to validate the interaction between mutant or p53 WT and biotinylated proteins. ResultsThe fusion protein's cellular localization and function were consistent with those of previous studies on the corresponding p53. Comparative profiles of R175H versus WT showed that most of the interacting proteins belonged to the intracellular organelle lumen, and the pathways involved were metabolism and genetic information processing. Comparative profiles of R175P versus WT suggested that the majority of the interacting proteins belonged to the intracellular organelle lumen and the extracellular membrane-bounded organelle, and the pathways involved were metabolism and genetic information processing pathways. The comparison between R175H and R175P revealed that most interacting proteins belonged to the organelle lumen, and pathways involved were genetic information processing pathways. Finally, the mutation of p53 significantly altered the interaction with the target proteins were confirmed. ConclusionWe verified the reliability of the miniTurbo system and obtained candidate targets of mutant p53, which provided new thoughts on the mechanism of mutant p53 gain-of-function and new potential targets for cancer therapy.