Mutation 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.

Abstract Background: Although smoking increases risk for many tumor types, no malignancy is more closely linked to tobacco exposure than small cell lung cancer (SCLC); 90% of SCLC patients are smokers. Carcinogenic compounds in cigarettes increase cancer susceptibility due to formation of DNA adducts, leading to oncogenic mutations. Etoposide in combination with cisplatin or carboplatin is the standard chemotherapy for SCLC. However, most SCLC patients eventually die of chemotherapy-refractory disease. Mutation in the tumor suppressor gene TP53 is one of the main causes for resistance, and occurs in more than 70% of SCLC patients (http://p53.free.fr). APR-246 (PRIMA-1MET) is the first clinical-stage small molecule that reactivates mutant p53 by inducing its wild-type conformation triggering apoptosis. It has been tested in a First in Human clinical trial in hematological malignancies with encouraging results (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study in combination with platinum-based therapy in recurrent ovarian cancer is ongoing (AACR abstract #CT204, 2015). We have previously observed strong synergy with APR-246 and platinum compounds in TP53-mutant drug-resistant ovarian cancer cells (Mohell et al. Cell Death and Disease 6, 2015). The aim of the present study was to investigate whether APR-246 synergizes with standard chemotherapy in SCLC cells carrying smoking-associated and/or lung cancer-specific TP53 mutations. Methods: Cell viability was tested using CellTiter-Glo assay, TP53 status with Sanger sequencing and single strand conformation analysis, and p53 protein level with Western blotting. Combination Index (CI) was calculated according to Additive model. Results: We observed synergistic (CI<0.8) or strong synergist effect (CI<0.5) with APR-246 and cisplatin in SCLC cell lines carrying smoking-associated (often G>T transversion) and/or lung cancer specific homozygous TP53 mutations, including NCI-H2195 (V157F, 4.3% of all SCLC tumors), NCI-H1048 (R273C, 1.78%) and NCI-H889 (C242S, 1.07%). Synergy was also observed in H196 cells with hotspot R175H mutation (2.14%), while additive effect (CI = 0.8-1.2) was found in NCI-H1882 (R273L, 1.78%) and NCI-H187 (S241C, 0.36%) cells. Synergy was also observed with etoposide in NCI-H2195 cells. Moreover, APR-246 sensitized the NCI-H2195 cells to cisplatin; the IC50 value decreased about 2-fold at clinically relevant concentration of APR-246. All tested SCLC cell lines had medium or high level of p53. Further studies with other conventional drugs are ongoing. Conclusions: Treatment with APR-246 in combination with cisplatin or etoposide resulted in synergy or strong synergy in SCLC cells carrying lung cancer-specific and/or smoking-related TP53 mutations. Our results suggest that combination treatment with APR-246 and standard chemotherapy can provide significantly improved treatment of TP53-mutant SCLC. Citation Format: Nina Mohell, Åsa Fransson, Jessica Alfredsson, Mikael von Euler, Ulf Björklund, Lars Abrahmsen. Synergistic effect with APR-246 and standard chemotherapy in small cell lung cancer cells carrying smoking-associated TP53 mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 270.

APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53. This phase Ib part of a proof of concept study aims to determine the recommended phase II dose (RP2D) of APR-246 in combination with carboplatin and pegylated liposomal doxorubicin (PLD) in platinum sensitive High Grade Serous Ovarian Cancer (HGSOC). Despite high response rates from carboplatin in combination with paclitaxel in first-line treatment of ovarian cancer, most patients relapse and develop resistance. Partially platinum sensitive patients relapse between 6 and 24 months and are commonly treated with second -line carboplatin and PLD (Pujade-Lauraine et al. JCO, 2010). The mechanisms of platinum resistance are multifactorial; two of the main causes are mutations in p53 and increased levels of intracellular glutathione. Like the analog PRIMA-1, APR-246 is a pro-drug that is converted to the active form MQ, which restores wild type conformation to mutant p53 (Lambert et al. Cancer Cell, 2009). In addition, APR-246 has been shown in vitro to reduce glutathione levels, resensitize cancer cells to platinum drugs, and induce ROS levels and ER stress (Mohell et al. Abstract #1801, AACR 2014; Lambert et al. Oncogene, 2010). In the first-in-human phase Ia study, APR-246 monotherapy was found to have a satisfactory safety and pharmacokinetic profile allowing it to be combined with full dose chemotherapy (Lehmann et al., JCO, 2012). The ongoing phase Ib/II study is enrolling patients with recurrent platinum sensitive HGSOC with positive p53 staining on immunohistochemistry. The phase Ib study has a 3+3 dose escalation design with 3 planned dose levels. APR-246 is administered as a 6h i.v. infusion on 4 consecutive days every 4 weeks. On day 4, APR-246 is given concomitantly with carboplatin AUC 5 and PLD 30 mg/m2. In the phase II part, 164 patients will be randomized to standard chemotherapy with or without APR-246. To date patients have been enrolled to all 3 dose cohorts. One DLT of ruptured diverticulum occurred at the 2nd dose level. No new safety concerns have emerged. The pharmacokinetic profile has not indicated any interaction between APR-246 and the chemotherapy. The first 3 patients have completed their therapy and are now in follow up. All 3 had partial response (PR) by RECIST 1.1 and 2/2 evaluable also had PR by GCIC. In conclusion, early results from the ongoing clinical study are encouraging and support the continued development of APR-246 in the phase II part of the study comparing platinum based standard chemotherapy with or without APR-246 in patients with HGSOC with mutant p53. Preliminary results from all three dose levels and the RP2D will be presented at the meeting. Citation Format: Mikael von Euler, Klas G. Wiman, Hani Gabra, James D. Brenton, Bristi Basu, Ignace Vergote, Charlie Gourley, Austin Smith, Jessica Alfredsson, Nina Mohell, John A. Green. Preliminary results from PiSARRO, a phase Ib/II study of APR-246, a mutant p53 reactivating small molecule, in combination with standard chemotherapy in platinum-sensitive ovarian cancer. [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 CT204. doi:10.1158/1538-7445.AM2015-CT204

Abstract 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

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-1(MET)) 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.

Abstract Background: Platinum compounds have been used as first-line treatment for many solid tumors including non small cell (NSCLC) and small cell (SCLC) lung cancer. However, patients with lung cancer often develop resistance to platinum compounds and eventually die of chemotherapy refractory disease. Mutation in the tumor suppressor protein p53 is common in lung cancer, ranging from 33% in adenocarcinomas to 70% in SCLC (The p53 website, http://p53.free.fr), and is one of the main causes for resistance to chemotherapy. APR-246 (PRIMA-1MET) is the first compound in clinical development that reactivates mutant p53 by inducing its wild type conformation thus triggering apoptosis (Lambert et al. Cancer Cell 15, 2009). APR-246 has yielded promising results in a first-in-human clinical trial in patients with hematological malignancies and prostate cancer (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing. Previously we have shown strong synergy with APR-246 and platinum compounds in p53-mutant drug-resistant ovarian cancer cells (AACR abstract # 3448, 2013). Moreover, APR-246 completely restored the sensitivity of cisplatin to resistant p53-mutant ovarian cancer cells (AACR abstract #1801, 2014). The aim of the current study was to investigate whether strong synergistic effect can also be observed in p53-mutant lung cancer cells. Methods: Cell viability was determined with FMCA or Cell Titer-Glo assay, p53 gene status by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Combination Index (CI) was calculated according to Additive model. Results: We observed strong synergistic effect (CI<0.5) with APR-246 and cisplatin in lung cancer cell lines carrying homozygous p53 hotspot mutations; NCI-H1770 (R248W), NCI-H1975 (R273H), NCI-H596 (G245C), PC-14 (R248W) and PC-14/CDDP (R248W). All these cell lines expressed a high level of p53. Synergistic (CI<0.8) or strong synergistic effect was found in lung cancer cell lines NCI-H378 (Y163C) and NCI-2087 (V157F) with homozygous mutations that occur frequently but are not hotspot mutations. NCI-2087 cells with the smoking-related V157F mutation express a high level of p53, while NCI-H378 cells express lower level. Mixed antagonist/additive/synergistic effects were observed in the p53 null cell line HOP-62, which does not express p53. Conclusions: Treatment with APR-246 in combination with cisplatin resulted in strong synergistic effect in both NSCL and SCLC cancer cells. Strongest synergies were observed in lung cancer cells with p53 hotspot mutations expressing a high level of p53. Studies to investigate the molecular mechanisms underlying the synergistic effects are ongoing. Our results suggest that combination treatment with APR-246 and platinum drugs may allow an improved therapy of p53-mutant lung cancer. Citation Format: Nina Mohell, Åsa Fransson, Jessica Alfredsson, Mikael von Euler, Ulf Björklund, Lars Abrahmsen. Strong synergistic effects with APR-246 and cisplatin in p53-mutant lung 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 2523. doi:10.1158/1538-7445.AM2015-2523

Abstract Background: Platinum-based drugs are since decades used as first-line treatment for many solid tumors. Patients with ovarian cancer often respond well to platinum compounds but a majority of patients rapidly develop resistance and die of chemotherapy refractory disease. The mechanisms underlying resistance are multifactorial, but two of the main causes are mutations in the tumor suppressor p53 and elevated intracellular glutathione (GSH) levels. Mutations in p53 occur in about 60% of ovarian tumors. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53. APR-246 is a prodrug that accumulates in cancer cells and is converted to the active form MQ, a Michael acceptor that binds to mutant p53, refolds it to wild type conformation and triggers apoptosis (Lambert et al. Cancer Cell 15, 2009). APR-246 has been tested in a Phase I/IIa clinical trial with promising results (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study with platinum-based combination therapy in recurrent p53 mutant ovarian cancer is underway. Methods: Cell viability was assessed with WST-1, MTT or FMCA assay. p53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Intracellular GSH levels were assessed with GSH kit (Cayman). Results: We have previously shown outstanding synergistic anticancer effects with APR-246 in combination with platinum compounds in p53 mutant solid cancer cell lines, including cisplatin resistant ovarian cancer cells. Synergistic effects were also observed ex vivo as well as in vivo in mice carrying human tumor xenografts. Here we show that APR-246 not only reactivates mutant p53 but also decreases intracellular GSH levels in a dose-dependent manner, presumably via adduct formation between MQ and GSH. APR-246 resensitized cisplatin resistant p53 mutant ovarian A2780-CP20 carcinoma cells to cisplatin, as shown by a decrease in the IC50 value from 52 to 2.9 µM, similar to the IC50 in parental A2780 cells. APR-246 also restored the sensitivity of resistant A2780ADR cells to doxorubicin. A2780-CP20 and A2780ADR were developed from the parental A2780 line with wild type p53 by chronic exposure to the respective drug. Moreover, APR-246 resensitized the p53 mutant OVCAR-3 cell line, established from a drug resistant patient, to cisplatin. Conclusions: Our results show that APR-246 not only reactivates mutant p53 but also decreases intracellular glutathione levels. We propose that this unique dual mechanism of action accounts for the resensitization and strong synergistic effects with APR-246 and platinum drugs. Our results provide strong rationale for the planned clinical study in ovarian cancer and suggest that combination treatment with APR-246 and DNA damaging drugs could have broad applicability in the treatment of drug resistant p53 mutant human tumors. Citation Format: Nina Mohell, Jessica Alfredsson, Åsa Fransson, Vladimir Bykov, Mikael von Euler, Klas Wiman, Ulf Björklund. APR-246, a clinical-stage mutant p53-reactivating compound, resensitizes ovarian cancer cells to platinum compounds and doxorubicin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1801. doi:10.1158/1538-7445.AM2014-1801

Abstract Background: The tumor suppressor protein p53 is frequently mutated in cancer, and cancer cells carrying defects in p53 are generally more resistant to conventional chemotherapy. About 60% of patients with ovarian cancer have p53 mutations. Thus, restoration of wild type function of p53 is a promising strategy for cancer therapy. APR-246 (PRIMA-1MET) belongs to a class of small molecules (quinuclidinones) that reactivate mutated or otherwise non-functional p53 by promoting its correct wt folding thus triggering apoptosis (Lambert et al. Cancer Cell 15, 2009). In various in vitro, ex vivo and in vivo cancer models APR-246 has shown good antitumor activity and a unique pharmacological profile. In a Phase I/II clinical dose-finding study on hematological malignancies and prostate cancer APR-246 had a good safety profile, and both biological and clinical responses were observed (Lehmann et al. J Clin Oncol 30, 2012). A Phase II Proof of Concept study in p53 mutant ovarian cancer patients is currently under way. Here results from combination studies with APR-246 and platinum compounds in p53 mutant ovarian cancer cell lines, and primary cells from patients, are presented. Methods: Cell viability/proliferation was assessed with WST-1, MTT and/or FMCA assay. p53 gene status was determined with Sanger sequencing and single strand conformation analysis. p53 expression was determined with Western immunoblotting. Results: In the ovarian cancer cell line OVCAR-3, with homozygous “hot spot” p53 core domain mutation (R248Q), strong/outstanding synergistic effects with APR-246 and cisplatin were observed (CI < 0.5). This cell line was established from a patient resistant to clinically relevant concentrations of cisplatin, doxorubicin and melphalan. The OVCAR-3 cell line expresses a high level of p53. In the in vitro cisplatin resistant ovarian cancer cell lines (A2780-CP20, IGROV-1/CDDP, IGROV-1/Pt-1) harboring heterozygous frequently occurring p53 core domain mutations, synergistic (CI < 0.8) or strong synergistic effects were observed. Also these cell lines express p53 but at lower level. These resistant cell lines have been developed from parental cisplatin sensitive cell lines derived from untreated patients with wt p53, by chronic in vitro exposure to cisplatin. Carboplatin and doxorubicin showed cross-resistance in cisplatin resistant cell lines, while the sensitivity of APR-246 did not change. Primary ovarian cancer cells were also investigated and strong synergistic effect was found in all samples. In vivo efficacy (xenograft) combination studies using cisplatin resistant ovarian cancer cell lines in nude mice are ongoing. Conclusion: These results further support the Phase II Proof of Concept study of APR-246 in p53 mutated ovarian cancer patients who are candidates for further platinum-based chemotherapy. Citation Format: Nina Mohell, Jessica Alfredsson, Maria Uustalu, Åsa Fransson, Vladimir J.N. Bykov, Klas G. Wiman, Ulf Björklund. Strong synergistic effects with cisplatin and APR-246, a novel compound reactivating mutant p53, in ovarian cancer cell lines and primary cells from patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3448. doi:10.1158/1538-7445.AM2013-3448

Neuropeptide Y and peptide YY receptor type 2 (Y2) is involved in appetite regulation and several other physiological processes. We have investigated the structure of the human Y2 receptor. Computational modeling of receptor-agonist interactions was used as a guide to design a series of receptor mutants, followed by binding assays using full-length and truncated peptide agonists and the Y2-specific antagonist BIIE0246. Our model suggested a hydrogen bond network among highly conserved residues Thr2.61, Gln3.32, and His7.39, which could play roles in ligand binding and/or receptor structure. In addition, the C-terminus of the peptide could make contact with residues Tyr5.38 and Leu6.51. Mutagenesis of all these positions, followed by binding assays, provides experimental support for our computational model: most of the mutants for the residues forming the proposed hydrogen bond network displayed reduced peptide agonist affinities as well as reduced hPYY3-36 potency in a functional assay. The Ala and Leu mutants of Gln3.32 and His7.39 disrupted membrane expression of the receptor. Combined with the modeling, the experimental results support roles for these hydrogen bond network residues in peptide binding as well as receptor architecture. The reduced agonist affinity for mutants of Tyr5.38 and Leu6.51 supports their role in a binding pocket surrounding the invariant tyrosine at position 36 of the peptide ligands. The results for antagonist BIIE0246 suggest several differences in interactions compared to those of the peptides. Our results lead to a new structural model for NPY family receptors and peptide binding.

The neuropeptide Y-family peptides and receptors are involved in a broad range of functions including appetite regulation. Both the peptide genes and the receptor genes are known to have duplicated in early vertebrate evolution. The ancestral jawed vertebrate had 7 NPY receptors but the number varies between 4 and 7 in extant vertebrates. Herein we describe the identification of an additional NPY receptor in two fish species, zebrafish and medaka. They cluster together with the Y2 receptors in phylogenetic analyses and seem to be orthologous to each other that is why we have named them Y2-2. Their genes differ from Y2 in having introns in the coding region. Binding studies with zebrafish Y2-2 receptors show that the three endogenous peptides NPY, PYYa and PYYb have similar affinities, 0.15–0.66 nM. This is in contrast to the Y2 receptor where they differed considerably from one another. N-terminally truncated NPY binds poorly and the Y2 antagonist BIIE0246 binds well to Y2-2, results that are reversed in comparison to Y2. Zebrafish Y2-2 mRNA was detected by PCR in the intestine and the eye, but not in the brain. In conclusion, we have found a novel Y2-like NPY/PYY receptor that probably arose in early teleost fish evolution.

The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T3.40 was selected based on sequence alignments both between subtypes and between species and G2.68, L4.60 and Q6.55 also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3–36), NPY(13–36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3–36) increased for the mutants T3.40I and Q6.55A. Increased affinity was also observed for PYY to Q6.55A. PYY(3–36) displayed decreased affinity for G2.68N and L4.60A whereas binding of NPY(13–36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T3.40I, L4.60A and Q6.55A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.

TPS183 Background: APR-246 belongs to a new class of small molecules that is currently being tested in a phase I/IIa clinical trial. The goal of the study is to evaluate safety and pharmacokinetic (PK) properties of APR-246, but efficacy parameters will also be included. APR-246 has been shown to restore nonfunctional p53 by promoting its correct folding (Lambert et al. Cancer Cell 15, 2009). This represents a new strategy with potential to treat patients resistant to conventional chemotherapy. APR- 246 has been tested in a full nonclinical safety package and has shown no toxicity at predicted therapeutic plasma levels. This profile is further supported by the tumor specificity of APR-246 demonstrated in ex vivo studies with primary cells. Methods: The ongoing phase I/IIa trial was initiated in June 2009 and is an open labeled, dose-escalating study on refractory hematological malignancies and prostate cancer. The study will comprise approximately 25 patients, and is performed at 7 sites in Sweden. Each patient receives APR-246 treatment on 4 consecutive days as a 2 h daily intravenous infusion. The treatment phase is followed by a 17-day follow-up phase to reveal any late adverse events. The total duration of one treatment cycle is 21 days. The primary objective is to determine the highest feasible dose of intravenously administered APR-246. As secondary endpoints tumor burden and apoptosis are measured. The study is estimated to be reported in Q3 2010. In parallel with the ongoing phase I/IIa clinical study, planning for a proceeding phase II, proof-of-concept, study is ongoing. As additive and synergistic effects have been observed in various cell lines and primary cells from patients, the design of the phase II study will most probably involve combination treatment with APR-246 and conventional chemotherapy. This will allow for lower doses of chemotherapy resulting in less severe side effects. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Apera AB, Aprea AB Aprea AB Aprea AB Aprea AB

Abstract Abstract 1806 The tumor suppressor protein p53 is a transcription factor involved in cell cycle arrest, senescence and apoptosis. The p53 gene is frequently mutated in cancer, and cancer cells carrying defects in p53 are generally more resistant to conventional chemotherapy. Thus, restoration of wild type function of p53 is a promising novel strategy for cancer therapy. APR-246 belongs to a new class of small molecules (quinuclidinones) that reactivates non-functional p53 by promoting its correct folding and triggering apoptosis (Lambert et al. Cancer Cell 15, 2009). The lead compound of APR-246, PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis) was identified by a cellular screen of a NCI (National Cancer Institute) library, and an optimization program led to the discovery of the analog APR-246 (PRIMA-1MET). In various in vitro,ex vivo andin vivo cancer models, APR-246 has shown good antitumor activity. It reduces cell viability and/or induces apoptosis in a large number of human cancer cell lines with different p53 status, including leukemia, lymphoma and myeloma cell lines (Mohell et al. Blood 114, 2009). Ex vivo efficacy of APR-246 alone and in combination with conventional chemotherapeutic drugs has been shown in primary cells from patients with acute myeloid leukemia (AML) (Jonsson-Videsater et al. Blood 114, 2009). Ex vivo efficacy of APR-246 has also been shown in primary cells from patients with chronic lymphocytic leukemia (CLL). APR-246 was 4–8 fold more potent in killing malignant than normal lymphocytes, whereas common cytostatics often have negative ratio (Mohell et al. Blood 114, 2009). In vivo efficacy of APR-246/PRIMA-1 has been demonstrated in xenograft studies using many solid tumor cell lines (Mohell et al. Blood 114, 2009). Here we present results from studies with APR-246 using in vivo systemic and metastasic xenograft model with the human AML primary cell line AML-PS. This model was established by Giovazzi et al. (Int. J. Cancer 61, 1995) and is considered as a predictive in vivo model for human AML. In addition, some key results from preclinical safety and toxicology studies are reported. Briefly, SCID (severe combined immunodeficiency) mice were inoculated i.v. with 5×106 human AML-PS primary cells. Three days after inoculation treatment with i.v. injections of APR-246 (200 and 300 mg/kg), twice daily for 10 days, was initiated. Mice were monitored daily for health status and mortality. Blood samples were collected for determination of the percentage of circulating human leukemia cells by FACS analysis. Human leukemic cells were detected using a fluorescent antibody against the major histocompatibility complex (HLA). In parallel, pharmacokinetic experiments to measure the concentration of APR-246 in the blood were performed. We found that APR-246 had a statistically significant antitumor effect by decreasing the percentage of circulating human AML-PS cells and increasing the survival time of the mice (P=0.0024, n=10). A good correlation between increase in survival time and decrease in circulating tumor cells in the blood was observed. Further in vivo efficacy studies using various treatment schedules and combinations with conventional cytostatics are ongoing. APR-246 was also investigated in pivotal toxicology studies using single and repeat-dose regimen. In dogs, APR-246 was well tolerated when administered as 2 h infusion with NOAEL (no observed adverse effect level) of 200 mg/kg/day (4000 mg/m2/day). In both dogs and mice, Cmax levels less than 100 μg/ml did not induce any toxicity, regardless of the administration protocol. No systemic target organ toxicity was observed in mice or dogs, including blood and bone-marrow parameters. In conclusion, APR-246 has in various efficacy models demonstrated significant antitumor activity and a unique pharmacological profile. In preclinical safety/toxicity studies no toxicity at predicted therapeutic plasma concentrations was observed. Thus, APR-246 appears to be a promising novel anticancer compound to treat patients resistant to common chemotherapy. Currently, APR-246 is investigated in a dose escalating Phase I/IIa First-in-Man study for refractory hematological malignancies and prostate cancer. The Phase II Proof of Concept study is planned to start in 2011. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Uhlin:Aprea AB: Employment. Linderholm:Aprea AB: Consultancy. Wiman:Aprea AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Neuropeptide Y (NPY) and peptide YY (PYY) share ∼70% of their 36 amino acids and bind to the same three human receptor subtypes, Y1, Y2 and Y5, even though these receptors only share ∼30% sequence identity. Based on our previous investigation of human Y1 we describe here a mutagenesis study of three corresponding positions in human Y2, i.e. Tyr2.64, Val6.58 and Tyr7.31. Pharmacological characterization was performed with the four peptide agonists PYY, NPY, PYY(3–36) and NPY(13–36) as well as the non-peptide antagonist BIIE0246. Results from mutants where Tyr2.64 has been substituted by Ala suggest that Tyr2.64 is involved in the interaction with all investigated ligands whereas position Tyr7.31 seems to be more important for interaction with the truncated peptide PYY(3–36) than with intact NPY. Surprisingly, substitution of Tyr7.31 with His, the corresponding residue in Y1, resulted in total loss of binding of iodinated porcine PYY. The third position, Val6.58, did not influence binding of any of the ligands. These findings differ from those obtained for Y1 where Ala substitution resulted in lost or changed binding for each of the three positions. Although Tyr2.64 and Tyr7.31 in Y2 are involved in ligand binding, their interactions with the peptide ligands seem to be different from the corresponding positions in Y1. This suggests that the receptor–ligand interactions have changed during evolution after Y1 and Y2 arose from a common ancestral receptor.

Abstract Abstract 3773 Poster Board III-709 Introduction The tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. Results The lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. Conclusion In relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

Peptides with agonist activity at the vasopressin V(2) receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. Of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V(1b) receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V(2) receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT((R))), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V(2) receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V(2) receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V(2) receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V(2) receptor agonist deficiency.

Interactions of the human NPY (neuropeptide Y) receptor Y1 with the two endogenous agonists NPY and peptide YY and two non-peptide antagonists were investigated using site-directed mutagenesis at 17 positions. The present study was triggered by contradictions among previously published reports and conclusions that seemed inconsistent with sequence comparisons across species and receptor subtypes. Our results show that Asp287, at the border between TM (transmembrane) region 6 and EL3 (extracellular loop 3) influences peptide binding, while two aspartic residues in EL2 do not, in agreement with some previous studies but in disagreement with others. A hydrophobic pocket of the Y1 receptor consisting of Tyr100 (TM2), Phe286 (TM6) and His298 (EL3) has been proposed to interact with the amidated C-terminus of NPY, a theory that is unsupported by sequence comparisons between Y1, Y2 and Y5. Nevertheless, our results confirm that these amino acid residues are critical for peptide binding, but probably interact with NPY differently than proposed previously. Studies with the Y1-selective antagonist SR120819A identified a new site of interaction at Asn116 in TM3. Position Phe173 in TM4 is also important for binding of this antagonist. In contrast with previous reports, we found that Phe173 is not crucial for the binding of BIBP3226, another selective Y1 receptor antagonist. Also, we found that position Thr212 (TM5) is important for binding of both antagonists. Our mutagenesis results and our three-dimensional model of the receptor based on the high-resolution structure of bovine rhodopsin suggest new interactions for agonist as well as antagonist binding to the Y1 receptor.