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Background: Cutaneous melanoma is the most serious skin malignancy and new therapeutic strategies are needed for advanced melanoma. TP53 mutations are rare in cutaneous melanoma and hence activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma. Here, we investigated the WIP1 inhibitor, GSK2830371, and MDM2–p53 binding antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells. Methods: A panel of three p53WT (A375, WM35 and C8161) and three p53MUT (WM164, WM35-R and CHL-1) melanoma cell lines were used. The effects of MDM2 and WIP1 inhibition were evaluated by growth inhibition and clonogenic assays, immunoblotting, qRT–PCR gene expression profiling and flow cytometry. Results: GSK2830371, at doses (⩽10 μM) that alone had no growth-inhibitory or cytotoxic effects on the cells, nevertheless significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53WT but not p53MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner. The siRNA-mediated knockdown of p53 provided further evidence to support the p53 dependence. GSK2830371 increased p53 stabilisation through Ser15 phosphorylation and consequent Lys382 acetylation, and decreased ubiquitination and proteasome-dependent degradation when it was combined with MDM2 inhibitors. These changes were at least partly ATM mediated, shown by reversal with the ATM inhibitor (KU55933). GSK2830371 enhanced the induction of p53 transcriptional target genes, cell cycle arrest and apoptosis. Conclusions: GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors by increasing phosphorylation, acetylation and stabilisation of p53 in cutaneous melanoma cells in a functional p53-dependent manner.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous hematologic malignancy. In approximately 90% of cases the TP53 gene is in its wildtype state at diagnosis of this malignancy. As mouse double-minute-2 homolog (MDM2) is a primary repressor of p53, targeting this protein is an attractive therapeutic approach for non-genotoxic reactivation of p53. Since the discovery of the first MDM2 inhibitor, Nutlin-3a, newer potent and bioavailable compounds have been developed. In this study we tested the second-generation MDM2 inhibitor, RG7388, in patient-derived CLL cells and normal cells, examining its effect on the induction of p53-transcriptional targets. RG7388 potently decreased viability in p53-functional CLL cells, whereas p53-non-functional samples were more resistant to the drug. RG7388 induced a pro-apoptotic gene expression signature with upregulation of p53-target genes involved in the intrinsic (PUMA, BAX) and extrinsic (TNFRSF10B, FAS) pathways of apoptosis, as well as MDM2 Only a slight induction of CDKN1A was observed and upregulation of pro-apoptotic genes dominated, indicating that CLL cells are primed for p53-dependent apoptosis. Consequently, RG7388 led to a concentration-dependent increase in caspase-3/7 activity and cleaved poly (ADP-ribose) polymerase. Importantly, we observed a preferential pro-apoptotic signature in CLL cells but not in normal blood and bone marrow cells, including CD34+ hematopoietic cells. These data support the further evaluation of MDM2 inhibitors as a novel additional treatment option for patients with p53-functional CLL.

Introduction Recent studies indicate that E7107, a spliceosome inhibitor, causes altered splicing of key genes in CLL. We evaluated the effect of E7107 on cell viability and key proteins in the p53 pathway. Material and methods Eight leukaemia/lymphoma cell lines and eight primary CLL samples (6 wild-type and 2 mutant for SF3B1) were exposed to E7107 (H3 Biomedicine) for 72 and 48 hours. Cell viability was assessed by XTT assay. To understand the effect of splicing modulation on key proteins in the p53 pathway, including p21 and MDM2, five B-cell lines were treated with E7107 for 24 hours. Results and discussions E7107 decreased cell viability at low nanomolar concentrations in all CLL samples (mean LC50=10.5±2.0 nM; but >300 nM in two healthy PBMC controls). No correlation between drug sensitivity and SF3B1 status was observed in CLL samples (p=0.5). Six out of eight cell lines were sensitive to E7107 (mean GI50=6±1.8 nM). The GI50 values were 60.2 and 203.5 nM for the resistant HEL and HAL-01 cells, respectively. The most frequently mutated regions of the SF3B1 gene, exons 14, 15 and 16, had no mutations detected by Sanger sequencing. There was no correlation between drug sensitivity and TP53 status. Western immunoblot revealed a marked decrease of MDM2 protein level in all cell lines, accompanied by a reciprocal concentration-dependent increase in p53. The normal molecular weight p21 disappeared at higher doses of E7107, with concomitant appearance of a high molecular weight p21 isoform (~30 kDa) in TP53 wild-type cells. In addition, a lower molecular weight p53 isoform was seen in OCI-Ly3 cells treated with 50 nM E7107, likely due to alternative splicing. RT-PCR of p21 mRNA, spanning exons 3 to 4, revealed an additional longer transcript in OCI-Ly3 cells treated with E7107(50 nM, 24 hours), which sequencing confirmed was due to intron retention, predicted to result in a high molecular weight protein with an alternative c-terminal sequence. RT-PCR of MDM2 mRNA spanning exons 1 to 11 detected an altered MDM2 transcript with exons 3–10 spliced out. Conclusion E7107 treatment resulted in decreased viability of B-cell lines and primary CLL samples independently of SF3B1 status. This was associated with the production of an aberrant high molecular weight isoform of p21 due to intron retention, and a short isoform of MDM2 missing exons 3–10. Loss of normal MDM2 was accompanied by increased p53. Further investigation is needed to understand the contribution of abnormal isoforms to cell fate.

Cutaneous melanoma is the most serious skin malignancy. The current study aimed to investigate the WIP1 inhibitor GSK2830371 and MDM2-p53 antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells. A panel of three p53WT (A375, WM35, C8161) and three p53MUT (WM164, WM35-R5R1, CHL-1) melanoma cell lines were used. GSK2830371 (≤10 μM) alone had no growth-inhibitory or cytotoxic effects on the cells, measured by sulforhodamine B (SRB) and clonogenic assays. In combination treatment GSK2830371 significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53WT but not p53MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner (Table). Western blotting demonstrated GSK2830371 increased p53 stabilization through Ser15 phosphorylation and consequent Lys382 acetylation when it was combined with MDM2 inhibitors. These changes were ATM-mediated, shown by reversal with the ATM inhibitor (KU55933). Furthermore, GSK2830371 was demonstrated to slow down p53 degradation when de-novo protein synthesis was inhibited by cycloheximide. In qRT-PCR, nutlin-3 or RG7388 induced p53 transcriptional target genes (CDKN1A, MDM2, BAX, FAS, PUMA, TNFBSF10B, TP53INP1) and GSK2830371 enhanced the induction in p53WT but not p53MUT cells. In conclusion, GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors, by increasing phosphorylation, acetylation, and stabilization of p53 in cutaneous melanoma cells in a functional p53-dependent manner. Further studies in vivo are warranted to investigate the efficacy of this combination treatment. GI50 by sulforhodamine B (SRB) and LC50 by clonogenic assays in p53WT melanoma cellsCell linesGI50 (Mean + SEM)A375WM35C8161GSK2830371-+-+-+Nutlin-3 (µM)5.1 + 0.61.5 + 0.48.3 + 1.55.3 + 1.41.7 + 0.31.0 + 0.5p value0.0120.0100.049RG7388 (nM)228 + 3962 + 2377 + 58169 + 5746 + 015 + 5p value0.0250.0280.009HDM201 (nM)167 + 2541 + 9165 + 9877 + 5981 + 1149 + 10p value0.00460.1048< 0.001LC50 (Mean + SEM)A375WM35C8161GSK2830371-+-+-+Nutlin-3 (µM)2.0 + 0.90.8 + 0.50.5 + 0.10.4 + 0.10.9 + 0.20.4 + 0.1p value0.0400.07140.003RG7388 (nM)166 + 9554 + 37186 + 7868 + 2350 + 2221 + 10p value0.0060.0700.026HDM201 (nM)198 + 7015 + 411 + 38 + 3139 + 8519 + 5p value Citation Format: Chiao-En Wu, Arman Esfandiari, Yi-Hsuan Ho, Colin Shepherd, Ahmed Khairallah Mahdi, Erhan Aptullahoglu, John Wen-Cheng Chang, Penny Lovat, John Lunec. Inhibition of WIP1/PPM1D phosphatase by GSK2830371 potentiates the growth inhibitory and cytotoxic activity of MDM2 antagonists (nutlin-3, RG7388 and HDM201) in cutaneous melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2151. doi:10.1158/1538-7445.AM2017-2151

Introduction. Current treatment protocols for chronic lymphocytic leukemia (CLL), including FCR (fludarabine, cyclophosphamide and rituximab), have dramatically improved response rates. However, since none of these therapies is curative, continued preclinical studies on innovative therapeutic strategies are warranted. In particular, the identification of new agents that interfere with the survival of CLL cells by promoting their apoptosis is one approach to improve therapeutic outcome. Taking into account that p53 deletions and/or mutations in CLL are restricted to 10%-15% of patients at diagnosis, we have investigated the efficacy of the second-generation MDM2 antagonist RG7388, in primary CLL samples, examining its ability to disrupt the p53-MDM2 interaction, resulting in expression of p53 transcriptional targets that trigger apoptosis. Methods. Heparinized peripheral blood samples were obtained from CLL patients (n=42) with informed consent, in accordance with institutional guidelines and the Declaration of Helsinki. Mononuclear cells were purified by Lymphoprep density-gradient centrifugation. Cells were resuspended in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin. Cells were treated with increasing concentrations of RG7388 and incubated at 37°C, 5% CO2. Ex vivo cytotoxicity was assessed in all samples 48 hours after treatment using an XTT assay. A pilot time-course experiment identified 6 hours after treatment as the most informative time point for gene expression analysis. Therefore, in 8 samples cells were treated for 6 hours and RNA was extracted using standard methods. qRT-PCR on a panel of nine p53 transcriptional targets (CDKN1A, MDM2, PUMA, BAX, FAS, FDXR, ZMAT3, TP53INP1, DR5) was performed using SYBR Green chemistry. The functional integrity of p53 was assessed by western blot following treatment with MDM2 antagonists in 20 samples. Apoptosis was assessed using a Caspase 3/7 luminescent assay. Results. Treatment with RG7388 significantly decreased cell viability (LC50 < 1µM) in 28/42 CLL samples (mean LC50 = 0.40 ± 0.05 µM). As expected, the 8 CLL samples that displayed non functional p53 on western blot and/or mutated TP53 by sequencing were more drug-resistant (mean LC50 = 5.22 ± 1.13 µM) than those sensitive responders (LC50 < 1µM) with a functional p53 (mean LC50 = 0.39 ± 0.05 µM, n=12). Interestingly, among p53 functional samples, we identified a small subset that showed intermediate response (1 µM 10 µM, n=3) to RG7388 treatment. RG7388 significantly upregulated the mRNA expression of MDM2 and of p53 transcriptional targets involved in the intrinsic pathway of apoptosis (PUMA and BAX), and the extrinsic pathway of apoptosis (death receptor 5 (DR5)) (Figure 1A, n=5 samples). Interestingly, only a slight upregulation of CDKN1A was observed and upregulation of pro-apoptotic genes dominated. Overall, in p53 functional CLL samples, exposure to 3 µM RG7388 for 6 hours led to a 7.8-fold increase in MDM2, a 7-fold increase in PUMA, a 4.9-fold increase in DR5, a 4.5-fold increase in BAX, and a 3.3-fold increase in CDKN1A (Figure 1A). Conversely, no significant upregulation of p53 target genes was seen in p53 non-functionalCLL samples (Figure 1B, n=3 samples). Treatment with RG7388 led to a concentration-dependent increase in caspase 3/7 activity in p53 functional CLL samples (Figure 1C). No significant difference in caspase 3/7 activity was observed in p53 non-functional samples after RG7388 treatment (Figure 1D). Interestingly, when we analysed samples taken two years apart from a fludarabine- and chlorambucil-resistant CLL patient, we found that treatment with RG7388 decreased cell viability, induced p53 accumulation and upregulation of p53-target genes, suggesting that inhibiting the p53-MDM2 interaction might be a promising treatment strategy in chemo-resistant CLL patients that show a functional p53. Conclusion. Taken together, our data indicate that RG7388 induces a characteristic dominantly pro-apoptotic gene expression profile of p53-target genes in primary p53-functional CLL samples. RG7388 showed a consequent potent apoptotic effect on CLL cells, which was dependent on the presence of a functional p53. Identification of patients in whom functional p53 activation drives apoptosis may translate into improved outcomes for patients treated with non-genotoxic MDM2 antagonists. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Today's conventional technology has to overcome many challenging aspects for integrating expressional analysis of a mutation into the cellular function of a living cell. It is very striking to detect the expressional status of a mutation in a living cell without compromising its cellular function as well as its viability that allows to perform subsequent functional analysis. This has been a long time goal for most of the biological studies. Myeloproliferative neoplasms (MPN) originate at the level of hematopoietic stem cell with a single nucleotide mutation in the cytosolic tyrosine kinase, Janus kinase 2 (JAK2) named as JAK2V617F which facilitates the constitutive intracellular activation of JAK2 and found in 95% of Polycythemia Vera (PV) patients. Detection of this mutation is a major criteria for diagnosis of the malignancy and changes in the expression levels of JAK2V617F is valued for the prognosis as well as response to the treatment. Here, we have utilized the nanotechnology's new development, entitled as "multiplexed nanoflares", a gold nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are highly oriented oligonucleotide sequences, which are hybridized to a reporter dye such as Cy3 and Cy5 and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra and intracellular environments. Thus, this signal could be easily detected by flow cytometry or confocal microscopy. We have employed JAK2 V617F specific nanoflare probes labeled with Cy3 (JAK2V617F-Cy3) and JAK2 wild type (wtJAK2) nanoflare probe labeled with Cy5 (JAK2-Cy5) including negative and positive control probes to distinguish and isolate mRNA's of JAK2V617F and wtJAK2 in live cells by measuring subsequent fluorescence with flow cytometry. The initial testing of the nanoflares has been performed by treating cell lines such as K562 (wtJAK2), SET-2 (JAK2V617F heterozygote), HEL (JAK2V617F homozygote) cells with nanoflare probes which did not alter cell viability and morphology with the applied concentrations during 18 hours of cell culture. We measured the levels of two mRNA targets, JAK2V617F and JAK2, and detected 90±6 % positivity with JAK2V617F-Cy3 treated HEL cells and no positivity in K562 cells, meanwhile wtJAK2 probe treated cells succeeded staining of SET and K562 cells. Real time reverse transcription PCR (RT-PCR) was performed on the same cells to compare the relative amounts of target RNA in each cell type by calculating relative changes in mRNA expression using the ΔΔCt (cycle threshold) method and comfirmed the results of flow cytometry with sensitivity of 0.001. Subsequently, we have tested mononuclear cells (MNC) received from peripheral blood of 5 patients with PV, one of which has JAK2V617F heterozygote allele (patient 5) and 4 patients had JAK2V617F homozygote allele (patients 1-4)which were tested with nested allel specific PCR. Flow cytomeyric analysis of the patients` MNC treated with the wtJAK2 and JAK2V617F probes demonstrated distinct populations of wtJAK2+ and JAK2V617F+. Populations of wtJAK2+/JAK2V617F+ (2.9%), wtJAK2-/JAK2V617F- ( 21.4%), wtJAK2+/JAK2V617F-( 0%), wtJAK2-/JAK2V617F+ ( 79%) of patient 5 and populations of JAK2V617F+ (36,8%± 5 ), JAK2V617F- of patients 1-4 were sorted by cell sorter with ~ 90% purity following detection and analysis of each populations for relative changes in mRNA expressions . The data revealed distinct ΔCt values between each populations. In this study, we have aimed for a new approach by utilizing nanotechnology aligned with the conventional techniques in order to save time, money and outcome. The capability of in-cell detection and isolation of the JAK2V617F and wtJAK2 first time by performing the expression analysis, changes the routine protocols backwards, enables the detecting direct effect of the mutation to the cells as well as other genes. This study creates a platform where functional analysis regarding to the more detailed understanding of the initiation of malignancy as well as responses to the therapies could be performed. Disclosures No relevant conflicts of interest to declare.