P53 Mutant Cell Lines Research Articles

Abstract 3022: GPR109A activation limits cell growth by inducing p53 and its targets in human colon cancer cells

Abstract Chemical carcinogens or xenobiotics present in the diet play a significant role in colon cancer (CC) formation. p53, a tumor suppressor and multi-functional protein, provides an important barrier against the initiation and maintenance of CC. p53 mutation or inactivation is observed in about 60% of CCs and about 90% of all cancers. Therefore, therapeutic avenues that will rejuvenate wild-type p53 or target mutant p53 should have a significant impact on the prevention and treatment of CC. GPR109A, a G-protein coupled receptor for the FDA-approved lipid-lowering drug Niacin (NA), bacterial metabolite Butyrate (BTR), and ketone body β-hydroxybutyrate (BHB), functions as a novel tumor suppressor in the colon by providing the molecular link between colonic bacteria and p53 function. GPR109A activation limits cAMP by inhibiting adenylate cyclase (AC), which induces RAS and its downstream growth-promoting signaling. cAMP also enhances the binding of MDM2 to p53 and induces the proteasomal degradation of p53. p53 is an NAD+-dependent molecule and deficiency of NA, the precursor of NAD+, impairs p53 function. We tested the role of GPR109A and its agonists (NA, BTR, and BHB) in normal immortalized colon epithelial cell lines (CCD33 and CCD81) and various CC lines such as HCT116-p53+/+, HCT116-p53−/−, DLD1 (p53 point mutation), VACO4A (p53 insertion mutation), and SW498 (p53 deletion) and measured cell proliferation, cell cycle, apoptosis, and analyzed the expression of GPR109A and p53 (total, phosphorylated, and acetylated), MDM2, p21, Bax, and Puma in the presence and absence of GPR109109 agonists. We found that GPR109A expression is significantly reduced in CC cell lines, especially in p53 mutant cell lines, when compared to normal immortalized colonic epithelial cell lines. Treatment of CC cell lines with GPR109A agonists-induced apoptosis in wild-type p53 expressing CC by activating p53 and its targets. However, some lesser extent in p53 mutant or p53 null cell lines. Lentiviral-mediated GPR109A expression in CC cell lines itself induced apoptosis in both wild-type and mutant p53 expressing CC cell lines and GPR109A agonists treatment induced further. Interestingly, GPR109A agonists BTR and BHB significantly increased apoptosis in p53 mutant and p53 null CC cell lines in a p53-independent manner. Overall, our results provide evidence that GPR109A activation can be used as an effective therapeutic strategy for the treatment of CC. Citation Format: Muthusamy Thangaraju, Nanditi N. Thangaraju, Lauren Gilstrap, Madison Chimenti, Tulshi Patel, Sathish Sivaprakasam, Nikhil Patel, Santhakumar Manicassamy, Puttur D. Prasad, Vadivel Ganapathy. GPR109A activation limits cell growth by inducing p53 and its targets in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3022.

Abstract 6588: Discovery of ASTX295, a potent, next-generation small molecule antagonist of MDM2 with differentiated pharmacokinetic profile

Abstract In response to cellular stress, the tumor suppressor p53 is activated to modulate cell cycle progression, DNA repair, and apoptosis. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 prevents its degradation and can reactivate p53 to elicit an anti-cancer effect. Targeting the p53-MDM2 interaction therefore remains a promising strategy for cancer therapy. However, development of first generation MDM2 antagonists has been challenged by dose-limiting, on-target bone marrow toxicities. Understanding of differential effects of p53 pathway activation in normal hematopoietic versus cancer cells (to be presented in a separate abstract) together with our expertise in structure-based drug design have led to the discovery of ASTX295, a potent MDM2 antagonist with differentiated pharmacokinetic profile aimed at sparing bone marrow toxicities and increasing the therapeutic index. Here, we present the first disclosure of the structure and pre-clinical characterisation of ASTX295. ASTX295 exhibits potent activity (IC50<1 nM) against MDM2 in an ELISA-based in vitro assay and induces significant growth reduction in p53 wild-type, MDM2-amplified SJSA-1 cells (GI50=27 nM). Antiproliferative activity of ASTX295 was further demonstrated in a panel of 219 p53 wild-type cell lines, with 143 cell lines showing GI50 values less than 1 μM and 50 showing values less than 0.1 μM. Effects of ASTX295 are shown in cell lines carrying functional p53 as confirmed in three p53 wild-type and mutant cell line pairs (SJSA1 and SN40R2, A2780 and A2780CP, HCT116 and HCT116 p53−/−). In addition to inhibiting cell cycle progression and cell proliferation, ASTX295 also potently induces apoptosis following 24-48 hour treatment. Further in vitro analyses of ASTX295 demonstrated an increase in the levels of p53 (EC50=10 nM after 2 hours) and its transcriptional targets such as p21 and MDM2. In vivo, ASTX295 shows robust induction of p53 and its target genes at 3 and 6 hours after oral administration together with dose-dependent inhibition of tumour growth in the SJSA-1 xenograft model. Importantly, ASTX295 exhibits optimised pharmacokinetic and pharmacodynamic profiles with relatively short duration of pathway modulation and a desired predicted human half-life of 2-8 hours. Based on our pre-clinical hypothesis on differential time-dependent sensitivities of normal versus cancer cells to p53 activation, achieving such a profile while maintaining potency may increase the therapeutic index. These data highlight the therapeutic potential of ASTX295, which is currently being tested in a Phase 1/2 clinical trial in advanced solid tumors with wild-type p53 (NCT03975387). We plan to present preliminary clinical data in a separate abstract at this meeting. Citation Format: Maria Ahn, Luke Bevan, Ildiko Buck, Celine Cano, Juan Castro, Ben Cons, Jane Endicott, Lynsey Fazal, Nicola Ferrari, Ian Hardcastle, Keisha Hearn, Rhian Holvey, Steven Howard, Chris Johnson, Claire Jennings, Justyna Kucia-Tran, Suzanne Kyle, John Lunec, John Lyons, Duncan Miller, David Rees, Martin Noble, David R. Newell, Judith Reeks, Harpreet Saini, Jeffrey St. Denis, Emiliano Tamanini, Huw Thomas, Neil Thompson, Mladen Vinkovic, George Ward, Nicola Wallis, Hugh Walton, Stephen Wedge, Pamela Williams, Elaine Willmore, Nicola Wilshire, Yan Zhao, Gianni Chessari. Discovery of ASTX295, a potent, next-generation small molecule antagonist of MDM2 with differentiated pharmacokinetic profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6588.

NEAT1 repression by MED12 creates chemosensitivity in p53 wild-type breast cancer cells.

Breast cancer is often treated with chemotherapy. However, the development of chemoresistance results in treatment failure. Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been shown to contribute to chemoresistance in breast cancer cells. In studying the transcriptional regulation of NEAT1 using multi-omics approaches, we showed that NEAT1 is up-regulated by 5-fluorouracil in breast cancer cells with wild-type cellular tumor antigen p53 but not in mutant-p53-expressing breast cancer cells. The regulation of NEAT1 involves mediator complex subunit 12 (MED12)-mediated repression of histone acetylation marks at the promoter region of NEAT1. Knockdown of MED12 but not coactivator-associated arginine methyltransferase 1 (CARM1) induced histone acetylation at the NEAT1 promoter, leading to elevated NEAT1 mRNAs, resulting in a chemoresistant phenotype. The MED12-dependent regulation of NEAT1 differs between wild-type and mutant p53-expressing cells. MED12 depletion led to increased expression of NEAT1 in a wild-type p53 cell line, but decreased expression in a mutant p53 cell line. Chemoresistance caused by MED12 depletion can be partially rescued by NEAT1 knockdown in p53 wild-type cells. Collectively, our study reveals a novel mechanism of chemoresistance dependent on MED12 transcriptional regulation of NEAT1 in p53 wild-type breast cancer cells.

Decitabine induces IRF7-mediated immune responses in p53-mutated triple-negative breast cancer: a clinical and translational study.

p53 is mutated in half of cancer cases. However, no p53-targeting drugs have been approved. Here, we reposition decitabine for triple-negative breast cancer (TNBC), a subtype with frequent p53 mutations and extremely poor prognosis. In a retrospective study on tissue microarrays with 132 TNBC cases, DNMT1 overexpression was associated with p53 mutations (P = 0.037) and poor overall survival (OS) (P = 0.010). In a prospective DEciTabinE and Carboplatin in TNBC (DETECT) trial (NCT03295552), decitabine with carboplatin produced an objective response rate (ORR) of 42% in 12 patients with stage IV TNBC. Among the 9 trialed patients with available TP53 sequencing results, the 6 patients with p53 mutations had higher ORR (3/6 vs. 0/3) and better OS (16.0 vs. 4.0 months) than the patients with wild-type p53. In a mechanistic study, isogenic TNBC cell lines harboring DETECT-derived p53 mutations exhibited higher DNMT1 expression and decitabine sensitivity than the cell line with wild-type p53. In the DETECT trial, decitabine induced strong immune responses featuring the striking upregulation of the innate immune player IRF7 in the p53-mutated TNBC cell line (upregulation by 16-fold) and the most responsive patient with TNBC. Our integrative studies reveal the potential of repurposing decitabine for the treatment of p53-mutated TNBC and suggest IRF7 as a potential biomarker for decitabine-based treatments.

CCRG-06. PP2A DEFICIENCY INDUCES CELLULAR SENESCENCE AND SENSITIZES P53 MUTANT TUMORS TO SENOLYTIC THERAPY

Abstract Loss of p53 can promote tumor progression by preventing induction of senescence, a critical tumor-suppressive mechanism that limits tumor proliferation. Promotion of senescence in a p53-independent manner can be a novel strategy to overcome treatment resistance in p53-mutant brain tumors. Protein-Phosphatase-2A (PP2A), a ubiquitous serine/threonine phosphatase, regulates various cellular processes including cell-cycle progression. We hypothesize that PP2A-deficiency can induce tumor senescence independent of p53-mutational-status and sensitize tumors to senolytic therapy. CRISPR-KO of PP2A was generated in multiple p53-mutant tumor cell lines: D425 (human medulloblastoma), SB28 and GL261 (murine glioma). Hallmarks of senescence such as b-galactosidase, p21, and p16 were assessed via flow cytometry, RT-PCR and Western-Blot. Secretion of senescence-associated secretory phenotype (SASP), such as IL8, was assessed through ELISA. b-galactosidase, p21, p16 and IL8 were dramatically increased in PP2A-KO compared to WT at baseline. The effect was further enhanced when combined with radiation therapy (RT), a known inducer of senescence, demonstrating PP2A-deficiency synergizes with RT and significantly increases cellular senescence. Expression of CD1d, a MHC1-like molecule associated with senescence and required for invariant NKT cell-activation (iNKT), was dramatically increased in PP2A-KO in comparison to WT via flow-cytometry. In-vitro, sensitivity to WT and PP2A-KO cells to senolytic treatments, such as ABT-737 and iNKT-cell therapy, was tested using an XCelligence-assay system and flow-cytometry-based cytotoxicity assay. PP2A-deficient cells were sensitized to ABT-737, a BCL-xL inhibitor that preferentially targets and kills senescent cells. Co-culture revealed increased cytotoxicity of iNKT-cells against PP2A-deficient tumors compared to WT. In vivo, D425 WT or PP2A-KO cells were injected intracranially into nude NU/J mice, radiated 2 days post-injection, and monitored till endpoint. At baseline, PP2A-KO mice survived longer than WT, and those with PP2A-KO and RT survived the longest. In conclusion, we report that PP2A-deficiency in tumor cells can induce p53-independent senescence and sensitize brain tumors to senolytic therapy.

Wee1 inhibition by MK1775 potentiates gemcitabine through accumulated replication stress leading to apoptosis in biliary tract cancer

Patients with advanced biliary tract cancer (BTC) have a poor prognosis, and novel treatments are needed. Gemcitabine, the standard of care for BTC, induces DNA damage; however, the ability of cancer cells to repair DNA dampens its effects. To improve the efficacy of gemcitabine, we combined it with MK1775, a Wee1 inhibitor that prevents activation of the G2/M checkpoint. BTC cell lines were treated with gemcitabine only or in combination with MK1775 to determine the therapeutic potential of BTC. Gemcitabine inhibited the growth and induced the apoptosis of four BTC cell lines to a greater extent when added with MK1775 than when added alone. The effects of the combination treatment were observed in both p53 wild-type and p53 mutant cell lines and were unaffected by knockdown of wild-type p53. The combination treatment increased the percentage of apoptotic cells and decreased the percentage of cells synthesizing DNA, suggesting that it caused DNA-damaged cells to accumulate and possibly die in S phase. It did not induce apoptosis when cells were arrested in mitosis using nocodazole. In a xenograft mouse model, gemcitabine plus MK1775 (but not either alone) inhibited the growth of tumors generated from inoculated BTC cells. Our results show that MK1775 highly enhances gemcitabine cytotoxicity in BTC regardless of p53 status. We suggest that the combination treatment elicits a DNA damage response and consequent apoptosis. Our preclinical study provides a basis for future clinical trials of gemcitabine plus MK1775 in patients with BTC.

The p53 reactivator PRIMA-1MET synergises with 5-fluorouracil to induce apoptosis in pancreatic cancer cells.

Pancreatic cancer (PC) is one of the deadliest malignancies; p53 is mutated in approximately 75% of PC patients. Hence, the protein derived from mutant/wild-type TP53 may represent a therapeutic target. Interestingly, a p53 reactivator (PRIMA-1MET) showed promise in clinical trials of haematological malignancies; therefore, it warrants an in vitro evaluation in PC cell lines. To evaluate the antiproliferative effects of PRIMA-1MET, either alone or combined with the common chemotherapy 5-fluorouracil (5-FU), against mutated and wild-type p53 PC cell lines. This study involved p53-mutant (AsPC-1) and p53-wild type (Capan-2) PC cell lines. The cytotoxicity of PRIMA-1MET alone or in combination with 5-FU was evaluated by MTT assay. Synergism was assessed by calculating the combination index (CI) via CalcuSyn software. Fluorescence microscopy was used to analyse apoptosis following acridine orange/ethidium bromide (AO/EB) staining. Morphological changes were investigated with an inverted microscope. Quantitative reverse transcription PCR (RT‒qPCR) was used to measure gene expression. Both PC cell lines were sensitive to PRIMA-1MET monotherapy. Furthermore, PRIMA-1MET and 5-FU had a synergistic effect (CI < 1), reflected by significant enhancement of apoptosis and morphological changes in the combination vs. monotherapy treatments. Moreover, the RT‒qPCR results indicated increased expression of the NOXA and TP73 genes in combination-treated cells. Our data suggested that PRIMA-1MET, whether alone or combined with 5-FU, has an antiproliferative effect on PC cell lines regardless of p53 mutational status. The synergism of the combination was associated with significant apoptosis induction through p53-dependent and p53-independent pathways. Preclinical confirmation of these data in in vivo models is highly recommended.

Relationship between efficacy of phase I/II drug ispinesib in medulloblastoma, molecular subtypes, and p53-mutant status.

e14001 Background: KIF11, a mitotic kinesin, is responsible for assembly and maintenance of mitotic spindle during mitosis. Tumor cells can upregulate KIF11 to enhance tumor proliferation. Many cancers overexpress KIF11, including medulloblastoma (MB), the most common malignant brain tumor in children. MB is molecularly classified into 4 molecular subtypes: sonic-hedgehog (SHH)-activated MB, Wingless (WNT)-activated, and less characterized Group 3 and 4 subtypes. Group 3 and 4 MBs are the most aggressive subtypes and Group 3 MBs have the worst survival outcome. p53 mutation defines a high-risk group in MB patients. We evaluated the efficacy of a Phase I/II KIF11 inhibitor, ispinesib on MB. Methods: We employed in-vitro proliferation assays (CCK), flow cytometry (Annexin V, Propidium Iodide), western blot, Hoechst staining, and two patient-derived orthotopic xenograft (PDOX) mouse models for in-vivo drug testing. Results: Ispinesib effectively inhibited proliferation at nanomolar concentrations for all 5 MB cell lines. A comparison of IC50 (day 4) indicated that p53-mutational-status did not influence sensitivity to ispinesib. In all five cell lines, regardless of p53 mutational status, ispinesib led to G2/M arrest. However, different p53-mutational-status led to different modes of cell death. High levels of necrotic cell death were observed in p53-mutant, while apoptosis was the predominant mode of cell death in p53-wildtype, on flow cytometry. Apoptotic nuclei were more significantly observed in p53-wildtype (p = 0.002) while p53-mutant cell line demonstrated more micronuclei (p = 0.0302) and multi-nucleation (p= 0.0161), compared to respective DMSO-control cells on Hoechst staining. Ispinesib has demonstrated effective blood brain barrier penetration in brain xenografts. We compared 2 PDOX models of MB. Ispinesib significantly improved survival outcome of Group 3 MB (Control=6, Treated=7, Log-rank p=0.0003), but worsened the survival outcome of p53-mutant SHH MB (Control=14, Treated=11, Log-rank p=0.169). This differential efficacy of ispinesib was recapitulated in the matched 3D-neurosphere cultures of both PDOX models. Cancer-stem-cell (CSC)-enriched neurospheres derived from aggressive Group 3 subtype demonstrated susceptibility to 7-day in-vitro treatment with ispinesib (AUC=0.7743). Monolayer tumor cells demonstrated similar susceptibility (AUC=0.7757). Area under curve (AUC)&gt;0.7 was the significant cut-off. In contrast, both CSC-enriched neurospheres (AUC=0.379) and monolayer tumor cells (AUC=0.491) derived from p53-mutant SHH MB did not demonstrate susceptibility to ispinesib, consistent with matching PDOX models. Conclusions: Preclinically, our results indicate feasibility of ispinesib treatment in MB, specifically aggressive Group 3 MB. Importantly, p53-mutant SHH MB represents a poor molecular-subtype candidate for ispinesib therapy.

Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells

TP53 (p53) and MYC are amongst the most frequently altered genes in cancer. Both are thus attractive targets for new anticancer therapies. Historically, however, both genes have proved challenging to target and currently there is no approved therapy against either. The aim of this study was to investigate the effect of the mutant p53 reactivating drug, COTI-2 on MYC. Total MYC, pSer62 MYC and pThr58 MYC were detected using Western blotting. Proteasome-mediated degradation was determined using the proteasome, inhibitor MG-132, while MYC half-life was measured using pulse chase experiments in the presence of cycloheximide. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Treatment of 5 mutant p53 breast cancer cell lines with COTI-2 resulted in dose-dependent MYC degradation. Addition of the proteasome inhibitor, MG132, rescued the degradation, suggesting that this proteolytic system was at least partly responsible for the inactivation of MYC. Using cycloheximide in pulse chase experiments, COTI-2 was found to reduce the half-life of MYC in 2 different mutant p53 breast cancer cell lines, i.e., from 34.8 to 18.6 min in MDA-MB-232 cells and from 29.6 to 20.3 min in MDA-MB-468 cells. Co-treatment with COTI-2 and the MYC inhibitor, MYCi975 resulted in synergistic growth inhibition in all 4 mutant p53 cell lines investigated. The dual ability of COTI-2 to reactivate mutant p53 and degrade MYC should enable this compound to have broad application as an anticancer drug.

Inclusion of Nitrofurantoin into the Realm of Cancer Chemotherapy via Biology-Oriented Synthesis and Drug Repurposing.

Structural modifications of the antibacterial drug nitrofurantoin were envisioned, employing drug repurposing and biology-oriented drug synthesis, to serve as possible anticancer agents. Eleven compounds showed superior safety in non-cancerous human cells. Their antitumor efficacy was assessed on colorectal, breast, cervical, and liver cancer cells. Three compounds induced oxidative DNA damage in cancer cells with subsequent cellular apoptosis. They also upregulated the expression of Bax while downregulated that of Bcl-2 along with activating caspase 3/7. The DNA damage induced by these compounds, demonstrated by pATM nuclear shuttling, was comparable in both MCF7 and MDA-MB-231 (p53 mutant) cell lines. Mechanistic studies confirmed the dependence of these compounds on p53-mediated pathways as they suppressed the p53-MDM2 interaction. Indeed, exposure of radiosensitive prostatic cancer cells to low non-cytotoxic concentrations of compound 1 enhanced the cytotoxic response to radiation indicating a possible synergistic effect. In vivo antitumor activity was verified in an MCF7-xenograft animal model.

Extracellular Matrix Modulates Outgrowth Dynamics in Ovarian Cancer.

Ovarian carcinoma (OC) forms outgrowths that extend from the outer surface of an afflicted organ into the peritoneum. OC outgrowth formation is poorly understood due to the limited availability of cell culture models examining the behavior of cells that form outgrowths. Prompted by immunochemical evaluation of extracellular matrix (ECM) components in human tissues, laminin and collagen-rich ECM-reconstituted cell culture models amenable to studies of cell clusters that can form outgrowths are developed. It is demonstrated that ECM promotes outgrowth formation in fallopian tube non-ciliated epithelial cells (FNE) expressing mutant p53 and various OC cell lines. Outgrowths are initiated by cells that underwent outward translocation and retained the ability to intercalate into mesothelial cell monolayers. Electron microscopy, optical coherence tomography, and small amplitude oscillatory shear experiments reveal that increased ECM levels led to increased fibrous network thickness and high shear elasticity of the microenvironment. These physical characteristics are associated with outgrowth suppression. The low ECM microenvironment mimicks the viscoelasticity of malignant peritoneal fluid (ascites) and supports cell proliferation, cell translocation, and outgrowth formation. These results highlight the importance of the ECM microenvironment in modulating OC growth and can provide additional insights into the mode of dissemination of primary and recurrent ovarian tumors.

WEE1 kinase inhibitor MK-1775 sensitizes oral tongue squamous cell carcinoma cells to radiation irrespective of TP53 status.

Oral tongue squamous cell carcinoma (OTSCC) frequently harbors non-functional p53 and depends on G2/M checkpoint mediated by WEE1. WEE1 suppression has been identified as a promising anti-tumor strategy. This study investigated the capacity of WEE1 kinase inhibitor (MK-1775) and its underlying mechanisms in enhancing radiation responses of OTSCC cells in vitro. WEE1 kinase expression and its downstream target (CDK1) were investigated in OTSCC versus normal oral tissue. A synergistic combination of MK-1775 with radiation on OTSCC cell lines with different p53 statuses was assessed by viability assay. The radio-sensitizing effects of MK-1775 on apoptosis, cell cycle, DNA damage, and mitotic entry were also determined. Irradiation enhanced CDK1 expression in all tested cell lines, though the effect was far more pronounced in p53 mutated cell lines. MK-1775 exhibited inhibitory effects against the survival of all cell lines and enhanced their response to the radiation. These effects were strongly elicited by induction of apoptosis and lethal mitosis, but less likely by abrogation of radiation-induced G2 arrest. These results demonstrate the efficacy of MK-1775 in enhancing the radiation effect on OTSCC in vitro associated with a significant apoptotic death rate, identifying WEE1 inhibitor as a potent radiosensitizer in OTSCC irrespective of p53 mutational status.

Abstract 1826: Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers

Abstract Background: Disulfiram (DSF) in combination with copper pills has emerged as a safe and exciting anticancer strategy and is undergoing clinical trials in multiple human cancers. However, DSF breaks down in the gut or blood immediately and chances of cupric diethyldithiocarbamate (Cu-DDC), the active cytotoxic principle being formed at tumor sites is very low. Therefore, we have found that synthetic Cu-DDC is directly and potently cytotoxic against p53 mutant cancers. We report here for the first time the differential effects of Cu-DDC in robustly eliminating the mutant p53 proteins from tumor cells in contrast to its augmentation of the wild-type p53 protein and activation. Destabilization of Gain Of Function (GOF) p53 mutant proteins for overcoming oncogenic addiction and improving the therapeutic efficacy has been a long-sought, but unmet anticancer avenue. Methods: A panel of 7 P53 mutant cell lines (R248W, R273H,R173H), and p53 wt tumor and normal cells (HPBLs, dental pulp stem cells, IMR90 and HUVEC) were used. Immunoprecipitation, Western blotting, Annexin-V apoptotic assays, flow cytometry, immunocytochemistry were used to quantitate p53 protein. Mia-Paca2 xenografts developed in nude mice were administered Cu-DDC/BSA. Results: Cu-DDC generated peroxides but not superoxides in tumor cells. The GOF mutant p53 proteins, R248W and R273H in three different tumor cell lines were eliminated after Cu-DDC treatment in a time- and concentration-dependent manner. The loss of mutant p53 proteins was confirmed by three different procedures. The degradation occurred rapidly in 8-10 h at 100 nM Cu-DDC and was inhibited by proteasome blockers. The expression of the GRO-α oncogene, positively regulated by GOF R248W was decreased in Cu-DDC treated Mia-Paca2 cells. Cellular proteasome activity was not inhibited by Cu-DDC. Ubiquitination of mutant p53 proteins was not altered by Cu-DDC. In contrast, the wild-type p53 present in HCT116 and MCF-7 cells, and normal cell counterparts (HPBL, IMR-90 lung cells) was significantly upregulated and activated with enhanced p21cip1 expression. Cu-DDC triggered strong apoptosis irrespective of the p53 status, although the mutant p53 containing cells were 2 to 3-fold more sensitive in cytotoxicity assays (IC50 range 0.25-0.66 µM). A significant tumor growth delay accompanied by loss of mutant p53 protein was observed in MiaPaca2 xenografts. Conclusions: In moving the DSF anticancer repurposing forward, we describe the unique ability of Cu-DDC to rapidly eliminate oncogenic p53 mutant proteins and confer potent antitumor efficacy. The degradation was unrelated to the proteostasis involving the inactivation of NPL4 by Cu-DDC described previously (supported by a Cancer Prevention and Research Institute of Texas [CPRIT] grant RP170207 to KSS). Citation Format: Viswanath Arutla, Kalkunte S. Srivenugopal. Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human 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 1826.

Abstract 5387: Investigating the role of p53 in doxorubicin-mediated lysosome membrane permeabilization in breast cancer cells

Abstract Background: Doxorubicin (DOX) is a chemotherapeutic characterized by multiple mechanisms. Wild-type p53 (WTp53) may be activated in the presence of either DNA damage or ROS. Upon activation, WTp53 may upregulate the transcription of pro-apoptotic Bcl-2 family members capable of triggering mitochondrial membrane permeabilization or lysosome membrane permeabilization (LMP), ultimately resulting in apoptosis. We have previously reported of DOX-induced LMP between WTp53 and mutant p53 (MUTp53) breast cancer cells. The goals of this study were to establish (1) the role of p53 in the initiation of DOX-mediated LMP and (2) whether p53 is critical to the onset of cytotoxicity. Methods: Studies were conducted using DOX at a range of concentrations and durations in both WTp53 (MCF-7 and MDA-MB-361) and MUTp53 (MDA-MB-231 and T47D) breast cancer cells. LMP was demonstrated using fluorescence microscopy as the cytosolic release of a 10 kDa AlexaFluor-488 Dextran conjugate that was preloaded into lysosomes. Viability was determined by MTT. Cytotoxicity was determined by trypan blue. Experiments were conducted in the presence of pifithrin-α p53 inhibitor to establish the importance of p53 towards DOX cytotoxicity. Results: Significant LMP was determined as early as 0.5 h in both of the WTp53 cell lines at 10 μM. MDA-MB-361 cells demonstrated a greater degree of LMP with 1 μM DOX showing significant LMP by 2 h, an effect that was not observed in MCF-7 cells until 6 h. The earliest onset of notable LMP in MUTp53 MDA-MB-231 cells occurred at 6 h with 10 μM DOX only. T47D cells did not demonstrate notable LMP at this time. By 24 h, both WTp53 cell lines demonstrated LMP across all of the tested concentrations of DOX, while both MUTp53 cell lines only demonstrated notable LMP for 10 μM DOX. At this same time, notable reductions in cell viability for the tested range of concentrations were observed for all cell lines except for T47D cells. Overall, the inhibition of p53 reversed notable LMP in both MCF-7 and MDA-MB-361 cells across all of the tested conditions. Similarly, reduced viability was reversed following the inhibition of p53. Conclusions: These results indicate a role of p53 in LMP in WTp53 cells. Furthermore, the combination of p53 and LMP seem necessary for the induction of cytotoxicity in WTp53 cells. Under circumstances of MUTp53 expression, cells promote a pro-oncogenic response with a lesser potential for LMP, MMP, and cytotoxicity. Citation Format: Rachel Nicoletto, Clyde M. Ofner. Investigating the role of p53 in doxorubicin-mediated lysosome membrane permeabilization in breast 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 5387.

Abstract 136: Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which lacks HER2 overexpression, as well estrogen and progesterone receptor expression. TNBC constitutes 10-15% of all breast cancers but has a worse prognosis due to limited treatment options and high rates of metastatic recurrence. Doxorubicin (dox) remains one of the most active chemotherapy agents for the treatment of TNBC, although de novo and acquired resistance to chemotherapy remains a major challenge. The purpose of this work was to characterize dox resistant cell phenotypes and investigate treatment-induced senescence in p53-mutated (Mut) and wildtype (WT) TNBC cell lines. Methods: A panel of 12 TNBC cell lines (p53 WT and p53 Mut) were exposed to dox (0-5 uM) for 72 hours and cellular proliferation was determined using the Cell Titer Glo assay. A subset of sensitive and resistant cell lines were treated with dox (0.1, 0.25 uM) or vehicle control and apoptosis was determined by flow cytometry (Annexin-V) at 24 and 48 hrs. Cells were treated with dox or control for 24 hrs and western blotting was performed for mediators of apoptosis. Senescence was analyzed by ß-galactosidase staining following treatment with dox for 3-14 days. shRNA knockdown (KD) of p53 was performed in the CAL-51 (p53 WT cell line) and cells were subject to investigations above. Results: Doxorubicin treatment resulted in decreased cellular proliferation and increased apoptosis as assessed by Annexin-V expression in a subset of p53 WT and p53 Mut cell lines. Treatment with dox resulted in an increase in the pro-apoptotic proteins BID and p21, as well as a decrease in VAMP in sensitive cell lines. In a subset of cell lines resistant to dox treatment, we observed an increase in cells demonstrating phenotypic features of senescence and ß -galactosidase staining. We observed a decrease in p16 with dox treatment in the CAL-51 (p53 WT cell line) compared to an increase following treatment in p53 Mut cell lines. KD of p53 resulted in an increase in senescent cells following treatment with low dose dox. Conclusions: Treatment with doxorubicin resulted in different terminal cell phenotypes in TNBC cell lines with apoptosis observed in p53 WT and Mut cell lines. Senescence was observed in resistant cells and KD of p53 increased dox-induced senescence, confirming a role for p53 in mediating terminal cell fate. Efforts are ongoing to understand the role of mutant p53 in mediating terminal cell fate in response to dox and rational combinations to overcome dox-induced senescence may be clinically active in metastatic TNBC. Citation Format: Stephen G. Smoots, Anna R. Schreiber, Betelehem Yacob, Adrian Dominguez, Cecilia Levandowski, Todd M. Pitts, Jennifer R. Diamond. Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines [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 136.

A phase 1/2 dose-escalation and dose-expansion study of ZN-c3 in combination with gemcitabine in adult and pediatric subjects with relapsed or refractory osteosarcoma.

TPS11584 Background: Osteosarcoma (OS) is the most common primary bone malignancy of childhood and adolescence with 5-year survival rates of 65-70% for localized disease and &lt; 30% for de novo metastatic disease or recurrent disease. Pooled analysis of previous phase 2 trials by the Children’s Oncology Group has determined a 4-month event-free survival (EFS) of 12%. The Wee1 kinase helps regulate DNA damage repair at the G2-M checkpoint. In the presence of DNA damage, the Wee1 kinase is activated, arresting cells in the G2 phase and preventing entry into the M phase. Inhibition of the Wee1 kinase abrogates the G2-M checkpoint, forcing cancer cells to undergo unscheduled mitosis even in the presence of DNA damage, leading to mitotic catastrophe. However, the Wee1 kinase is often upregulated in OS, preserving the G2-M checkpoint and allowing tumor growth and metastases. Additionally, up to 90% of OS tumors have alterations in p53, a critical protein in the regulation of the G1-S checkpoint, especially in relapsed or refractory cases. With a dysfunctional G1-S checkpoint, cancer cells further rely on G2-M checkpoint to repair DNA damage and preserve genomic integrity. Prior studies have demonstrated that pharmacologic inhibition of the Wee1 kinase produced cell death in OS cell lines and patient-derived xenografts. While p53 mutational status appeared to modulate efficacy of the Wee1 kinase inhibitor, activity was observed in p53 wild type, mutant and null cell lines. Combination therapy studies have also been performed, demonstrating potential synergism with gemcitabine. As expected, by precipitating DNA damage, susceptibility to inhibition of the G2-M checkpoint is further increased. Methods: NCT04833582 is an ongoing, open label, multicenter, phase 1/2 clinical trial to evaluate the activity of ZN-c3, an oral Wee1 inhibitor, in combination with gemcitabine in subjects ≥12 years and ≥40 kg, with relapsed, refractory OS. Subjects are dosed once daily, continuously with ZN-c3 and receive gemcitabine 1000 mg/m2 on days 1 and 8 of 21-day cycles. Up to 18 subjects are expected to enroll in the phase 1 portion based on a typical 3 + 3 escalation design; ̃60 subjects will be enrolled in the phase 2 portion, consisting of three stages: futility, promising clinical activity, and improved precision for clinical activity. The first two stages follow a Simon two-stage optimal design with 30 subjects, to differentiate an EFS rate at 18 weeks between 12% and 36% (which may be considered a more suitable endpoint for OS, compared with radiographic response). Tumor and skin punch biopsies are incorporated into the trial to identify potential biomarkers of treatment response. Subjects must be able to swallow oral tablets and have measurable disease by RECIST v1.1; prior exposure to gemcitabine is allowed. Global enrollment began August 1, 2021, and is ongoing. Clinical trial information: NCT04833582.

Abstract GS1-09: Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by lack of ER, PR, and Her2. Up to 85% of TNBCs have a p53 mutation as their oncogenic driver. TP53 is a tumor suppressor known as the “guardian of the genome” for its roles in regulating growth and death after genomic insult. Due to this high frequency of p53 mutations in TNBCs, targeting p53 mutants in a clinical setting is highly attractive. However, there are currently no FDA-approved drugs that can directly target p53 mutant TNBCs. We propose pathways exist that, when inhibited, will induce the specific death of p53-mutant breast cancer cells, but not p53-wild type breast cells. To investigate this hypothesis, we performed high-throughput drug screening to identify drugs that induce death in p53-mutant TNBCs. We then characterized the identified drugs for mechanism of death induction and in vivo drug effect. Methods: In vitro and in silico drug screens were conducted with small-molecule libraries of drugs with known protein targets, and screens were integrated and common drugs identified. P53-wild type and mutant cells were treated with identified drugs and stained with DAPI and DRAQ7 to identify growth-suppressive and death-inductive effects. One of these drugs, the GPX4 inhibitor ML-162, was selected for further study. Mechanism of death induction by ML-162 was determined with AnnexinV/PI, caspase cleavage, and ferroptosis assays. To confirm these results, GPX4 was knocked out and replicated the effect of ML-162. To determine the reliance of this phenotype on p53 mutational status, a series of inducible p53-mutant cell lines were created in ER+ and TNBC cell lines and then treated with small molecule inhibitors. ML-162 in vivo effect was demonstrated by treating p53-mutant TNBC xenografts in nude mice. Results: Integration of in vitro and in silico screens identified 6 common drugs, representing cell cycle inhibitors, cell division inhibitors, a proteasome inhibitor, and a peroxidase inhibitor. Identified drugs were demonstrated to reduce growth or induce death of p53-mutant TNBC cell lines. The GPX4 inhibitor ML-162 was further characterized, and found to induce death through ferroptosis, and not apoptosis or necroptosis. GPX4 knockout in p53-mutant TNBC cell lines induced ferroptosis and can be blocked with an anti-ferroptosis drug. Inducible p53-mutations in ER+ and TNBC cell lines were treated with ML-162. P53 mutations in TNBC, and not ER+, cell lines showed increased sensitivity to ML-162. To demonstrate in vivo effect of ML-162, TNBC cell line xenografts were grown in nude mice and treated with ML-162. This treatment significantly reduced tumor volume and also induced lipid peroxidation, a hallmark of ferroptosis. Conclusion: Our high-throughput screening demonstrated several of the identified drugs suppress growth or induce death preferentially in p53-mutant breast cancers. One of these drugs, ML-162, induces death of p53-mutant triple-negative breast cancer cells through induction of ferroptosis, both in vitro and in vivo. These studies provide the basic science foundation to further develop ferroptosis inducers for the targeted treatment of p53-mutant breast cancers. This work was funded by the John Charles Cain Endowment. Citation Format: William M Tahaney, Jing Qian, Reid Powell, Cassandra L Moyer, Yanxia Ma, Nghi Nguyen, Jamal Hill, Clifford Stephan, Abhijit Mazumdar, Peter JA Davies, Powel H Brown. Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-09.

Abstract P1-02-05: A genome-wide functional genomics screen reveals unique co-driver mutations of mutant TP53 promoting cellular heterogeneity during breast cancer progression

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks three major drug-targetable receptors, ER, PR, and HER2. TNBC patients have much worse 5-year survival rates (60%) in contrast to 90% for other breast cancer subtypes and display highly heterogeneous molecular profiles, cellular phenotypes, and drug responses, which poses major challenges in patient treatments. The tumor suppressor gene TP53 is mutated in 30% of breast tumors overall and but highly prevalent (~80%) in TNBC. Unlike mutations in other tumor suppressor genes that are predominantly loss-of-function deletions or truncations, TP53 mutations occur mostly as &amp;gt;100 different missense mutations within the DNA binding domain, implying that the mutant proteins may exert both loss-of-function activities and gain of distinct neomorphic functions, thus contributing to phenotypic heterogeneity of TNBC. When we characterized systematically a panel of MCF10A cell lines expressing 10 most prevalent missense mutant p53 proteins, the cell lines indeed displayed highly diverse neomorphic cellular phenotypes distinct from those of p53-knockdown cells. To investigate molecular mechanisms underlying the heterogeneity, we then performed RNA-Seq and pathway analysis and identified the key pathways, such as the Hippo/YAP pathway, that were dysregulated correlatively with phenotypic aggressiveness of the mutant p53 cell lines. In addition, ChIP-Seq analysis revealed that promoter binding capacity and preference of mutant p53 proteins associated with more aggressive phenotypes were more severely affected, especially for the genes in the dysregulated pathways identified from RNA-Seq analysis. These demonstrated collectively that different missense p53 mutations lead to heterogeneous phenotypes by exerting distinct neomorphic molecular functions. Further, given that TP53 mutations by themselves cannot drive full cancer progression, these imply that cells with different p53 missense mutations need distinct sets of additional “co-driver” mutations and alteration of cellular programs specific to each mutation for full cancer progression, representing potential molecular targets for personalized therapies. Supporting this hypothesis, when we performed genome-wide in vitro CRISPR screens in search of co-driver mutations specific to different p53 mutations, a unique set of hits was identified for each mutant p53-expressing cell lines. However, in in vivo mouse xenograft models, even the cells expressing aggressive p53 mutants such R273C failed to develop tumors upon transducing gene-deleting CRISPR gRNA libraries at high MOI. Based on reasoning that development of tumor requires mutations in both tumor suppressors and oncogenes, we then performed CRISPR screens on the p53-R273C cells overexpressing MYC, a known oncogene for TNBC, and observed tumor formation within 9 weeks, only after the CRISPR library transduction. By next-generation sequencing of the gRNA cassettes amplified from the tumors, &amp;gt;20 novel co-driver candidates in addition to known tumor suppressors such as NF2 and PTEN were identified. Interestingly, ARAF, a proto-oncogene, was one of the top candidates found in multiple tumors, and the targeted sequencing confirmed out-of-frame deletions resulting in truncated proteins with only the N-term Ras-binding domain. We are currently validating the functional relevance of these findings in conjunction with the dysregulated pathways identified from RNA-Seq and ChIP-Seq analysis. Taken together, our integrated approach of utilizing phenotyping, multi-omics bioinformatics analysis, and screening has revealed the molecular mechanisms underlying phenotypic and molecular heterogeneity and potential molecular targets of TNBC. Citation Format: Dustin Grief, Anasuya Pal, Laura Gonzalez-Malerva, Seron Eaton, Chenxi Xu, Grant Christensen, Joy Blain, Nicholas Mellor, Jason Steel, Chitrak Gupta, Ellen Streitwieser, Abhishek Singharoy, Jin Park, Joshua LaBaer. A genome-wide functional genomics screen reveals unique co-driver mutations of mutant TP53 promoting cellular heterogeneity during breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-05.

P53-Induced Autophagy Regulates Chemotherapy and Radiotherapy Resistance in Multidrug Resistance Cancer Cells.

BackgroundMultidrug resistance (MDR), a major problem in oncology therapy, limits the effectiveness of anticancer drugs. Although p53 functions as a tumor suppressor, the associations between p53 status, autophagy, and MDR are complicated and conditional.Method In this report, p53-null human ovarian cancer cell line SKOV3 and its MDR phenotype SKVCR and human leukemia cell line CEM and its MDR phenotype CEM-VLB) (p53 mutant cell line) were used.Results Compared to parental SKOV3, the mRNA and protein levels of MAPLC3-II and Beclin1 were higher in SKVCR cells. The inhibition of autophagy by 3-MA significantly sensitized SKVCR to VCR. Conversely, in drug-resistant leukemic cells CEM-VLB, the expressions of Beclin1 and MAPLC3-II were lower than CEM. CEM and CEM-VLB cells were treated with VLB .01 or 0.5 μg/mL, respectively, and the expression of p53 and autophagy up-regulated after VLB (.01 μg/mL) treatment in CEM cells. The percentage of S-phase and G2/M phase cells up-regulated significantly by .01 μg/mL VLB in CEM, which may relate to the status of p53 of CEM cells. A combination of radiation with 3-MA significantly increased apoptosis in CEM-VLB cells.Conclusion Our discovery found that p53 is an important regulator controlling the balance between autophagy and MDR, as a potential drug target for ovarian cancer and leukemia.

Combination of Compound Kushen Injection and cisplatin shows synergistic antitumor activity in p53-R273H/P309S mutant colorectal cancer cells through inducing apoptosis

Ethnopharmacological relevanceColorectal cancer (CRC) is one type of worldwide popular and refractory tumors. Compound Kushen Injection (CKI) is a frequently applied traditional Chinese medicine formula as an adjuvant drug for the chemotherapy of CRC. P53 is the most commonly mutated gene in CRC, accounting for the development, malignant and prognosis progression of CRC. However, effect of CKI on the therapeutic efficacy of p53-mutant CRC remains understood. Besides, the combined efficacy of different chemotherapeutics drugs in combination with CKI for CRC treatment is rarely concerned. Aim of studyTo investigate the combined efficacy of the CKI-derived combination strategies in the p53-mutant CRC. Materials and methodsTwo CRC cell lines HCT116 and SW480 cells, which respectively harbor wild-type p53 and p53-R273H/P309S mutant, were applied. Cisplatin (Cis) and 5-fluorouracil (5FU) were combined chemotherapeutics drugs of CKI-derived combination strategies in this article. In vitro antitumor activity was detected by sulforhodamine B (SRB) assay and colony formation assay. Combenefit soft was applied to evaluate the synergetic/antagonistic effect of drug combination. Lentivirus-mediated overexpression method was used to generate a set of p53-mutant and wild-type CRC cell lines harboring identical genomes. Transcriptomics and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were applied to predicate the underlying mechanism of synergetic interaction between drug combination. Western blot was performed to verify predicated pathways contributing to the synergy of drug combination. ResultsCKI preferentially combined with Cis but not 5FU, to produce a synergistical antitumor efficiency for p53-R273H/P309S mutant, rather than wild-type p53 harboring CRC cells. The combination of CKI and Cis strongly reprogrammed the transcriptional profiles of SW480 cells. Cytokine-cytokine receptor interaction pathway was a key pathway involved in cooperativity between CKI and Cis in SW480 cells. Mechanistically, compared to that Cis individually triggered necroptosis, the co-treatment of CKI and Cis reinforced the cell death of SW480 cells in a possible synergistic manner by inducing extrinsic apoptosis pathway. ConclusionThis article provides a novel perspective into the precision clinical application of CKI-derived combination therapy programs of CRC based on genetic variation and the classes of chemotherapeutics drugs.