Two main causes of platinum resistance are mutation in the tumor suppressor gene TP53 and drug-induced increase in intracellular glutathione concentration. Mutations in TP53 occur in about 50% of human tumors. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 and induces apoptosis. APR-246 is a prodrug that is converted to the active compound methylene quinuclidinone (MQ), a Michael acceptor that binds to cysteine residues in mutant p53 and restores its wild-type conformation. Here, we show that MQ also binds to cysteine in glutathione, thus decreasing intracellular free glutathione concentration. We also show that treatment with APR-246 completely restores the cisplatin and doxorubicin sensitivity to p53-mutant drug-resistant ovarian cancer cells. We propose that this unique ability of APR-246/MQ to bind to cysteines in both mutant p53 and glutathione has a key role in the resensitization as well as in the outstanding synergistic effects observed with APR-246 in combination with platinum compounds in ovarian cancer cell lines and primary cancer cells. However, MQ binding to cysteines in other targets, for example, thioredoxin reductase, may contribute as well. Strong synergy was also observed with the DNA-damaging drugs doxorubicin and gemcitabine, while additive effects were found with the taxane docetaxel. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with platinum-based therapy in patients with p53-mutant recurrent high-grade serous (HGS) ovarian cancer. More than 96% of these patients carry TP53 mutations. Combined treatment with APR-246 and platinum or other DNA-damaging drugs could allow dramatically improved therapy of a wide range of therapy refractory p53 mutant tumors.

Mast cells are found in tissues throughout the body where they play important roles in the regulation of inflammatory responses. One characteristic feature of mast cells is their longevity. Although it is well established that mast cell survival is dependent on stem cell factor (SCF), it has not been described how this process is regulated. Herein, we report that SCF promotes mast cell survival through inactivation of the Forkhead transcription factor FOXO3a (forkhead box, class O3A) and down-regulation and phosphorylation of its target Bim (Bcl- 2 IntroductionMast cells are long-lived multifunctional effector cells of the immune system originating from the hematopoietic CD34 ϩ stem cells found in the bone marrow. 1 From the bone marrow, mast cell precursors enter the circulation where they are recruited into peripheral tissues to mature and express their final phenotype under the influence of stem cell factor (SCF) and other locally produced cytokines. 2 Although best known for their role in allergic reactions, mast cells are now also recognized as cells of importance in both innate immunity and in the onset and severity of chronic inflammations. 3,4 The versatile effector mechanisms mast cells have been endowed with can be deduced from their capability to release a wide variety of inflammatory mediators such as histamine, proteases, and cytokines that are preformed and stored in granules and prostaglandins, leukotrienes, and cytokines that are secreted upon activation. 5 The number of tissue mast cells is normally relatively constant, but during an acute or chronic inflammation the number can increase substantially. 6 The regulation of mast cell numbers is most likely regulated by proliferation, migration, and apoptosis or survival. The mechanisms that regulate the viability of mature mast cells or promote mast cell apoptosis are poorly investigated. SCF is a cardinal growth factor in mast cell biology, regulating mast cell growth, differentiation, adhesion, migration, and survival. 7 The number of tissue mast cells is at least in part regulated by SCF produced by resident stromal cells. SCF rescues mast cells from spontaneous apoptosis in vitro, whereas inhibition of SCF synthesis in vivo leads to mast cell apoptosis. [8][9][10] Although it is accepted that SCF is a prosurvival factor for mast cells, it remains largely unclear how SCF promotes survival in these cells.The B-cell lymphoma-2 (Bcl-2) family, which contains both prosurvival and proapoptotic proteins, are essential regulators of cell survival and apoptosis. 11 The levels and interactions of prosurvival versus proapoptotic Bcl-2 family proteins determine whether a cell survives or will undergo apoptosis. During apoptosis induced by proapoptotic Bcl-2 family members, cytochrome c is released from the mitochondria and a caspase cascade is activated that induces DNA fragmentation. 12,13 The prosurvival Bcl-2 family members include Bcl-2, Bcl-X L , Bcl-w, Mcl-1 (myeloid cell For personal use only. on May 12, 2018. by guest www.bloodjournal.org From leuk...

Mast cells play critical roles in the regulation of acute and chronic inflammations. Apoptosis is one of the mechanisms that limit and resolve inflammatory responses. Mast cell survival can be controlled by growth factors and activation of the IgE-receptor FceRI. Members of the Bcl-2 protein family are critical regulators of apoptosis and our study provides evidence that the proapoptotic BH3-only family member Bim is essential for growth factor deprivation-induced mast cell apoptosis and that Bim levels increase upon FceRI activation. Bim deficiency or Bcl-2 overexpression delayed or even prevented cytokine withdrawal-induced mast cell apoptosis in culture. The prosurvival protein Bcl-X L and the proapoptotic Bim were both induced upon FceRI activation. These results suggest that Bim and possibly also other BH3-only proteins control growth factor withdrawal-induced mast cell apoptosis and that the fate of mast cells upon FceRI activation depends on the relative levels of pro-and antiapoptotic Bcl-2 family members.

BackgroundMutation in the tumor suppressor gene TP53 is an early event in the development of high-grade serous (HGS) ovarian cancer and is identified in more than 96 % of HGS cancer patients. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 protein by refolding it to wild type conformation, thus inducing apoptosis. APR-246 has been tested as monotherapy in a Phase I/IIa clinical study in hematological malignancies and prostate cancer with promising results, and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing. In the present study, we investigated the anticancer effects of APR-246 in combination with conventional chemotherapy in primary cancer cells isolated from ascitic fluid from 10 ovarian, fallopian tube, or peritoneal cancer patients, 8 of which had HGS cancer.MethodsCell viability was assessed with fluorometric microculture cytotoxicity assay (FMCA) and Combination Index was calculated using the Additive model. p53 status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by western blotting.ResultsWe observed strong synergy with APR-246 and cisplatin in all tumor samples carrying a TP53 missense mutation, while synergistic or additive effects were found in cells with wild type or TP53 nonsense mutations. Strong synergy was also observed with carboplatin or doxorubicin. Moreover, APR-246 sensitized TP53 mutant primary ovarian cancer cells, isolated from a clinically platinum-resistant patient, to cisplatin; the IC50 value of cisplatin decreased 3.6 fold from 6.5 to 1.8 μM in the presence of clinically relevant concentration of APR-246.ConclusionThese results suggest that combination treatment with APR-246 and DNA-damaging drugs could significantly improve the treatment of patients with TP53 mutant HGS cancer, and thus provide strong support for the ongoing clinical study with APR-246 in combination with carboplatin and pegylated liposomal doxorubicin in patients with recurrent HGS cancer.

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IntroductionMast cells are potent effector cells displaying versatile functions during immune responses and as regulators of inflammation. [1][2][3] Many of these functions are executed via activation of the high affinity IgE receptor (Fc⑀RI) and subsequent release of regulatory factors stored in granules. 4 In addition, receptor stimulation initiates signaling cascades, which result in activation of specific genes encoding cytokines and growth factors. 5 In some cases, the activated transcription has been shown to be mediated by members of the NF-B and/or NFAT transcription factor families. 6 These transcription factors are sequestered in an inactive state in the cytosol of resting cells, and after cell stimulation they are translocated to the nucleus where they bind target DNA sequences and activate transcription.In contrast to granulocytes and certain other hematopoietic cells, mature mast cells are not recruited from the bloodstream in response to inflammatory signals. Instead, long-lived mast cells are located in the tissues, and their relative abundance and increase during inflammation are regulated at the level of cell migration within the tissue and the control of survival/apoptosis. 7,8 We and others have previously demonstrated that, after stimulation of the high affinity IgE receptor, Fc⑀RI, mast cell survival is substantially enhanced. [9][10][11] These cells can therefore undergo a new round of activation and thus contribute again to an inflammatory response. [12][13][14] The activation-induced survival effect is attributed to the specific up-regulation of the antiapoptotic Bcl-2 family member A1/Bfl-1 gene. 10,15 Accordingly, mast cells from A1-deficient mice do not exhibit activation-induced survival after Fc⑀RI crosslinking. 10 A1 is expressed and exerts its antiapoptotic function not only in mast cells but also in endothelial cells, T and B lymphocytes, neutrophils, and macrophages. [16][17][18][19][20][21] In these cell types, expression of the A1 gene is induced by diverse stimuli, such as inflammatory cytokines, lipopolysaccharide (LPS), CD40-activation, and antigen receptor (TCR or sIg) receptor activation. The increased transcription of the A1 gene in lymphocytes has been demonstrated to be dependent on the NF-B transcription factor pathway. [22][23][24][25] It was shown that antigen receptor crosslinking-mediated A1 induction is abolished in NF-B-deficient cells and that enforced NF-B overexpression increases A1 levels. Moreover, a functional NF-B binding site has been mapped within the A1 promoter.Knowledge of the mechanisms leading to A1 induction after Fc⑀RI activation could identify possible ways to interfere with this pathway and thereby control mast cell survival and its downstream effects. In this report, we examine these signaling pathways in mast cells and show that, in contrast to other cell types and stimuli, NF-B is not responsible for the IgE receptor activation-mediated induction of A1. Instead, this study indicates that in mast cells, a member of the NFAT class of transcript...

Activation of the high-affinity IgE-receptor, FceRI, expressed on mast cells can result in either enhanced survival or apoptosis depending on the circumstances. In this study, we have analysed signalling pathways involved in the regulation of mast cell survival and apoptosis. FceRI cross-linking induces phosphorylation of Akt and its downstream target forkhead transcription factors. In addition, Bad, GSK-3b and IkB-a also become phosphorylated. A1, a prosurvival Bcl-2 homologue transcriptionally controlled by NFkB transcription factors, is upregulated upon FceRI activation. These events have prosurvival effects on the mast cells. Moreover, FceRI activation upregulates the levels of the proapoptotic protein Bim and induces a rapid, but transient, phosphorylation of Bim. Thus, FceRI activation of mast cells leads to both prosurvival and proapoptotic signalling events where the outcome most likely depends on the balance between these signals.

Background: Mutations in the TP53 gene occur in at least 60% of ovarian tumors and are associated with chemoresistance and poor prognosis. APR-246 (PRIMA-1MET) is the first mutant p53-reactivating compound in clinical development and has been tested as monotherapy in hematological malignancies and prostate cancer with promising results (Lehmann et al. J Clin Oncol 30, 2012). The aim of this study was to investigate the anticancer effects of APR-246 in combination with conventional chemotherapy in cancer cells isolated from ascites fluid from ovarian cancer patients. Methods: Ascites cells were purified by Ficoll and viably frozen, and the quality and purity were confirmed by May Grünwald/Giemsa staining. For some samples, immunocytochemical stainings with anti-Ber-EP4 and anti-calretinin antibodies were used to distinguish between mesothelial and cancer cells. Cell viability was assessed with FMCA assay and Combination Index (CI) calculated using Additive model. CI < 0.8 indicates synergy and CI < 0.5 strong synergy. TP53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Results: Eight of ten samples tested were from patients with recurrent ovarian cancer previously treated with platinum drugs. Cancer cells from seven patients possessed TP53 core domain missense mutations L111Q, C135Y, P151H, Y163H, C238F, P278R and R280K, respectively; two had nonsense mutations E346* and E204*, and one was wild type. All the missense mutations have been predicted to severely affect p53 tumor suppressor function. Missense mutant p53 proteins were expressed at high levels while no p53 expression was detected in cells with wild type or nonsense mutant p53. Synergistic or strong synergistic effects with APR-246 and cisplatin were observed in all ten samples tested. Synergy was also observed with the platinum analogue carboplatin and the anthracycline doxorubicin. The IC50 values for cisplatin ranged from 3 to 40 μM and for APR-246 from 5 to 37 μM. We also tested the ability of APR-246 to sensitize the primary ovarian cancer cells carrying Y163H mutant p53, to cisplatin; the IC50-value of cisplatin decreased from 10 to 2.6 μM in the presence of 6 μM APR-246. Conclusions: We observed striking synergy with APR-246 and platinum drugs or doxorubicin. These results are consistent with our previous results in ovarian cancer cell lines showing synergy not only in p53 mutant but also in p53 null cancer cell lines. In these cells, the synergy may be related to the fact that APR-246 decreases intracellular glutathione level. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with carboplatin and doxorubicin in patients with recurrent ovarian cancer and suggest that combination treatment with APR-246 and DNA-damaging drugs could allow significantly improved treatment for ovarian cancer carrying mutant p53. Citation Format: Åsa Fransson, Daria Glaessgen, Jessica Alfredsson, Klas G. Wiman, Svetlana Bajalica Lagercrantz, Nina Mohell. Strong synergy with APR-246 and DNA-damaging drugs in primary ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2015-1639