P53 Wild-type Cells Research Articles

The Doxorubicin-Induced G2/M Arrest in Breast Cancer Cells Modulated by Natural Compounds Naringenin and Hesperidin

Doxorubicin as the common drug for breast cancer has been widely proposed to use in combine with a natural compound in order to overcome its side effects such as cardiotoxicity and resistance. Previously, we reported that naringenin and hesperidin, the abundant flavanons in citrus fruit peel, increased cytotoxic and apoptosis activities of doxorubicin in doxorubicin resistant breast cancer cells (T47D and MCF-7 cells). Since doxorubicin arrests G2/M phase in most cancer cells, both flavanons are speculated to affect the similar phase in breast cancer cells. Cell cycle distributions were determined by flowcytometry using propidium iodide (PI) to stain DNA of the cells. Combination of naringenin or hesperidin with doxorubicin increased accumulation of T47D cells in G2/M phase, while in MCF-7 cells, accumulated cells in G2/M phase were decreased, accompanying with slightly increased in G1 phase. Naringenin itself had no effect on cell cycle of both cells. Whereas, hesperidin arrested G2/M and G1 phases in T47D and MCF-7 cells, respectively. The different effect of naringenin and hesperidin in T47D and MCF-7 cells is most likely caused by difference of p53 status. In p53 mutant, T47D cells, naringenin and hesperidin supported mechanism of doxorubicin to arrest at G2/M that to be considered via p53-independent pathway. Whereas, in p53 wild-type MCF-7 cells, naringenin and hesperidin decreased G2/M arrest, suggesting that both flavanons do not utilize cell cycle arrest for their anticancer activity with doxorucibin. This study revealed that potential co-chemoterapeutic agents, naringenin and hesperidin distinctly modulated cell cycle arrest induced by doxorubicin according to the characteristic of breast cancer cells.Keywords: naringenin, hesperidin, doxorubicin, cell cycle, breast cancer cells.

Severe cellular stress activates apoptosis independently of p53 in osteosarcoma

Apoptosis induced by doxorubicin, bortezomib, or paclitaxel, targeting DNA, 26S proteasome, and microtubules respectively, was assessed in two osteosarcoma cells, p53 wild-type U2OS and p53-null MG63 cells. Doxorubicin-induced apoptosis only occurred in U2OS, not in MG63. In contrast, bortezomib and paclitaxel could drive U2OS or MG63 toward apoptosis effectively, suggesting that apoptosis induced by bortezomib or paclitaxel is p53-independent. The expressions of Bcl2 family members such as Bcl2, Bcl-xl, and Puma could be seen in U2OS and MG63 cells with or without doxorubicin, bortezomib, or paclitaxel treatment. In contrast, another member, Bim, only could be observed in U2OS, not in MG63, under the same conditions. Bim knockdown did not affect the doxorubicin-induced apoptosis in U2OS, suggested that a BH3-only protein other than Bim might participate in apoptosis induced by doxorubicin. Using a BH3-mimetic, ABT-263, to inhibit Bcl2 or Bcl-xl produced a limited apoptotic response in U2OS and MG63 cells, suggesting that this BH3-mimetic cannot activate the Bax/Bak pathway efficiently. Significantly, ABT-263 enhanced doxorubicin- and bortezomib-induced apoptosis synergistically in U2OS and MG63 cells. These results implied that the severe cellular stress caused by doxorubicin or bortezomib might be mediated through a dual process to control apoptosis. Respectively, doxorubicin or bortezomib activates a BH3-only protein in one way and corresponding unknown factors in another way to affect mitochondrial outer membrane permeability, resulting in apoptosis. The combination of doxorubicin with ABT-263 could produce synergistic apoptosis in MG63 cells, which lack p53, suggesting that p53 has no role in doxorubicin-induced apoptosis in osteosarcoma. In addition, ABT-263 enhanced paclitaxel to induce moderate levels of apoptosis.

Valproic Acid Enhanced Temozolomide-Induced Anticancer Activity in Human Glioma Through the p53-PUMA Apoptosis Pathway.

Glioblastoma (GBM), the most lethal type of brain tumor in adults, has considerable cellular heterogeneity. The standard adjuvant chemotherapeutic agent for GBM, temozolomide (TMZ), has a modest response rate due to the development of drug resistance. Multiple studies have shown that valproic acid (VPA) can enhance GBM tumor control and prolong survival when given in conjunction with TMZ. However, the beneficial effect is variable. In this study, we analyzed the impact of VPA on GBM patient survival and its possible correlation with TMZ treatment and p53 gene mutation. In addition, the molecular mechanisms of TMZ in combination with VPA were examined using both p53 wild-type and p53 mutant human GBM cell lines. Our analysis of clinical data indicates that the survival benefit of a combined TMZ and VPA treatment in GBM patients is dependent on their p53 gene status. In cellular experiments, our results show that VPA enhanced the antineoplastic effect of TMZ by enhancing p53 activation and promoting the expression of its downstream pro-apoptotic protein, PUMA. Our study indicates that GBM patients with wild-type p53 may benefit from a combined TMZ+VPA treatment.

Apigenin enhances apoptosis induction by 5-fluorouracil through regulation of thymidylate synthase in colorectal cancer cells

Although effective drugs have been developed, including 5-fluorouracil (5-FU), advanced colorectal cancer (CRC) shows low therapeutic sensitivity resulting from the development of 5-FU resistance. Thymidylate synthase (TS) is a target protein of 5-FU, and elevated TS lowers the 5-FU sensitivity of CRC cells. Here, we tested the efficacy of several candidate phytochemicals against human CRC-derived HCT116 cells expressing wild-type tumor suppressor protein P53 and HT29 cells expressing mutant P53. Among them, we found that apigenin enhanced the inhibitory effect of 5-FU on cell viability. In addition, apigenin inhibited the upregulation of TS induced by 5-FU. Apigenin also potentiated 5-FU-induced apoptosis of HCT116 cells and enhanced cell cycle disruption. Furthermore, apigenin increased reactive oxygen species production, intracellular and intramitochondrial Ca2+ concentrations, and mitochondrial membrane potential upon cotreatment with 5-FU. Knockdown of forkhead box protein M, a transcription factor modulating 5-FU sensitivity, enhanced the potentiation of apoptosis by apigenin in HCT116 cells. Moreover, apigenin suppressed TS expression and inhibited the viability of 5-FU-resistant HCT116 cells. Therefore, apigenin may improve the therapeutic efficacy of 5-FU against CRC by suppressing TS, but apoptosis induction is mainly dependent on functional P53.

A-24, a steroidal saponin from Allium chinense, induced apoptosis, autophagy and migration inhibition in p53 wild-type and p53-deficient gastric cancer cells

Allium chinense is a vegetable with nutrition and unique flavor, and it is used as traditional Chinese medicine. We previously reported that the active compound A-24 induces apoptosis and autophagy in p53 wild-type gastric cancer cells through the PI3K/Akt/mTOR pathway. Our present work indicates that A-24 also has a significant proliferation inhibition effect on p53-deficient KATO-III cells, and the p53 status did not affect A-24 induced migration inhibition, but negatively controlled the occurrence of autophagy. We also found that the accumulation of reactive oxygen species (ROS) mediated A-24 induced apoptosis is p53-independent. Besides, p-Akt was not downregulated by A-24 in p53-deficient gastric cancer cells. Taken together, our results indicate that A-24 induced apoptosis and autophagy via the ROS-PI3K/Akt/mTOR pathway in p53 wild-type gastric cancer cells and through the ROS-mTOR pathway in p53-deficient gastric cancer cells. Our study recommended A-24 as a promising future phytotherapeutic candidate for gastric cancer treatment.

Acquisition of aneuploidy drives mutant p53-associated gain-of-function phenotypes

p53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.

P53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth.

Cholesterol is essential for membrane biogenesis, cell proliferation, and differentiation. The role of cholesterol in cancer development and the regulation of cholesterol synthesis are still under active investigation. Here we show that under normal-sterol conditions, p53 directly represses the expression of SQLE, a rate-limiting and the first oxygenation enzyme in cholesterol synthesis, in a SREBP2-independent manner. Through transcriptional downregulation of SQLE, p53 represses cholesterol production invivo and invitro, leading to tumor growth suppression. Inhibition of SQLE using small interfering RNA (siRNA) or terbinafine (a SQLE inhibitor) reverses the increased cell proliferation caused by p53 deficiency. Conversely, SQLE overexpression or cholesterol addition promotes cell proliferation, particularly in p53 wild-type cells. More importantly, pharmacological inhibition or shRNA-mediated silencing of SQLE restricts nonalcoholic fatty liver disease (NAFLD)-induced liver tumorigenesis in p53 knockout mice. Therefore, our findings reveal a role for p53 in regulating SQLE and cholesterol biosynthesis, and further demonstrate that downregulation of SQLE is critical for p53-mediated tumor suppression.

TRAIL-receptor 2\u2014a novel negative regulator of p53

TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2) can induce apoptosis in cancer cells upon crosslinking by TRAIL. However, TRAIL-R2 is highly expressed by many cancers suggesting pro-tumor functions. Indeed, TRAIL/TRAIL-R2 also activate pro-inflammatory pathways enhancing tumor cell invasion, migration, and proliferation. In addition, nuclear TRAIL-R2 (nTRAIL-R2) promotes malignancy by inhibiting miRNA let-7-maturation. Here, we show that TRAIL-R2 interacts with the tumor suppressor protein p53 in the nucleus, assigning a novel pro-tumor function to TRAIL-R2. Knockdown of TRAIL-R2 in p53 wild-type cells increases the half-life of p53 and the expression of its target genes, whereas its re-expression decreases p53 protein levels. Interestingly, TRAIL-R2 also interacts with promyelocytic leukemia protein (PML), a major regulator of p53 stability. PML-nuclear bodies are also the main sites of TRAIL-R2/p53 co-localization. Notably, knockdown or destruction of PML abolishes the TRAIL-R2-mediated regulation of p53 levels. In summary, our finding that nTRAIL-R2 facilitates p53 degradation and thereby negatively regulates p53 target gene expression provides insight into an oncogenic role of TRAIL-R2 in tumorigenesis that particularly manifests in p53 wild-type tumors.

Mechanistic insights into p53-regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells.

Late‐stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post‐translational modifications (PTMs). While the relevance of p53 C‐terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild‐type p53 or p53‐negative human CRC cells, cells with acetylation‐defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase‐1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan‐treated p53‐positive CRC cells. This specifically relies on the C‐terminal acetylation of p53 by CREB‐binding protein/p300 and the presence of C‐terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C‐terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53‐proficient CRC.

Comparable radiation sensitivity in p53 wild-type and p53 deficient tumor cells associated with different cell death modalities

Studies of radiation interaction with tumor cells often take apoptosis as the desired results. However, mitotic catastrophe and senescence are also promoted by clinically relevant doses of radiation. Furthermore, p53 is a well-known transcription factor that is closely associated with radiosensitivity and radiation-induced cell death. Therefore, we aimed to investigate the involvement of radiosensitivity, cell death modalities and p53 status in response to carbon-ion radiation (CIR) here. Isogenic human colorectal cancer cell lines HCT116 (p53+/+ and p53−/−) were irradiated with high-LET carbon ions. Cell survival was determined by the standard colony-forming assay. 53BP1 foci were visualized to identify the repair kinetics of DNA double-strand breaks (DSBs). Cellular senescence was measured by SA-β-Gal and Ki67 staining. Mitotic catastrophe was determined with DAPI staining. Comparable radiosensitivities of p53+/+ and p53−/− HCT116 colorectal cells induced by CIR were demonstrated, as well as persistent 53BP1 foci indicated DNA repair deficiency in both cell lines. Different degree of premature senescence in isogenic HCT116 colorectal cancer cells suggested that CIR-induced premature senescence was more dependent on p21 but not p53. Sustained upregulation of p21 played multifunctional roles in senescence enhancement and apoptosis inhibition in p53+/+ cells. p21 inhibition further increased radiosensitivity of p53+/+ cells. Complex cell death modalities rather than single cell death were induced in both p53+/+ and p53−/− cells after 5 Gy CIR. Mitotic catastrophe was predominant in p53−/− cells due to inefficient activation of Chk1 and Chk2 phosphorylation in combination with p53 null. Senescence was the major cell death mechanism in p53+/+ cells via p21-dependent pathway. Taken together, p21-mediated premature senescence might be used by tumor cells to escape from CIR-induced cytotoxicity, at least for a time.

P300 Participates in Ionizing Radiation-Mediated Activation of Cathepsin L by Mutant p53.

Our previous studies have shown that cathepsin L (CTSL) is involved in the ability of tumors to resist ionizing radiation (IR), but the specific mechanisms responsible for this remain unknown. We report here that mutant p53 (mut-p53) is involved in IR-induced transcription of CTSL. We found that irradiation caused activation of CTSL in mut-p53 cell lines, whereas there was almost no activation in p53 wild-type cell lines. Additionally, luciferase reporter gene assay results demonstrated that IR induced the p53 binding region on the CTSL promoter. We further demonstrated that the expression of p300 and early growth response factor-1 (Egr-1) was upregulated in mut-p53 cell lines after IR treatment. Accordingly, the expression of Ac-H3, Ac-H4, AcH3K9 was upregulated after IR treatment in mut-p53 cell lines, whereas histone deacetylase (HDAC) 4 and HDAC6 were reciprocally decreased. Moreover, knockdown of either Egr-1 or p300 abolished the binding of mut-p53 to the promoter of CTSL. Chromatin immunoprecipitation assay results showed that the IR-activated transcription of CTSL was dependent on p300. To further delineate the clinical relevance of interactions between Egr-1/p300, mut-p53, and CTSL, we accessed primary tumor samples to evaluate the relationships between mut-p53, CTSL, and Egr-1/p300 ex vivo. The results support the notion that mut-p53 is correlated with CTSL transcription involving the Egr-1/p300 pathway. Taken together, the results of our study revealed that p300 is an important target in the process of IR-induced transcription of CTSL, which confirms that CTSL participates in mut-p53 gain-of-function. SIGNIFICANCE STATEMENT: Transcriptional activation of cathepsin L by ionizing radiation required the involvement of mutated p53 and Egr-1/p300. Interference with Egr-1 or p300 could inhibit the expression of cathepsin L induced by ionizing radiation. The transcriptional activation of cathepsin L by p300 may be mediated by p53 binding sites on the cathepsin L promoter.

Abstract 1386: The role of MDM2 inhibition in the radiosensitization of ER+ breast cancers

Abstract Background​: Radiation therapy (RT) is standard in the treatment of many women with breast cancer (BC). Despite this, women with estrogen receptor positive (ER+) BC respond heterogeneously to RT. Radiosensitization methods for aggressive ER+ disease are needed. We performed a radiosensitizer screen paired with transcriptomic and proteomic data from ER+ models treated +/-RT to identify potential mediators of RT resistance. Methods​: Clonogenic survival assays were used to determine RT sensitivity of 21 BCC lines as well as radiosensitization with drug treatment. IC50 values were determined for 130 clinical compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Microarray and RPPA data was used for differential gene/protein expression and pathway analysis. AlamarBlue was used to determine IC-50 values of the MDM2 inhibitor AMG-232. Western blot analysis of Cleaved PARP and Annexin V staining for FLOW was used to measure apoptosis and Cyclins A, E, B and p-Histone H3 and flow cytometry to measure cell cycle progression. yH2AX immunofluorescence was used to measure dsDNA breaks. Results​: A MDM2 inhibitor (JNJ-26854165) was nominated as an effective drug in treatment for RT-resistant BC cell lines (R2​ = 0.43, p-value <0.01) in our novel radiosensitizer screen. Differential gene expression and pathway analysis in multiple non-overlapping ER+ BC cell lines treated +/-RT identified apoptosis, cell cycle, and p53 signaling as the top pathways induced in ER+ cell lines by RT. Within these MDM2 was significantly overexpressed after RT+ compared to RT- in ER+ p53 wild-type (WT) cells. In p53 mutant (MT) cell lines, however, MDM2 was not differentially expressed. This suggests MDM2 may mediate radioresistance in a p53 dependent manner. Cell growth in the p53​ ​WT cell lines MCF-7 and ZR-75-1 was inhibited by AMG-232, an MDM2 inhibitor (IC-50 values of 554nM and 264nM). p53 MT ER+ cell lines were not sensitive to MDM2 inhibition with this drug (IC-50> 10uM). Clonogenic survival assays demonstrated that at sub-IC50 doses MDM2 inhibition leads to radiosensitization in p53 WT ER+ cell lines (MCF-7 rER: 1.17-2.13; ZR751 rER: 1.30-1.65), however, p53 MT ER+ cells were not radiosensitized (T47D rER: 0.94-1.11; CAMA-1 rER: 0.88-0.95). AMG-232 and RT combined led to an increase in apoptosis compared to RT alone in ER+ p53 WT cells but not p53 MT cells. Combination treatment led to differential cyclin and p-Histone H3 expression in p53 WT cells but not p53 MT cells. ​G1 cell cycle arrest was a secondary effect of MDM2 inhibition and radiation. Experiments investigating the role of dsDNA breaks in radiosensitization are ongoing. Conclusions​: Our novel radiosensitizer screen identifies MDM2 as a potential mediator of radioresistance in ER+ BC in a p53-dependent manner and suggests that MDM2 targeting concurrent with RT may represent a tractable clinical strategy in women with locally advanced ER+, p53 WT BC. Citation Format: Cassandra Lynne Ritter, Benjamin C. Chandler, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Tanner Ward, Amanda Zhang, Mattia Cremona, Lori J. Pierce, Bryan Hennessy, Corey W. Speers. The role of MDM2 inhibition in the radiosensitization of ER+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1386.

Abstract 3066: DNA ligase IV inhibitor and X-ray exert a synthetic lethal in loss-of-function p53 cells

Abstract The majority of lung cancer patients receive radiotherapy during the treatment process. Dysfunction of DNA ligase IV impaired non-homologous end joining (NHEJ) and sensitized cells to X-ray. p53 regulated DNA damage repair and was mutated in various lung cancer cells. This study aimed to investigate the synergic effects of DNA ligase IV inhibitor SCR-7 and radiation on both wild-type and p53-deficient lung cancer cells. In the p53 wild-type cell, X-ray and SCR-7 triggered G2/M checkpoint, induced BRCA1, RAD18 activation, and promoted PCNA ubiquitination, facilitating homologous recombination (HR) and translesion synthesis (TLS) rather than NHEJ. In p53-deficient cells, SCR-7 and X-ray exerted a synthetic lethal via inducing more DNA damage, muting TLS, and impairing G2/M checkpoint as dysfunction of Cdc2 and Cyclin B1. Moreover, many cells were collapsed in mitosis catastrophe as a large number of disintegrating chromosomes, and apoptosis was significantly increased. Furthermore, the synergic effects of SCR-7 and X-ray were diminished by exogenous p53 overexpression. Therefore, we hypothesized p53 should be a major regulator of HR and TLS activation when HNEJ deficiency. Our studies indicated that patients with a loss function of p53 might benefit from SCR-7 and radiotherapy with fewer side effects. Citation Format: Tingting Lin, Zhilian Zhou, Lifeng Zhu, Yandan Fan, Xiaofen Ding, Yingming Sun. DNA ligase IV inhibitor and X-ray exert a synthetic lethal in loss-of-function p53 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3066.

Abstract 1277: Discovery of novel CDC7 inhibitors that disrupt cell cycle dynamics and show anti-proliferative effects in cancer cells

Abstract Introduction: CDC7 is a serine/threonine protein kinase that phosphorylates the MCM2-7 helicase complex, a required step in DNA replication initiation. CDC7 has emerged as an attractive target for cancer treatment because of high expression in a number of tumors (e.g. ovarian, lung, and oral) which is thought to be linked to their proliferative capacity and ability to bypass DNA damage responses. Consistent with this, disruption of CDC7 activity in cancer cells results in delayed DNA replication, mitotic abnormalities and cell death whereas non-transformed, p53 wildtype cells are protected from cytotoxicity due to G1 cell cycle arrest. Due to the low ATP Km of CDC7, very potent inhibitor molecules are required to effectively block CDC7 activity and drive cancer cells into apoptosis. We have identified novel potent and selective CDC7 inhibitors targeting the ATP binding site that are active in biophysical, biochemical and cellular assays as well as in vivo CDX models. Results: Our lead compounds show potent picomolar (pM) inhibition of CDC7 in a biochemical kinase activity assay, pM affinity in SPR assay and complete inhibition of MCM2 (S53) phosphorylation in COLO205, A427, MV-4-11 and SW48 cancer cell lines. In a broad kinase selectivity panel, the novel inhibitors showed good selectivity for CDC7 kinase. Mechanistic studies show that our CDC7 inhibitors induced apoptosis, disrupted DNA replication and cell cycle dynamics with accumulation of polyploid cells after 48 h of treatment of cancer cells with minimal effects on human fibroblast cell lines. Our compounds have shown potent anti-proliferative and cytotoxic effects in a panel of more than a 100 cancer cell lines of varying origin including COLO205, SW48, A427, MOLM-13, and SUM149. Comparison of CDC7 inhibitors with other oncology drugs in a panel of cancer cell lines revealed a unique mechanism of action. In vivo, our compounds reduced tumor cell MCM2 (S53) phosphorylation in the mouse COLO205 xenograft model and showed strong tumor growth inhibition. We have also examined the effect of CDC7 inhibitors on cancer cell proliferation in combination with other anti-cancer agents, including other DNA damage response (DDR) targeting agents. Conclusions: We have identified novel potent ATP-competitive CDC7 inhibitors that show target engagement in cells and CDX tumors and have shown strong inhibition of cancer cell proliferation in vitro and tumor growth in vivo. CDC7 inhibitors show promise for use in combination with other targeted therapies for the treatment of cancers of varying origin. Citation Format: Lyuben Tsvetkov, Adam Levinson, Xianhai Huang, Sayan Mondal, Jeff Bell, Lin Tang, Robert Pelletier, Karen Dingley, Nick Boyles, Jackson Chief Elk, Leah Frye, Alan Futran, Phani Ghanakota, Jeremy Greenwood, George Lai, Sarah Silvergleid, Wu Yin, Hamish Wright, Karen Akinsanya, Wayne Tang, Kristian Jensen. Discovery of novel CDC7 inhibitors that disrupt cell cycle dynamics and show anti-proliferative effects in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1277.

Abstract 1180: A novel synthetic lethality treatment strategy for metastatic breast cancer

Abstract Breast carcinomas commonly carry mutations in the tumor suppressor p53, while therapeutic efforts to target mutant p53 were largely unfruitful. Here we present preclinical data supporting a novel combination therapy strategy for treatment of p53-deficient cancers. Genomic data revealed a high expression activity of Base-Excision Repair (BER) pathways in p53-deficient breast cancers. However, we found that BER-mediated repair was significantly dysregulated in p53-mutant cancer cells. Treatment with deoxyuridine analogues induced accumulation of DNA damage in p53-mutant cells and inhibitors of poly (ADP-ribose) polymerase (PARPi) greatly enhanced this response. In contrast, normal cells responded to PARPi with activation of the p53-p21 axis and cell cycle arrest. Inactivation of either p53 or p21/CDKN1A in p53 wild-type cells conferred the p53-mutant phenotype. Preclinical breast cancer studies revealed that the combination of deoxyuridine analogue with PARPi was more effective in inhibition of tumor growth and metastases than either drug alone. This work illustrates a novel combination therapy strategy that may improve survival rates and outcomes for thousands of breast cancer patients. Citation Format: Justin Zonneville, Moyi Wang, Mohammed Alruwaili, Kevin Eng, Thomas Melendy, Ben Ho Park, Renuka Iyer, Christos Fountzilas, Andrei V. Bakin. A novel synthetic lethality treatment strategy for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1180.

Functional Implication of p53-Mediated DNA Damage Response on the B-Cell Lymphoma Microenvironment Interaction in Chemoimmunotherapy

Resistance against chemotherapy remains a challenge in the current treatment of B cell malignancies. In previous work we could show that macrophages are an essential element in the synergistic response to the administration of the DNA damaging agent Cyclophosphamide and monoclonal antibodies. The combination treatment strongly increases tumor clearance overall survival in the treatment refractory humanized BCL2-MYC-dependent double hit lymphoma hMB mouse model.Here we observed that the alkylating agent Cyclophosphamide is inducing the secretion of distinct cytokines like VEGF-A, CCL4, IL8, IL10 and TNFα by the leukemic cells. This in turn leads to a repolarization of tumor associated macrophages from a suppressive to an activated phenotypic state, which is designated as acute secretory activating phenotype (ASAP). We analyzed the functional implications of the DNA damage response pathway for the generation of the ASAP and synergy in chemoimmunotherapy. When disrupting DNA damage response pathway by knock-down of key elements like ATM, DNA-PK or p53 in the leukemic cells we could prevent the formation of the stimulatory ASAP effect on macrophage phagocytic capacity. Particularly p53 status seems to possess a central regulating role on macrophage mediated malignant cell depletion. TP53 activation via Nutlin-3A treatment of lymphoma cell enhances ADCP in in p53 wildtype cells, while not displaying enhancement in p53-deficient lymphoma cells. Utilizing the hMB model for modeling in vivo treatment of lymphoma bearing mice we could demonstrate diminished ASAP and ADCP for p53-deficient lymphoma treated with cyclophosphamide in vivo . Using primary human CLL patient cells comparing both wildtype and p53-deficient status, the p53-deficient CLL cells failed to induce the stimulatory, cytokine-mediated effect on macrophage phagocytosis in response to combination treatment as seen with the p53 proficient CLL cells. This observation is underlined by in vivo modeling of CLL therapy by treating Eµ-TCL1/p53wt/wt as well as Eµ-TCL1p53-/- mice. Here, low-dose cyclophosphamide treated Eµ-TCL1p53-/- mice failed to induce an antibody mediated stimulatory effect on macrophage phagocytosis capacity as seen with Eµ-TCL1/p53wt/wt mice.In this work, we are able to recapitulate the effect of ASAP mediated increase of macrophage mediated antibody dependent cellular phagocytosis in the Eµ-Tcl1 mouse model and therefore demonstrate that the ASAP is not only restricted to double hit lymphoma, but also shows functional relevance in low-grade lymphoma such as CLL. Moreover, we indicate that p53 functional status influences the generation of the ASAP in independent models in vitro and in vivo as in primary CLL patient cells. Mediated by this process p53 regulates the interraction with tumor asociated macrophages and the tumor microenvironment.Finally our ongoing research offers possibility to reveal and tailor new treatment approaches for chemorefractory patients. DisclosuresHallek:AbbVie: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding.

Downregulation of BMI-1 By the Small Molecule PTC596 Induces Mitochondrial Apoptosis in Mantle Cell Lymphoma

▪Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma typically associated with the t(11;14) translocation, resulting in overexpression of cyclin D1 (CCND1). Although the clinical approval of the first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a new treatment option, the great majority of patients with MCL remain incurable. Studies are needed to optimize the role of ibrutinib in MCL therapy and to address ibrutinib resistance. BMI-1 is crucially involved in cancer stem cell self-renewal and maintenance. BMI-1 has been shown to be up-regulated in MCL cells, especially in tumor-initiating side population (SP) cells (Teshima et al. Oncogene 2014). Importantly, BMI-1 levels are higher in refractory/relapsed patients than those at initial diagnosis. PTC-209 and PTC596 are recently developed novel small-molecule selective BMI-1 inhibitors with different modes of action (Kreso et al. Nat Med 2014). PTC-209 inhibits BMI-1 production while PTC596 accelerates BMI-1 degradation. We have reported anti-leukemia effects of the BMI-1 inhibitors PTC-209 and PTC596 (Cancer Sci 2015; Blood Cancer J 2017) in acute myeloid leukemia cell lines and patient-derived leukemia blasts. In this study, we investigated the effects of PTC-209 and PTC596 in MCL. Oncomine analysis showed that BMI1 mRNA levels were higher in MCL cells than in normal B cells. The levels were not specifically associated with CCND1 levels, ATM, CDKN2A or TP53 mutations, or patient prognosis, implying that BMI1 overexpression may be a fundamental abnormality in MCL. PTC-209 and PTC596 inhibited cell growth and induced apoptosis in 8 MCL cell lines in a dose- and time-dependent manner. Their IC50 values (concentration at which cell growth is inhibited by 50%) ranged from 1.5 to 11.2 µM for PTC-209 and from 68 to 340 nM for PTC596. ED50 values (effective concentration inducing 50% cell killing as measured by annexin V positivity) ranged from 2.7 to > 50 µM for PTC-209 and from 150 to 507 nM for PTC596. PTC596 was > 10 times more potent than PTC-209. IC50 and ED50 values of PTC-209 significantly correlated with those of PTC596 [r =0.94 (P= 0.0004) for IC50 and r =0.85 (P= 0.015) for ED50], supporting the idea that their anti-lymphoma activities depend on downregulation of BMI-1. In support of this, high BMI-1 protein levels predicted high susceptibility to PTC596 (r = -0.88; P=0.0039). The anti-lymphoma effects of PTC596 appeared to be p53-independent since IC50 and ED50 values were not significantly different against p53 wild-type and p53 defective cell lines. In addition, stable knockdown of p53 did not significantly affect MCL cell susceptibility to PTC596. Activity of PTC596 was independent of MCL cell susceptibility to ibrutinib. PTC596 induced mitochondrial apoptosis, as evidenced by loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation and phosphatidylserine externalization in MCL cells. PTC596 effectively decreased SP cells (by 97.3% at 150 nM, 99.8% at 300 nM and >99.9% at 600 nM PTC596) compared to ibrutinib that decreased SP cells by 33% even at 5 µM ibrutinib. Combination of PTC596 with bortezomib or doxorubicin showed additive anti-proliferative and apoptotic effects. The averaged combination index values were approximately 1, ranging from 0.95 to 1.18. Interestingly, PTC596 synergized with ibrutinib in inducing cell death, through modulation of the BCL-2 family protein MCL-1. Collectively, BMI-1 inhibition may offer a novel therapeutic strategy for MCL. DisclosuresLiangl:PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment. Branstrom:PTC Therapeutics: Employment.

BET Inhibitors in Chronic Lymphocytic Leukemia: JQ1 Synergizes with Venetoclax in Promoting Apoptosis

▪Background: The bromodomain and extra terminal BET family proteins (BRD2, BRD3, BRD4 and the testis-specific BRDT) play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Specifically, BET inhibitors block the transcription of key oncogenes (BCL2, C-MYC and CDK6) through the displacement of BRDs and other epigenetic modifiers from chromatin. However, while BET inhibitors were consistently described as highly effective drugs in in vitro experiments, few reports and clinical trials have already demonstrated the rapid emergence of BET inhibitors resistance.Aim: We examined the efficacy of the BET inhibitor JQ1 in CLL samples, upon short and prolonged treatments, to assess its efficacy and the potential development of resistances.Methods: MEC-1/EHEB CLL cell lines (p53 wild-type EHEB and p53-mutated MEC-1 cell lines) and primary CD19 positive lymphocytes from CLL patients were subjected to short and prolonged treatment with the BET inhibitor JQ1. Responses were measured in terms of proliferation, cell-cycle and apoptosis.Results: Treatment of CLL cell lines with the BET inhibitor JQ1 was associated with block of proliferation, cell cycle arrest and apoptosis. Molecularly, JQ1 treatment was associated to c-Myc down-modulation. Notably, treatment with JQ1 caused also loss of viability of primary patient-purified CLL cells. However, starting from 48 hours of JQ1 treatment in CLL cell lines, we observed a compensatory increase of BRDs mRNA levels, which was associated to the development of resistant clones after longer treatments (30 days). These data demonstrate that resistance to BET inhibitors can rapidly emerge and suggest that a combination therapy with other drugs should be explored. Interestingly, we observed that JQ1 was also able to modulate BCL-2 expression in CLL. We therefore assessed the effect of a combination therapy based on JQ1 in association with the BCL-2 inhibitor, Venetoclax. Intriguingly, co-treatment with JQ1 and Venetoclax significantly increased apoptosis demonstrating a synergic action.Conclusions: Our data demonstrated for the first time that: i) CLL may be effectively targeted by BET inhibitors; ii) short term treatment with BET inhibitors rapidly results in the upregulation of additional BRD family members; iii) prolonged treatment is associated with the development of resistance; IV) BET proteins modulate BCL-2 and c-MYC expression also in CLL; V) the combination therapy based of JQ1 administration in association with Venetoclax is extremely effective in CLLDiscussion: Here, we show that JQ1 treatment results in growth inhibition and apoptosis of CLL cells by down-modulating the expression of the pro-survival genes, C-MYC and BCL-2. Furthermore, our data show that treatment with JQ1 is only effective for a short time while the co-treatment with Venetoclax exerts a synergistic lethality effect in CLL. These data suggest the efficacy of a combination therapy with JQ1 and Venetoclax in refractory TP53 mutated CLL patients. DisclosuresSaglio:Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Gambogenic Acid Enhances Chemosensitivity of Multiple Myeloma to Bortezomib through p53/ROS/p38 MAPK Apoptotic Pathway

Background: Multiple myeloma (MM) is the second most prevalent hematological cancer (10%) with a five-year survival rate of only 49%. Approval of bortezomib, the first in-class proteasome inhibitor, has significantly improved outcomes of MM in the last few years; however, the almost inevitable refractory or resistance has made a cure of MM remains out of reach. Identification and validation of novel effective targeted agents are necessary. As one of the natural compounds isolated from Traditional Chinese Medicine gamboge, gambogenic acid (GNA) is a very promising antitumor agent and has been proved to have more potent anticancer effect and less systemic toxicity according to early investigations. In our previous studies, we have demonstrated that GNA could synergistically potentiate bortezomib-induced apoptosis of MM. In this study, our aim was to further explore and understand the concrete apoptotic action mechanism of GNA and bortezomib combination treatment against MM in pre-clinical models.Methods: We performed pre-clinical studies in myeloma cell lines and mouse models treated by GNA and bortezomib, alone and in combination. Cell counting kit-8 (CCK-8) assay, combination index (CI) isobologram, flow cytometry (FCM), western blot, xenograft tumor models, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemistry (IHC) and oxidation-sensitive fluorescent dye carboxy-DCFDA were applied in this study. The thiol antioxidant N-acetylcysteine (NAC) and p38 MAPK inhibitor SB203580 were used to detect the effect of GNA and bortezomib combination therapy on ROS, p53 and p38 MAPK.Results: Both p53 wild type cell line MM.1S and mutant type cell line RPMI 8226 myeloma cell lines were sensitive to GNA therapy. Interestingly, GNA only enhanced the chemosensitivity of bortezomib on p53 wild type cell line MM.1S. Both 0.51μM GNA combined with 2.3nM bortezomib and 0.90μM GNA combined with 4.0nM bortezomib to MM.1S cells showed a more markedly apoptotic rate of MM compared with that of the single agent group of each accordingly (p <0.01; p <0.01), and the apoptosis was presented in a dosage- and time-dependent manner. Increased apoptosis of MM.1S cells was found via the activation of PARP cleavage, p53, Caspase-3 cleavage and Bax and inhibition of Bcl-2 expression. An increased antitumor effect of combination therapy of GNA and bortezomib on MM.1S xenograft models were also observed. A significant generation of ROS was detected after exposure of GNA and bortezomib in MM.1S cells. The expression level of phospho-p38MAPK was upregulated by combination therapy compared with that of single agent (p<0.05; p<0.05). To characterize the mechanisms of action of GNA in combination with bortezomib, we pretreated MM.1S cells with NAC and SB203580. We found that the apoptosis of MM.1S cells could be inhibited by both NAC and SB203580 (p<0.01; p<0.01). However, the generation of ROS could only be inhibited by NAC (p<0.05), and the elevated expression of p53 could be inhibited neither by NAC nor SB203580. These results indicated that p53/ROS/p38 MAPK pathway is a pre-requisite for GNA to accomplish the enhancement of chemosensitivity of MM to bortezomib.Conclusions: We demonstrate that GNA could potentiate apoptosis of MM induced by bortezomib in vivo and in vitro, and p53/ROS/p38 MAPK pathway plays a central role of the apoptotic action. Thus, our data provide a persuasive rationale for the successful utilization of dual GNA and bortezomib for chemotherapy in MM patients in the future. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Anti-Lymphoma Activity of the BCL-2 BH4 Domain Antagonist Bda-366 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell non-Hodgkin lymphoma. MCL is genetically characterized by t(11;14) translocation, which leads to cyclin D1 overexpression. Despite the introduction of high-dose chemotherapy followed by autologous stem cell transplantation and novel agents including the Bruton's tyrosine kinase inhibitor ibrutinib, the proteasome inhibitor bortezomib and the immunomodulator lenalidomide, MCL still remains an incurable disease. BCL-2 is often amplified and/or overexpressed in MCL; high-level copy number gains of the BCL2 locus at 18q21.3 are most frequent. The novel BCL-2BH4 domain antagonist BDA-366 converts anti-apoptotic BCL-2 into the pro-apoptotic molecule (Han et al., Cancer Cell 2015). Considering the promising clinical activity of the BH3 mimetics venetoclax (Davids et al., JCO 2017), we investigated anti-lymphoma effects of BDA-366 in MCL. A total of eight MCL cell lines (Z-138, JVM-2, Granta-519, MINO, JeKo-1, REC-1, MAVER-1 and NCEB-1) were used. Z-138, Granta-519, MINO and NCEB-1 have high-level DNA amplification of BCL-2 . BDA-366 induced dose- and time-dependent phosphatidylserine externalization in MCL cells, with 72-h ED50 (effective concentration inducing 50% cell killing as measured by annexin V positivity) values ranging from 9.3 to 240 nM. 72-h IC50 values (concentration at which cell growth is inhibited by 50%) were also determined, which ranged from 5 to 30 nM. BDA-366 showed smaller differences in sensitivity than venetoclax, raising the possibility that BDA-366 may exhibit broader anti-lymphoma activity against heterogeneous MCL backgrounds. Basal levels of BCL-2 (determined by flow cytometry) did not significantly affect MCL cell susceptibility (ED50) to BDA-366 (r = -0.08, P = 0.85). The presence of high-level DNA amplification of BCL-2 was not associated with BDA-366 sensitivity either. BDA-366 did not increase total BCL-2 levels but it actively promoted the exposure of pro-apoptotic BH3 domain of BCL-2, supporting its proposed mode of action. BDA-366 did not significantly affect cell cycle distribution; S and G2/M phase cells appeared to be susceptible to BDA-366-induced apoptosis. BDA-366 induced loss of mitochondrial membrane potential, caspase-3 cleavage and BAX activation, indicating mitochondrial apoptosis. p53 knockdown (>85% knockdown efficiencies, as determined by Western blotting) desensitized p53 wild-type cell lines Z-138 and JVM-2 to BDA-366 (ED50 values of 29 nM and 10 nM in Z-138; 303 nM and 108 nM in JVM-2), implying the interference of p53 with BCL-2 signaling. BDA-366 synergized with venetoclax to induce apoptosis in venetoclax-sensitive Z-138 (averaged combination index (aCI) = 0.25) and MINO cells (aCI = 0.14), indicating that the combination may abrogate BCL-2-mediated survival signaling in MCL. The combination effects of BDA-366 with doxorubicin or AraC were additive (aCI ~ 1.0). BDA-366 induced apoptosis in primary leukemia cells from patients with B-cell chronic lymphoproliferative disorders. BDA-366 appeared to take longer time to fully induce apoptosis than ABT-199, perhaps reflecting the distinct mode of action of BDA-366 that changes BCL-2 conformation. From a clinical viewpoint, the slower pharmacodynamics in inhibiting BCL-2 and inducing MCL cell death may reduce the risk of tumor lysis syndromes in patients receiving BDA-366. Collectively, BCL-2 BH4 inhibition may offer a novel therapeutic strategy for MCL. DisclosuresNo relevant conflicts of interest to declare.