TP53-null Cell Research Articles

Thymidylate Synthase Inhibition with Capped F10 Is Effective in TP53 Null Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is an aggressive cancer of the myeloid linage characterized by progressive marrow failure and death. Responses to therapy are between 50 and 80% but relapse is common and salvage rates once relapsed are low. The risk of relapse is greatly influenced by specific somatic mutations. One of the highest risk mutations in AML involves loss of the tumor suppressor TP53. Patients with AML harboring loss of TP53 function have a dismal prognosis and there is a clear need for new approaches. To identify novel agents with activity against TP53 null AML, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 in a genetically defined murine AML cell line. Clonal isolates were generated by serial dilution and loss of detectable TP53 was confirmed by Western blotting. A high throughput screen of currently FDA approved agents was performed on these cell lines. When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents either directly targeted thymidylate synthase or its substrates via inhibition of the folate cycle. This finding suggests that thymidylate synthase (TS) is a key enzyme in TP53 null AML cells. To confirm and extend these results, an additional murine AML TP53 null/WT cell line pair was generated. Chemotherapy resistance was confirmed in the TP53 null cell lines when treated with doxorubicin (Figure 1). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that is a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (Figure 1). To extend this result TP53 null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly or doxorubicin 3mg/kg and cytarabine at 100 mg/kg daily for 5 days. CF10 provided a significant survival benefit in the TP53 null AML in this highly aggressive model where doxorubicin and cytarabine did not (p=0.0057, Figure 2). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and containing a GFP reporter. When the mixed population was treated with a titration of doxorubicin there was a highly significant increase in GFP+ cells indicating the involvement of TP53 in doxorubicin mediated cell death. In contrast when cells were treated with a titration of CF10 there was a no significant increase in GFP+ population consistent with CF10 inducing a TP53 independent mechanism of cell death. For CF10 to of use clinically there must be an acceptable therapeutic window. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg to mimic DPD deficiency. Animals were then treated with repeated doses of either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals maintained their weight compared to controls (data not shown). Taken together these data suggest that TS is a viable target in TP53 null AML and CF10 is well tolerated and provides a survival benefit in a highly aggressive, chemotherapy resistant model. Ongoing studies will explore the mechanisms involved in CF10 induced cell death.

Capped F10, a novel DNA directed fluoropyrimidine, in TP53 mutated acute myeloid leukemia (AML).

7046 Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by resistance to treatment and poor outcomes. One of the most devastating mutations in AML involves loss of the tumor suppressor TP53. AML harboring this loss carries a dismal prognosis and there is a clear need for new approaches. Methods: To identify new therapeutics, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 using a murine AML cell line. Clonal isolates were obtained and loss of detectable TP53 was confirmed by Western Blotting. A high throughput screen of currently FDA approved anticancer agents was performed. Results: When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents targeted the folate/thymidylate synthase axis. This suggests that thymidylate synthase (TS) is a vulnerability in TP53 null AML cells. To confirm TS as a target in TP53 null AML an additional murine AML TP53 null/WT cell line pair was generated using CRISPR/CAS9. When these cell lines were treated with doxorubicin the TP53 null cells were significantly more resistant (p≤0.001 at 10nM). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that has been shown to be a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (p = 0.31 at 50pM). To extend this result TP53 WT or null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly. CF10 provided a significant survival benefit in the TP53 null but not WT injected mice consistent with CF10 having activity against TP53 null AML (p = 0.035 vs 0.13). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and a GFP reporter. When the mixed population was treated with the IC90 concentration of 5-fluorouracil there was a highly significant increase in GFP+ cells indicating a TP53 mediated cell death (p = < 0.001). In contrast when cells were treated with the IC90 concentration of CF10 there was a non-significant increase in GFP+ population (p = 0.074) consistent with a mainly TP53 independent mechanism. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg alone or in combination with either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals did not have a significant change in weight compared to controls. Conclusions: Taken together these data suggest that CF10 is a promising new agent for TP53 null AML.

27 Entinostat restores responsiveness to olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 μM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 μM Entinostat, 10 μM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicletreated controls (p=0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p<0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer.

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

Abstract Introduction: Most cancers proliferate by activating telomerase (TA+) while 10% of cancers utilize alternate lengthening of telomeres (ALT). ALT has been associated with resistance to DNA damaging agents, p53 loss-of-function (p53LOF), ATRX mutations, and very poor survival. ATM kinase, which activates functional p53, is constitutively activated in ALT cancers (Science Translational Medicine 18:eabd5750, 2021). We hypothesized that the constitutive activation of ATM kinase in ALT cancers would confer high sensitivity to pharmacological reactivation of p53 function. Methods: We used patient-derived ALT (telomeric DNA C-circle positive) and TA+ neuroblastoma, sarcoma, colorectal, and breast cancer cell lines and xenografts treated with the clinical-stage p53 reactivator eprenetapopt (APR-246) and irinotecan. In vitro cytotoxicity was assayed by DIMSCAN digital imaging microscopy, ATM activation by immunofluorescence microscopy, and protein expression by western blotting. Results: ALT p53LOF cell lines were significantly (p&amp;lt;0.05) less sensitive to DNA-damaging agents relative to TA+ p53LOF comparators. Constitutive phosphorylation (activation) of ATM kinase and the DNA damage marker 53BP1 were observed at telomeres in ALT but not TA+ cell lines. APR-246 induced p53 targets p21 and NOXA and was significantly more cytotoxic (p&amp;lt;0.001) for ALT relative to TA+ cell lines, regardless of TP53 status. Overexpression of p53 in a TP53-null ALT cell line increased sensitivity (p&amp;lt;0.0001) to APR-246. Knockdown of p53 or ATM kinase in ALT TP53 mutated and wild-type cell lines antagonized (p&amp;lt;0.0001) APR-246 activity. Induction of telomere dysfunction in a TA+ p53LOF neuroblastoma cell line using dominant-negative TRF2 (a shelterin protein that blocks ATM activation at telomeres) activated ATM and sensitized cells to APR-246 (p&amp;lt;0.01). APR-246 enhanced the cytotoxicity of irinotecan (as SN38) in ALT cell lines in vitro to a higher degree than in TA+ p53LOF cell lines (p&amp;lt;0.05). Single-agent APR-246 significantly (p&amp;lt;0.0001) increased event-free survival (EFS) of mice with ALT xenografts relative to controls. APR-246 enhanced (p&amp;lt;0.0001) the activity of irinotecan in 3 neuroblastoma, 2 rhabdomyosarcoma, 1 colorectal, and 1 triple-negative breast ALT xenograft models with most mice (47/56) achieving complete responses and an EFS of &amp;gt;100 days (45/56) compared to no complete responses (median EFS ~ 34 days) in mice treated with only irinotecan (p&amp;lt;0.0001). APR-246 + irinotecan had no significant effect relative to irinotecan alone (p=0.08) on EFS of mice with TA+ p53LOF xenografts (median EFS ~ 40 days). Conclusion: ALT cancers of a variety of histologies are highly resistant to DNA damaging agents, have p53LOF, and constitutive activation of ATM kinase, which confers high sensitivity to p53 reactivation by APR-246. APR-246 warrants clinical testing in patients with ALT cancers. Citation Format: Shawn J. Macha, Balakrishna Koneru, Trevor Burrow, Charles Zhu, Dzmitry Savitski, Jonas Nance, Kristyn McCoy, Cody Eslinger, C Patrick Reynolds. APR-246, which restores p53 function, is highly active against alternative lengthening of telomere (ALT) cell lines and PDXs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6228.

Abstract 795: Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 µM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 µM Entinostat, 10 µM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicle-treated controls (p= 0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p&amp;lt;0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p&amp;lt; 0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer. Citation Format: Tyler Woodard, Viju Gupta, Simona Miceska, Bisiayo Fashemi, Wendy Zhang, Dineo Khabele. Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer 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 795.

Cas9 activates the p53 pathway and selects for p53-inactivating mutations.

Cas9 is commonly introduced into cell lines to enable CRISPR/Cas9-mediated genome editing. Here we studied the genetic and transcriptional consequences of Cas9 expression per se. Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon Cas9 introduction, specifically in TP53-WT cell lines. This was confirmed at the mRNA and protein levels. Moreover, elevated levels of DNA repair were observed in Cas9-expressing cell lines. Genetic characterization of 42 cell line pairs showed that Cas9 introduction can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53-WT/TP53-null cell lines. Lastly, Cas9 was less active in TP53-WT than in TP53-mutant cell lines, and Cas9-induced p53 pathway activation affected cellular sensitivity to both genetic and chemical perturbations. These findings may have broad implications for the proper use of CRISPR/Cas9-mediated genome editing.

The cancer-associated, gain-of-function TP53 variant P152Lp53 activates multiple signaling pathways implicated in tumorigenesis

TP53 is the most frequently mutated tumor suppressor gene in many cancers, yet biochemical characterization of several of its reported mutations with probable biological significance have not been accomplished enough. Specifically, missense mutations in TP53 can contribute to tumorigenesis through gain-of-function of biochemical and biological properties that stimulate tumor growth. Here, we identified a relatively rare mutation leading to a proline to leucine substitution (P152L) in TP53 at the very end of its DNA-binding domain (DBD) in a sample from an Indian oral cancer patient. Although the P152Lp53 DBD alone bound to DNA, the full-length protein completely lacked binding ability at its cognate DNA motifs. Interestingly, P152Lp53 could efficiently tetramerize, and the mutation had only a limited impact on the structure and stability of full-length p53. Significantly, when we expressed this variant in a TP53-null cell line, it induced cell motility, proliferation, and invasion compared with a vector-only control. Also, enhanced tumorigenic potential was observed when P152Lp53-expressing cells were xenografted into nude mice. Investigating the effects of P152Lp53 expression on cellular pathways, we found that it is associated with up-regulation of several pathways, including cell-cell and cell-extracellular matrix signaling, epidermal growth factor receptor signaling, and Rho-GTPase signaling, commonly active in tumorigenesis and metastasis. Taken together, our findings provide a detailed account of the biochemical and cellular alterations associated with the cancer-associated P152Lp53 variant and establish it as a gain-of-function TP53 variant.

ENHANCING EXPRESSION OF PRO-APOPTOTIC FACTORS THROUGH INHIBITION OF HDACS DRIVES P53-INDEPENDENT SYNERGY WITH VENETOCLAX FOR THE TREATMENT OF AML

Targeting BCL-2 with the BH3-mimetic venetoclax (VEN) has emerged as a highly efficacious treatment for elderly patients with acute myeloid leukaemia (AML), in combination with either low-dose Ara-C (Wei, JCO 2019) or hypomethylating agents (Di Nardo, Lancet Oncol 2018). Long-term survival outcomes remain poor among patients with adverse risk karyotype, who often harbour mutations in the TP53 gene. Therefore, identification of effective treatment options, with TP53 independent activity, represents an area of high unmet therapeutic need. We generated TP53-null AML cell lines using CRISPR-Cas9 gene editing and screened these cell lines against 48 compounds. The histone deacetylase inhibitors (HDACi) had substantial efficacy. Additional studies performed in factor dependent myeloid (FDM) cells demonstrated cell death from HDACi to be Bax/Bak dependent, but Bim and p53 independent. RNA-sequencing, following HDACi treatment, revealed a rapid upregulation of pro-apoptotic factors, including PMAIP1 (NOXA) and BBC3 (PUMA). These results indicate that treatment with HDACi upregulates pro-apoptotic factors, independently of P53, and led to our hypothesis that HDACi could sensitise tumor cells to BH3-mimetics. We then tested the activity of HDACi in combination with BH3-mimetics targeting BCL-2 against 61 primary AML patient samples. Overall, LC50 at 48h was reduced by >2 logs in 60% of cases. Importantly, TP53 mutant cases were sensitive to this combination. Patient-derived xenograft models confirmed that this combination was tolerable and able to suppress bone marrow AML in vivo. Our data supports the clinical use of combined HDAC and BCL-2 targeting for AML and represents a promising treatment option for patients with adverse risk genomic characteristics; in particular, those who have failed VEN treatment in combination with low dose chemotherapy, and cases defective in P53.

Effect of CHK1 Inhibition on CPX-351 Cytotoxicity in vitro and ex vivo

CPX-351 is a liposomally encapsulated 5:1 molar ratio of cytarabine and daunorubicin that recently received regulatory approval for the treatment of therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes based on improved overall survival compared to standard cytarabine/daunorubicin therapy. Checkpoint kinase 1 (CHK1), which is activated by DNA damage and replication stress, diminishes sensitivity to cytarabine and anthracyclines as single agents, suggesting that CHK1 inhibitors might increase the effectiveness of CPX-351. The present studies show that CPX-351 activates CHK1 as well as the S and G2/M cell cycle checkpoints. Conversely, CHK1 inhibition diminishes the cell cycle effects of CPX-351. Moreover, CHK1 knockdown or addition of a CHK1 inhibitor such as MK-8776, rabusertib or prexasertib enhances CPX-351-induced apoptosis in multiple TP53-null and TP53-wildtype AML cell lines. Likewise, CHK1 inhibition increases the antiproliferative effect of CPX-351 on primary AML specimens ex vivo, offering the possibility that CPX-351 may be well suited to combine with CHK1-targeted agents.

Clinical Impact and Therapeutic Opportunity of Insulin Receptor Substrates 1/2 in Acute Myeloid Leukemia

Background: The identification of biological and clinical prognostic factors in acute myeloid leukemia (AML) allowed the definition of patient subgroups and the realization of risk-adapted and targeted treatment strategies. Insulin-like growth factor 1 receptor/Insulin Receptor Substrates (IGF1R/IRS) pathway plays an important role in the development of neoplasia. IRS1/2 activates AKT/mTOR and MAPK pathways, through their interaction with PI3K and GRB2, culminating in increasing cell proliferation. NT157 is an allosteric inhibitor of IGF1R-IRS1/2 signaling that showed antineoplastic effects in preclinical studies of solid tumors. However, IRS1/2 clinical function and NT157 effects were not assessed in AML. Aims: To investigate IRS1 and IRS2 mRNA expression in AML patients and their impact in clinical outcomes, and to analyze the effects of the NT157 in AML cell lines. Material and methods: Comparison of IRS1 (probe nº 204686) and IRS2 (probe nº209184_s) expression from 581 AML patients and 8 CD34+ cells from healthy subjects were analyzed using data from Amazonia! Platform. For survival analysis, IRS1 and IRS2 mRNA expression levels from 173 AML patients (92 male - median age 58 years [range: 18-65]) were obtained from TCGA AML study available online on CBioPortal for Cancer Genomics. NB4, NB4-R2, Kasumi-1 and THP1 cell lines were submitted to NT157 (0.5, 1.0, 2.0, 4.0, 8.0 or 16 µM) 72 hours and evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), cell cycle (PI), ROS production (DCFDA), mitochondria staining (MitoTracker), and protein expression/activation (western blot). Bone marrow mononuclear cells (BMMC) were obtained from 4 AML patients at diagnosis and submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal-Wallis and Spearman correlation tests, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with the log-rank test. Cox regression analysis was also applied. Results: IRS1 expression, but not IRS2, predicted outcomes. Reduced IRS1 expression showed poorer disease-free survival (DFS) (survival median time [MT]: 10.1 months [mos] vs. 28.4 mos, P<0.001; Hazard ratio [HR]: 0.51 [CI95:0.32 - 0.79]) and overall survival (OS) (MT: 14.5 mos vs. 27.4 mos, P=0.009; HR: 0.61 [CI95:0.42 - 0.88]). IRS1 expression independently predicted poorer DFS (HR: 0.59 [CI95: 0.36 - 0.79]; P= 0.03) using cytogenetic risk stratification, age and leukocytes as confounders. Of note, IRS1 level was positively correlated with proapoptotic CD27 (r=0.51; P<0.001) and with IL17RA (r=0.62; P<0.001) related to CD34 cell differentiation. IRS2 expression was upregulated in AML harboring t(15;17) (n=36; P<0.01) and inv(16) (n=37; P=0.01) in comparison to CD34+. In NB4, NB4-R2 and Kasumi-1 cells, NT157≥0.5µM reduced cell viability (P<0.05) and increased apoptosis (P<0.05). The mean percentage of annexin V+ cells for control, NT157 2.5, 5 and 10µM were 11, 47, 73 and 75% for NB4, 11, 41, 69 and 75% for NB4-R2 and 17, 45, 61 and 64% for Kasumi-1, respectively. In TP53-null cell line THP1, NT157 reduced cell viability at doses higher than 2µM (P<0.05) and induced apoptosis at 10µM (9.1 vs. 25%; P<0.05). NT157 induced ROS production in NB4 (fold-increase of mean fluorescence intensity [MFI]: 25.8 and 24.8), NB4-R2 (MFI: 26.7 and 31.4), Kasumi-1 (MFI: 5.8 and 6.6) and THP1 (MFI: 1.8 and 4.1) at 5 and 10µM (all P<0.05) and increased mitochondrial mass in NB4 (MFI: 3.9 and 3.7), NB4-R2 (MFI: 2.6 and 2.9), Kasumi-1 (MFI: 3.2 and 4.7) and THP-1 (MFI: 2.6 and 2.2) (all P<0.05). NT157 also modulated cell cycle progression, as evidenced by G2/M arrest in THP-1 and sub-G0/G1 in other cell lines (P<0.05). The IGF1R-IRS1/2 inhibitor NT157 reduced activation/expression of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70), ERK1/2 (Thr185/Tyr187) and induced DNA damage (increased γH2AX). NT157 did not induce autophagy, as demonstrated by non-degradation of p62 and lack of conversion of LC3BI into LC3BII in cell lines tested. NT157≥0.5 µM reduced cell viability and induced apoptosis in BMMC from 4 AML patients in a dose dependent manner (P<0.05). Conclusions: In AML, downregulation of IRS1 predicted dismal prognosis and the IGF1R-IRS1/2 inhibitor NT157 exerted an antineoplastic activity, downregulated PI3K/AKT and MAPK signaling. IRS1/2 arises as a promising therapeutic target for AML patients. DisclosuresNo relevant conflicts of interest to declare.

TP53 is required for BECN1- and ATG5-dependent cell death induced by sphingosine kinase 1 inhibition

ABSTRACTThe bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the enzyme that produces it, SPHK1 (sphingosine kinase 1), regulate many processes important for the etiology of cancer. It has been suggested that SPHK1 levels are regulated by the tumor suppressor protein TP53, a key regulator of cell cycle arrest, apoptosis, and macroautophagy/autophagy. However, little is still known of the relationship between TP53 and SPHK1 activity in the regulation of these processes. To explore this link, we examined the effects of inhibiting SPHK1 in wild-type and TP53 null cancer cell lines. SK1-I, an analog of sphingosine and isozyme-specific SPHK1 inhibitor, suppressed cancer cell growth and clonogenic survival in a TP53-dependent manner. It also more strongly enhanced intrinsic apoptosis in wild-type TP53 cells than in isogenic TP53 null cells. Intriguingly, SK1-I induced phosphorylation of TP53 on Ser15, which increases its transcriptional activity. Consequently, levels of TP53 downstream targets such as pro-apoptotic members of the BCL2 family, including BAX, BAK1, and BID were increased in wild-type but not in TP53 null cells. Inhibition of SPHK1 also increased the formation of autophagic and multivesicular bodies, and increased processing of LC3 and its localization within acidic compartments in a TP53-dependent manner. SK1-I also induced massive accumulation of vacuoles, enhanced autophagy, and increased cell death in an SPHK1-dependent manner that also required TP53 expression. Importantly, downregulation of the key regulators of autophagic flux, BECN1 and ATG5, dramatically decreased the cytotoxicity of SK1-I only in cells with TP53 expression. Hence, our results reveal that TP53 plays an important role in vacuole-associated cell death induced by SPHK1 inhibition in cancer cells.

Abstract B026: SNX-2112 interferes with mitochondrial metabolism in TP53 mutant tumors

Abstract Introduction: SNX-5422 is an orally active prodrug of SNX-2112, a potent, highly selective inhibitor of Hsp90 with epigenetic character that has shown antitumor activity in clinical trials. K562 cells have loss of p53 expression by nonsense-mediated mRNA decay. Since both loss of function TP53 mutations and MYC-driven tumors have been linked to aberrant cancer metabolism, including increased glycolysis, the effect of SNX-2112 on oncometabolic networks in the TP53 mutant chronic myelocytic leukemia K562 cell line was examined. Methods: The 50% inhibition concentration (IC50) of SNX-2112 on cell viability of cancer cell lines was determined using CellTiter-Glo® Luminescent Cell Viability Assay after incubation at various concentrations. All cells were cultured in media supplemented with 10% fetal bovine serum at 37°C, 5% CO2 and 95% humidity. Culture medium was purchased from GIBCO or Sigma, USA. Data from transcriptomics of the K562 cell line treated with SNX-2112 were obtained, and affected genes were analyzed for their roles in metabolic processes. Results: The IC50 values in TP53 null and in MYC-rearranged tumor cell lines obtained are shown in the table. Transcriptomics analysis of the effect of SNX-2112 on K562 onco-metabolism revealed that several genes involved in glycolysis (including AK2 and PCK2) and in the tricarboxylic cycle (e.g., DLST) were downregulated. SNX-2112 impaired the catabolism of branched-chain amino acids, as indicated by downregulation of BCAT, for example. Reductions were also observed in the following: ribosomal genes (MRPLs, MRPSs) associated with RNA translation; regulators of purine biosynthesis/one-carbon cycle (e.g., MTHFD1); and effectors of oxidative phosphorylation, including CYC1 and CYCS. Mitochondrial genes participating in carbohydrate (e.g., MECR, AUH) and protein metabolisms (MIPEP, SCO2), as well as redox and detoxification (e.g., SOD2, SUOX) pathways were downregulated. Conclusion: SNX-2112 demonstrated significant antitumor activity in TP53 null tumors and in rearranged MYC (8q24) hematologic and selected solid tumors (e.g., hepatocellular carcinoma, mesothelioma). This activity appears to be, in part, the result of interference with onco-metabolic pathways. Further research into this mechanism is ongoing to support this target in clinical trials. Cell LineTumor TypeIC50 (nM)TP53 null cell linesHep3BHepatocellular carcinoma7KATO IIIGastric carcinoma12NCI-H1299NSCLC - Adenocarcinoma8NCI-H358NSCLC - Adenocarcinoma10HL-60Acute promyelocytic leukemia31SK-MES-1NSCLC - SQCC10MEC-1Chronic lymphocytic leukemia5NCI-H520NSCLC - SQCC18RERF-LC-AINSCLC - SQCC54MYC-rearranged cell linesNAMALWABurkitt lymphoma4ST486Burkitt lymphoma4DaudiBurkitt lymphoma6RajiBurkitt lymphoma8CA46Burkitt lymphoma14SU-DHL-6DLBCL GCB - Double hit lymphoma6DOHH-2DLBCL GCB - Double hit lymphoma2WSU-DLCL-2DLBCL GCB - Richter’s FCL4NB-4Acute promyelocytic leukemia6AMO-1Multiple myeloma6MsTo 211HMesothelioma9NCI-H2452Mesothelioma7 Citation Format: Everardus Orlemans. SNX-2112 interferes with mitochondrial metabolism in TP53 mutant tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B026.

Abstract PR13: p53-related protein kinase is a novel prognostic marker and therapeutic target in multiple myeloma

Abstract Background: Multiple myeloma (MM) is a disease characterized by the accumulation of malignant plasma cells in the bone marrow. The introduction of novel drugs, such as immunomodulators (IMIDs), proteasome inhibitors, monoclonal antibodies, and HDAC inhibitors has revolutionized the approach to treat patients. Nevertheless, a better characterization of MM pathogenesis and of the mechanisms of action of current medications is needed to improve patient selection and hence outcomes, by increasing responses to therapies and reducing side effects. To this end, we have developed an approach based on genetic screening and drug characterization that allowed us to identify p53-related protein kinase (TP53RK) as a novel therapeutic target in MM. Methods: Gene expression profiling analysis was performed in primary MM cells and cell lines using Affymetrix U133 Plus 2.0 chips. Knockdown of proteins (TP53RK, c-Myc, p53, RRM1) was carried out using siRNA and/or shRNA. Expression of RNA and protein was assessed by real-time qPCR and immunoblotting, respectively. Cell proliferation was determined by live cell count and MTT assay. Electrophoretic mobility shift assay was used for evaluation of E2F1 activity. Pomalidomide-conjugating beads were generated for pull-down assay. Nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry were used to confirm pomalidomide-Pom binding to TP53RK. Results: We identified a novel protein, called TP53RK, that is expressed at higher levels in malignant plasma cells compared to normal plasma cells. In newly diagnosed MM patients higher TP53RK level confers poor prognosis. Downregulation of TP53RK induces apoptosis and inhibits cellular growth in multiple MM cell lines. TP53RK knockdown inhibits phosphorylation of TP53 (Ser15), promoting downregulation of MYC and E2F1-dependent upregulation of BIM. Importantly, we demonstrated by standard affinity chromatography and nuclear magnetic resonance spectroscopy that lenalidomide and pomalidomide bind to TP53RK affecting its activity, with biologic sequelae similar to TP53RK knockdown. Lenalidomide binds to cereblon (CRBN), a substrate adaptor of the CRL4 E3 ubiquitin ligase, and induces the ubiquitination and degradation of IKZF1 and IKZF3. CRBN knockdown does not alter TP53RK, or p53 axis; conversely, TP53RK downregulation does not modulate CRBN expression, indicating that lenalidomide and pomalidomide independently bind and regulate TP53RK and CRBN. TP53RK knockdown-induced MM cell growth inhibition was observed even in TP53-mutated and TP53-null MM cell lines, suggesting additional molecules targeted by TP53RK. Indeed, we identified RRM1, p18, proteasome inhibitor subunits, and telomerases as potential downstream targets. Hence, our studies demonstrate that either genetic or pharmacologic inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and -independent pathways. Furthermore, TP53RK blockade may be one of the novel mechanisms of action responsible for IMIDs' effects in MM. Conclusions: Our results identify TP53RK as a marker of poor prognosis in MM, validate TP53RK as a novel therapeutic target in this patient subset, and delineate a novel mechanism whereby IMIDs bind TP53RK, inhibit its activity, and trigger apoptotic MM cell death. Ongoing efforts are directed to developing selective TP53RK inhibitors to improve patient outcome in this patient subgroup. This abstract is also being presented as Poster 04. Citation Format: Francesca Cottini, Teru Hideshima, Yoshihisa Nozawa, Hiroto Ohguchi, Teruhiro Utsugi, Kenneth C Anderson. p53-related protein kinase is a novel prognostic marker and therapeutic target in multiple myeloma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR13.

Characterization of the new human pleomorphic undifferentiated sarcoma TP53-null cell line mfh-val2.

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm2, a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.

High throughput RNAi screening identifies ID1 as a synthetic sick/lethal gene interacting with the common TP53 mutation R175H

The TP53 mutation (R175H) is one of the most common mutations in human cancer. It is a highly attractive strategy for cancer therapy to find the genes that lead the R175H-expressing cancer cells. The aim of this study was to identify the synthetic sick/lethal gene interacting with R175H. Using lentiviral bar-coded comprehensive shRNA library and a tetracycline-inducible R175H expressed in the SF126 human glioblastoma cell line (SF126-tet-R175H), we conducted high-throughput screening to identify the candidate genes that induce synthetic sickness/lethality in R175H-expressing cells. We identified 906 candidate gene suppressions that may lead to accelerated cell growth inhibition in the presence of R175H. Inhibitor of differentiation 1 (ID1) was one of the candidate genes, and its suppression by siRNA resulted in the acceleration of growth inhibition in cell lines both transiently and endogenously expressing R175H but not in TP53-null cell lines or other common p53 mutants (such as R273H). Flow cytometry analysis showed that ID1 suppression resulted in G1 arrest, and the arrest was accelerated by the expression of R175H. ID1 is a synthetic sick/lethal gene that interacts with R175H and is considered to be a novel molecular target for cancer therapy in R175H-expressing cells.

Attenuation of Soft-Tissue Sarcomas Resistance to the Cytotoxic Action of TNF-α by Restoring p53 Function

BackgroundIsolated limb perfusion with TNF-α and melphalan is used with remarkable efficiency to treat unresectable limb sarcomas. Here we tested the ability of TNF-α to directly induce apoptosis of sarcoma cells. In addition, we investigated the impact of p53 in the regulation of such effect.Methodology/Principal FindingsWe first analysed the ability of TNF-α to induce apoptosis in freshly isolated tumour cells. For this purpose, sarcoma tumours (n = 8) treated ex vivo with TNF-α were processed for TUNEL staining. It revealed substantial endothelial cell apoptosis and levels of tumour cell apoptosis that varied from low to high. In order to investigate the role of p53 in TNF-α-induced cell death, human sarcoma cell lines (n = 9) with different TP53 and MDM2 status were studied for their sensitivity to TNF-α. TP53Wt cell lines were sensitive to TNF-α unless MDM2 was over-expressed. However, TP53Mut and TP53Null cell lines were resistant. TP53 suppression in TP53Wt cell lines abrogated TNF-α sensitivity and TP53 overexpression in TP53Null cell lines restored it. The use of small molecules that restore p53 activity, such as CP-31398 or Nutlin-3a, in association with TNF-α, potentiated the cell death of respectively TP53Mut and TP53Wt/MDM2Ampl. In particular, CP-31398 was able to induce p53 as well as some of its apoptotic target genes in TP53Mut cells. In TP53Wt/MDM2Ampl cells, Nutlin-3a effects were associated with a decrease of TNF-α-induced NF-κB-DNA binding and correlated with a differential regulation of pro- and anti-apoptotic genes such as TP53BP2, GADD45, TGF-β1 and FAIM.Conclusion/SignificanceMore effective therapeutic approaches are critically needed for the treatment of unresectable limb sarcomas. Our results show that restoring p53 activity in sarcoma cells correlated with increased sensitivity to TNF-α, suggesting that this strategy may be an important determinant of TNF-α-based sarcomas treatment.

Abstract 2956: High throughput RNAi screening of synthetic lethal genes interacting with the common TP53 mutation R175H

Abstract The R175H mutation of p53 which account for approximately 6% of the missense mutations of identified in human cancer is one of the most common mutations, and cancer cells with this mutation stably express the R175H protein in the nucleus. Targeting R175H overexpressing cells by synthetic lethality is one of the extremely effective strategies for cancer therapy. To identify the synthetic lethal gene interacting with R175H, we conducted high throughput screening using a tetracycline-inducible R175H expression system in SF126 cells and comprehensive shRNA library carried by lentivirus. We identified 906 candidate gene suppressions that may lead to accelerated cell growth inhibition in the presence of R175H (p&amp;lt;0.05, n=3). Among these, we selected 50 genes (21 genes from the group with the smallest p-values, 20 genes from the group with the largest fold change, and 9 genes reproduced by different siRNA sequences) for further validation testing. By transfection of siRNA of these candidate genes into five R175H expressing cell lines and four TP53-null cell lines, we selected five candidate genes that significantly lead R175H expressing cells to strong cell growth inhibition. Inhibitor of differentiation 1 (ID1) was one of the five candidate genes, and its suppression by siRNA resulted in the acceleration of growth inhibition in cell lines expressing endogenous R175H; however, this was not observed in TP53-null cell lines. The transient expression of R175H in TP53-null cell lines (PC3) and suppression of ID1 and/or TP53 R175H in cell lines with endogenous R175H revealed that cell growth inhibition by ID1 suppression depended on the expression of R175H, and not that of other common p53 mutants (e.g., R273H). Flow cytometric analysis exhibited that ID1 suppression resulted in G1 arrest, and the arrest was accelerated by the expression of R175H. In conclusion, ID1 is a synthetic lethal gene that interacts with R175H and is considered to be a novel molecular target for cancer therapy in R175H expressing cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2956. doi:1538-7445.AM2012-2956

A novel loss-of-function mutation in TP53 in an endometrial cancer cell line and uterine papillary serous carcinoma model

The etiology of carcinoma of the uterine endometrium (ECa) is poorly understood. However, loss of apoptosis is one of the major factors that allow cancer cells to survive and progress. Hec50co, a poorly differentiated human ECa cell line, is widely used in the investigation of ECa. Previously, Hec50co xenograft tumor model in nude mice developed an advanced phenotype, similar to that of uterine papillary serous carcinoma (UPSC). Importantly, loss-of-function mutation in tumor suppressor TP53 was found in 20-30% of all ECa and >90% of UPSC. Thus, understanding the status of TP53 in Hec50co is essential for using Heco50co as a model for UPSC. To obtain an accurate genotype-phenotype status of TP53 in Hec50co, we performed mutation and functional analysis of TP53 gene of Hec50co by RT-PCR, genomic-PCR, and cloning and expression of mutant and wildtype TP53 alleles. We found a novel 42-bp deletion mutation in the exon6-intron6 splice junction of TP53 (TP53.del42bp) leading to a 113-bp exon6-deleted/skipped transcript was identified in Hec50co. In addition, the other TP53 allele in Hec50co is inactivated through a large deletion. Adenovirus (AD) harboring wildtype full-length TP53 cDNA induces caspase-dependent apoptosis; while the AD-TP53.del42bp allele does not. In addition, messenger RNA of TP53.del42bp allele is stable whereas the protein product of TP53.del42bp allele is made but not stable. Taken together, we demonstrate that Hec50co is a TP53-null cell line possessing one TP53.del42bp allele and the other lost allele and therefore provides an excellent model to dissect the molecular and cellular bases of UPSC and other p53-null cancers.

The Individual Cell Properties of Oral Squamous Cell Carcinoma and p53 Tumor Suppressor Gene Mutation

There is no consensus on the relationship between variations in TP53 mutations and tumor properties in oral squamous cell carcinoma (OSCC). To further the basic research required to eventually develop individualized (order-made) treatments and prognoses for OSCC, we established six human OSCC lines from patients within our department. Together with another nine cell lines derived from donations by other organizations, we determined the TP53 mutation and single nucleotide polymorphism (SNP) of codon 72 in a total of 15 cell lines, and examined in vitro cell invasion activity and anti-cancer drug sensitivity. The missense mutation at codon 248 was most abundant, and was noted in four cell lines, but other diverse mutation variations were also revealed. The cells which expressed the mutated p53 protein (the p53(+) group) showed slightly higher invasion activity than did the p53 (−) group. In p53(+) group, the 72R of SNP (72P/R) was higher than the 72P in invasion activity, although the difference was not significant. Surprisingly, an anti-cancer drug sensitivity test with four different types of drugs showed that the p53 (−) group was more resistant in other than CDDP, and that 72R was more sensitive than 72P in the p53(+) group. To clarify the characteristics of the R248Q mutation, which is the most abundant missense mutation, the gene was introduced with an expression plasmid vector into a TP53 null Saos-2 cell. The transformant of R248Q mutation gained higher activity of invasion, while its anti-cancer drug sensitivity also increased. Our findings suggest that it may be possible to estimate oral cancer cell characteristics and the malignancy level based on differences in the TP53 mutation.