Randomized Controlled Trial of the Gastrin/CCK2 Receptor Antagonist Netazepide in Patients with Barrett's Esophagus.

Hypergastrinemia has been associated with high-grade dysplasia and adenocarcinoma in patients with Barrett's esophagus, and experimental studies suggest proinflammatory and proneoplastic effects of gastrin on Barrett's esophagus. This is of potential concern, as patients with Barrett's esophagus are treated with medications that suppress gastric acid production, resulting in increased physiologic levels of gastrin. We aimed to determine whether treatment with the novel gastrin/CCK2 receptor antagonist netazepide reduces expression of markers associated with inflammation and neoplasia in Barrett's esophagus. This was a randomized, double-blind, placebo-controlled trial of netazepide in patients with Barrett's esophagus without dysplasia. Subjects were treated for 12 weeks, with endoscopic assessment at baseline and at end of treatment. The primary outcome was within-individual change in cellular proliferation as assessed by Ki67. Secondary analyses included changes in gene expression, assessed by RNA-sequencing, and safety and tolerability. A total of 20 subjects completed the study and were included in the analyses. There was no difference between arms in mean change in cellular proliferation (netazepide: +35.6 Ki67+ cells/mm2, SD 620.7; placebo: +307.8 Ki67+ cells/mm2, SD 640.3; P = 0.35). Netazepide treatment resulted in increased expression of genes related to gastric phenotype (TFF2, MUC5B) and certain cancer-associated markers (REG3A, PAX9, MUC1), and decreased expression of intestinal markers MUC2, FABP1, FABP2, and CDX1 No serious adverse events related to study drug occurred. The gastrin/CCK2 receptor antagonist netazepide did not reduce cellular proliferation in patients with nondysplastic Barrett's esophagus. Further research should focus on the biological effects of gastrin in Barrett's esophagus.Prevention Relevance: Treatment of patients with Barrett's esophagus with a gastrin/CCK2 receptor antagonist did not have obvious chemopreventive effects.

Effects of Rovalpituzumab Tesirine on Ventricular Repolarization in Patients With Small-Cell Lung Cancer.

Small cell lung cancer (SCLC) is a leading cause of cancer death worldwide, with few treatment options. Rovalpituzumab tesirine (Rova-T) is an antibody-drug conjugate that targets delta-like 3 on SCLC cells to deliver a cytotoxic payload directly to tumor cells. In this study, the cardiac safety profile of Rova-T was assessed by evaluating changes in QT interval, electrocardiogram (ECG) waveform, heart rate, and proarrhythmic adverse events (AEs) after treatment with Rova-T in patients with previously treated extensive-stage SCLC. Patients underwent ECG monitoring for 2 weeks after each of 2 i.v. infusions of 0.3 mg/kg Rova-T over 30 minutes, administered 6 weeks apart. Forty-six patients received at least one dose of Rova-T. At the geometric mean Rova-T maximum serum concentration of 7,940 ng/mL, ECG monitoring showed no significant changes in the Fridericia-corrected QT (QTcF) interval; the upper limit of the 2-sided 90% confidence interval did not exceed 10 msec for any time point. There were no clinically significant changes in QRS or PR intervals, ECG waveforms, or heart rate after Rova-T administration. All patients experienced a treatment-emergent AE (TEAE); 78% had a grade ≥ 3 TEAE, 59% had a serious TEAE, and 41% had a cardiac-related TEAE. The TEAEs that might signal proarrhythmia tendencies were uncommon. Confirmed partial responses were observed in 24% of patients. Based on the evaluation of ECG data collected in this study from patients treated with Rova-T at 0.3 mg/kg i.v. administered every 6 weeks, a QTcF effect of clinical concern can be excluded.

CBL0137 increases the targeting efficacy of Rovalpituzumab tesirine against tumour-initiating cells in small cell lung cancer.

Small cell lung cancer (SCLC) is characterised by high relapse rates. Tumour-initiating cells (TICs) are responsible for drug resistance and recurrence of cancer. Rovalpituzumab tesirine (Rova-T), a potent humanised antibody-drug conjugate, selectively targets delta-like protein 3, which is highly expressed in SCLC TICs. The experimental drug CBL0137 (CBL) inhibits the histone chaperone FACT (facilitates chromatin transcription), which is required for the expression of transcription factors that are essential for TIC maintenance. Rova-T and CBL each target SCLC TICs as single agents. However, acquired or intrinsic resistance to single agents is a major problem in cancer. Therefore, we investigated the potential effect of combining Rova-T and CBL in SCLC to eradicate TICs more effectively. Our preclinical studies report a novel and highly translatable therapeutic strategy of dual targeting TICs using Rova-T in combination with CBL to potentially increase survival of SCLC patients.

Gastric Corpus Mucosal Hyperplasia and Neuroendocrine Cell Hyperplasia, but not Spasmolytic Polypeptide-Expressing Metaplasia, Is Prevented by a Gastrin Receptor Antagonist in H+/K+ATPase Beta Subunit Knockout Mice.

Proton pump inhibitor use is associated with an increased risk of gastric cancer, which may be mediated by hypergastrinemia. Spasmolytic polypeptide-expression metaplasia (SPEM) has been proposed as a precursor of gastric cancer. We have examined the effects of the gastrin receptor antagonist netazepide (NTZ) or vehicle on the gastric corpus mucosa of H+/K+ATPase beta subunit knockout (KO) and wild-type (WT) mice. The gastric corpus was evaluated by histopathology, immunohistochemistry (IHC), in situ hybridization (ISH) and whole-genome gene expression analysis, focusing on markers of SPEM and neuroendocrine (NE) cells. KO mice had pronounced hypertrophy, intra- and submucosal cysts and extensive expression of SPEM and NE cell markers in the gastric corpus, but not in the antrum. Numerous SPEM-related genes were upregulated in KO mice compared to WT mice. NTZ reduced hypertrophia, cysts, inflammation and NE hyperplasia. However, NTZ neither affected expression of SPEM markers nor of SPEM-related genes. In conclusion, NTZ prevented mucosal hypertrophy, cyst formation and NE cell hyperplasia but did not affect SPEM. The presence of SPEM seems unrelated to the changes caused by hypergastrinemia in this animal model.

Phase I safety and pharmacokinetics study of rovalpituzumab tesirine in Japanese patients with advanced, recurrent small cell lung cancer.

OBJECTIVES: Rovalpituzumab tesirine (Rova-T™) is an antibody-drug conjugate that targets delta-like protein 3 (DLL3) on small cell lung cancer (SCLC) tumors, is internalized and releases the toxin pyrrolobenzodiazepine to induce cell death. This open label phase I study was the first study of Rova-T in Japanese patients. The aim of this study was to evaluate, safety, pharmacokinetics, and preliminary efficacy of Rova-T in Japanese patients with advanced recurrent SCLC. MATERIALS AND METHODS: Patients received Rova-T (0.2 or 0.3 mg/kg) by intravenous infusion on Day (D) 1 of each 6-week cycle for 2 doses and dexamethasone (8 mg BID oral) on D-1, D1, and D2 of each 6-week cycle. Retreatment with Rova-T was permitted for patients who tolerated their initial doses and then progressed after disease control (defined as stable disease or better) was observed for at least 12 weeks after their last dose of Rova-T. RESULTS: Rova-T exhibited toxicity that was generally manageable in Japanese patients (N = 29). No dose-limiting toxicities were experienced. The most common treatment-related adverse events (≥25% of patients, all grades) were platelet count decreased, pleural effusion, peripheral edema, aspartate aminotransferase increased, white blood cell count decreased, neutrophil count decreased, alanine aminotransferase increased, hypoalbuminaemia, anemia and decreased appetite. Safety and pharmacokinetics exposures were similar to previous observations in non-Japanese populations. Per investigator assessment of DLL3 high patients, 17% (3/18) had confirmed partial responses, and the disease control rate was 56%, mPFS was 2.9 months, and mOS was 7.4 months. CONCLUSIONS: These preliminary data support further exploration of Rova-T treatment in Japanese patients with SCLC in global studies. This trial was registered with ClinicalTrials.gov as NCT03086239.

Novel targeted strategies to overcome resistance in small-cell lung cancer: focus on PARP inhibitors and rovalpituzumab tesirine.

Introduction: Small-cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumour, and its outcome is strongly conditioned by the rapid onset of resistance to conventional chemotherapeutics. First-line treatment with a combination of platinum agents and topoisomerase inhibitors has been the standard of care for over 30 years, with disappointing clinical outcome caused by early-acquired chemoresistance. In this disheartening scenario, novel treatment strategies are being implemented in order to either revert or bypass resistance mechanisms. Areas covered: The general mechanism of action of the standard frontline treatment regimens for SCLC, as well as the known resistance mechanisms to these drugs, is reviewed. Moreover, we focus on the current preclinical and clinical evidence on the potential role of PARP inhibitors and rovalpituzumab tesirine (Rova-T) to tackle chemoresistance in SCLC. Expert opinion: Preliminary evidence supports PARP inhibitors and Rova-T as two promising approaches to either revert or bypass chemoresistance in SCLC, respectively. The identification of potential predictive biomarkers of response to these innovative treatments (SLFN11 and DLL3) has shortened the gap between SCLC and personalized targeted therapy. Further large-scale clinical studies are urgently needed for a better designation of PARP inhibitors and Rova-T in the therapeutic algorithm of SCLC patients.

Enhancement of cytotoxic effects of gemcitabine by Dclk1 inhibition through suppression of Chk1 phosphorylation in human pancreatic cancer cells.

Although gemcitabine (GEM) is frequently used in the treatment of pancreatic cancer, the effects are limited. To increase the inhibitory effect of GEM, the identification of a molecular target is needed. Recent studies have revealed that doublecortin-like kinase 1 (Dclk1) positively regulates tumor growth, invasion, metastasis, factors related to epithelial-mesenchymal transition (EMT), pluripotency, angiogenesis, and anti-apoptosis in pancreatic cancer cells. Therefore, Dclk1 is a potential therapeutic target for pancreatic cancer. However, the Dclk1-signaling pathway including its substrate proteins remains to be elucidated. To identify the candidate substrate proteins phosphorylated by Dclk1, we performed a cancer-related phosphorylated protein microarray using Dclk1-inhibited MIA Paca2 cells. Expression levels of phosphorylated cdc25A (p-cdc25A) and phosphorylated Chk1 (p-Chk1), belonging to the ATR pathway, were decreased by treatment with Dclk1 inhibitor LRRK2-IN-1 (LRRK), indicating Dclk1 involvement in the ATR pathway. Consistent with this finding, the GEM-induced p-Chk1 expression was significantly decreased by treatment with LRRK. Notably, combined treatment with GEM and LRRK allowed cell cycle progression without arresting at S phase, while individual treatment with GEM induced cell cycle arrest at S phase. In addition, combined treatment with GEM and LRRK increased the number of γ-H2AX-positive cells compared with that upon individual treatments. Moreover, LRRK alone, and combined treatment with GEM and LRRK, induced caspase-3 activation and PARP1 cleavage, in contrast to treatment with GEM alone. Finally, combined treatment with GEM and LRRK significantly reduced cell survival compared to individual treatment with GEM. These results indicate that Dclk1 inhibition in combination with GEM treatment offers a novel approach to treat pancreatic cancer cells.

Z-360 Suppresses Tumor Growth in MIA PaCa-2-bearing Mice via Inhibition of Gastrin-induced Anti-Apoptotic Effects.

BACKGROUND/AIM: The aim of the study was to evaluate the anti-tumor mechanism of Z-360, a gastrin/cholecystokinin-2 receptor (CCK2R) antagonist, in MIA PaCa-2 cells and in a subcutaneous xenograft mice model. MATERIALS AND METHODS: The anti-tumor effects of Z-360 and/or gemcitabine were monitored using a MIA PaCa-2 xenograft model. The effect of Z-360 on apoptosis in the model was examined by TUNEL staining and real-time PCR analysis and the effect in MIA PaCa-2 cells stably expressing human CCK2R was also evaluated by caspase-3/7 activity. RESULTS: In this xenograft model, Z-360 significantly reduced the tumor weight, increased TUNEL-positive cells and suppressed the expression of anti-apoptosis factors such as survivin, XIAP and Mcl-1, and these effects of Z-360 combined with gemcitabine were more effective. Furthermore, gastrin-17 and gastrin-34 inhibited apoptosis in vitro and Z-360 dose-dependently abrogated this effect. CONCLUSION: These results suggest that Z-360 exerts an anti-tumor effect through a reduction in anti-apoptosis factors by blocking CCK2R.

A randomized phase II study of gemcitabine plus Z-360, a CCK2 receptor-selective antagonist, in patients with metastatic pancreatic cancer as compared with gemcitabine plus placebo.

BACKGROUND: We investigated the efficacy and safety of 60, 120, or 240 mg of Z-360, which is a highly potent cholecystokinin2-receptor-selective antagonist, combined with gemcitabine in patients with metastatic pancreatic cancer. METHODS: Patients were randomly assigned in a 1:1:1:1 ratio to one of four treatment groups. Patients received 1000 mg/m2 gemcitabine for each cycle and Z-360 tablets of 60 mg (GZ 60 mg group), 120, 240 mg or placebo tablets (Gem group) orally twice daily. The primary endpoint was overall survival (OS). RESULTS: The median OS was 1.3 months longer in the GZ 60 mg group compared with the Gem group (8.5 vs. 7.2 months) and the risk of death was reduced by 19% compared with the Gem group, although there were no statistically significant differences. The study treatments were well tolerated. CONCLUSIONS: In this Phase II study, no statistically significant differences between the GZ groups and Gem group were detected in any analysis. However, Z-360 in dose of 60 mg tends to improve OS in patients with metastatic pancreatic cancer with low toxic effect. Further exploratory trials with other agents such as gemcitabine plus nab-paclitaxel might be beneficial.

Cardioprotective effects of intracoronary administration of 4-chlorodiazepam in small and large animal models of ischemia-reperfusion.

BACKGROUND: The Translocator Protein (TSPO) of the mitochondrial membrane has been recognized as a potential therapeutic target for mitigation of myocardial ischemia-reperfusion injury. Administration of 4-chlorodiazepam (4-CLD), a TSPO ligand, has been shown to confer acute cardioprotective effects in small animals; however, long-term studies and studies in clinically-relevant large animal models are lacking. In the present study we investigated a potential cardioprotective effect of intracoronary administration of 4-CLD in small and large animal models of ischemia-reperfusion. METHODS: Acute myocardial infarction was induced in 38 Wistar rats and 29 farm pigs by ligation of the left anterior descending coronary artery, followed by reperfusion. Animals were randomized to undergo intracoronary infusion of 2µM 4-CLD or vehicle just prior (pigs) or immediately after (rats) reperfusion. Infarcted rats were euthanized either after 1h of reperfusion (for histological assessment of the "no reflow" area) or after 60days (for serial evaluation of cardiac function and structure by echocardiography and assessment of infarct size). Infarcted pigs were euthanized after 2h of reperfusion for histological assessment of infarct size and "no reflow" area. RESULTS: In infarcted rats, intracoronary infusion of 4-CLD resulted in acute reduction of the "no reflow" area and conferred durable long-term structural and functional benefits (reduction in infarct size, attenuation of adverse remodeling, improvement in global systolic function). In infarcted pigs, intracoronary infusion of 4-CLD was well-tolerated from a hemodynamic standpoint and resulted in acute reduction in infarct size, reduction in "no reflow" area and more rapid resolution of ST-segment elevation. CONCLUSIONS: In a rat model of myocardial infarction, intracoronary administration of 4-CLD attenuated the "no reflow" phenomenon and produced long-term structural and functional benefits. In a porcine model of myocardial infarction intracoronary administration of 4-CLD did not raise safety concerns and conferred acute cardioprotective effects.

EGCG Suppresses ERK5 Activation to Reverse Tobacco Smoke-Triggered Gastric Epithelial-Mesenchymal Transition in BALB/c Mice.

Tobacco smoke is an important risk factor of gastric cancer. Epithelial-mesenchymal transition is a crucial pathophysiological process in cancer development. ERK5 regulation of epithelial-mesenchymal transition may be sensitive to cell types and/or the cellular microenvironment and its role in the epithelial-mesenchymal transition process remain elusive. Epigallocatechin-3-gallate (EGCG) is a promising chemopreventive agent for several types of cancers. In the present study we investigated the regulatory role of ERK5 in tobacco smoke-induced epithelial-mesenchymal transition in the stomach of mice and the preventive effect of EGCG. Exposure of mice to tobacco smoke for 12 weeks reduced expression of epithelial markers E-cadherin, ZO-1, and CK5, while the expression of mesenchymal markers Snail-1, Vimentin, and N-cadherin were increased. Importantly, we demonstrated that ERK5 modulated tobacco smoke-mediated epithelial-mesenchymal transition in mice stomach, as evidenced by the findings that tobacco smoke elevated ERK5 activation, and that tobacco smoke-triggered epithelial-mesenchymal transition was reversed by ERK5 inhibition. Treatment of EGCG (100 mg/kg BW) effectively attenuated tobacco smoke-triggered activation of ERK5 and epithelial-mesenchymal transition alterations in mice stomach. Collectively, these data suggested that ERK5 was required for tobacco smoke-triggered gastric epithelial-mesenchymal transition and that EGCG suppressed ERK5 activation to reverse tobacco smoke-triggered gastric epithelial-mesenchymal transition in BALB/c mice. These findings provide new insights into the mechanism of tobacco smoke-associated gastric tumorigenesis and the chemoprevention of tobacco smoke-associated gastric cancer.

A translocator protein 18 kDa ligand, Ro5-4864, inhibits ATP-induced NLRP3 inflammasome activation.

Ro5-4864 and PK11195, prototypical synthetic ligands of translocator protein 18 kDa (TSPO), have shown anti-inflammatory effects in several models of inflammatory diseases; however, their biochemical mechanisms remain poorly understood. Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation as a part of the innate immune system, has been implicated in a variety of inflammatory diseases. Here, we demonstrate for the first time that TSPO ligands, especially Ro5-4864, potently suppressed ATP-induced NLRP3 inflammasome activation in THP-1 and BMDM cells. Detailed action mechanism was further investigated in THP-1 cells. Ro5-4864 efficiently attenuated NLRP3 translocation to mitochondria, inflammasome assembly/oligomerization, activation of caspase-1, and subsequent secretion of the mature forms of interleukin-1ß and -18. Ro5-4864 also reduced the production of mitochondrial superoxide and preserved the mitochondrial membrane potential in ATP-treated cells, suggesting that Ro5-4864 may act on mitochondria or more upstream targets in NLRP3 inflammasome signaling. We also observed the distinct effects of the TSPO ligands between THP-1 monocytes and macrophages, which suggested different NLRP3 inflammasome signaling depending on cell type. Collectively, our novel findings demonstrate that Ro5-4864 effectively inhibited ATP-induced NLRP3 inflammasome activation through the prevention of mitochondrial perturbation. Our results indicate Ro5-4864 as a promising candidate for the treatment of NLRP3 inflammasome-related diseases.

Topotecan synergizes with CHEK1 (CHK1) inhibitor to induce apoptosis in ovarian cancer cells.

BACKGROUND: Topotecan (TPT) is a therapeutic option for women with platinum-resistant or -refractory ovarian cancer. However, the dose-limiting toxicity of TPT is myelosuppression. This led us to seek a combination treatment to augment TPT anti-cancer activity in a cancer-targeted manner. Ovarian serous cancers, a major subtype, show dysregulated DNA repair pathway and often display a high level of CHEK1 (CHK1), a cell cycle regulator and DNA damage sensor. CHEK1 inhibitors are a novel approach to treatment, and have been used as single agents or in combination chemotherapy in many cancers. METHODS: We evaluated the cellular effects of TPT in a panel of high grade serous (HGS) and non-HGS ovarian cancer cells. We then determined IC50s of TPT in the absence and presence of CHEK1 inhibitor, PF477736. Synergism between TPT and PF477736 was calculated based on cellular viability assays. Cytotoxic effect of the combined treatment was compared with apoptotic activities by Caspase3/7 activity assay and Western blotting of cleaved-PARP1 and γH2AX. RESULTS: Non-HGS ovarian cancer cells were generally more sensitive to TPT treatment compared to HGS ovarian cancer cells. When combined with CHEK1 inhibitor, TPT potently and synergistically inhibited the proliferation of HGS ovarian cancer cells. This dramatic synergism in cellular toxicity was consistent with increases in markers of apoptosis. CONCLUSIONS: Our findings suggest that the addition of CHEK1 inhibitor increases the response of ovarian cancer cells to TPT. Furthermore, reduced dosages of both drugs achieved maximal cytotoxic effects by combining TPT with CHEK1 inhibitor. This strategy would potentially minimize side effects of the drugs for extended clinical benefit.

BMS-871: a novel orally active pan-Notch inhibitor as an anticancer agent.

This Letter describes synthesis, SAR, and biological activity of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides as inhibitors of γ-secretase mediated signaling of Notch receptors. Optimization of this series led to the identification of BMS-871 (compound 30) which displayed robust in vivo efficacy in Notch-dependent leukemia and solid tumor xenograft models.

CHK1 overexpression in T-cell acute lymphoblastic leukemia is essential for proliferation and survival by preventing excessive replication stress.

Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Effect of netazepide, a gastrin/CCK2 receptor antagonist, on gastric acid secretion and rabeprazole-induced hypergastrinaemia in healthy subjects.

AIMS: To compare gastric acid suppression by netazepide, a gastrin/CCK2 receptor antagonist, with that by a proton pump inhibitor (PPI), and to determine if netazepide can prevent the trophic effects of PPI-induced hypergastrinaemia. METHODS: Thirty healthy subjects completed a double-blind, randomized, parallel group trial of oral netazepide and rabeprazole, alone and combined, once daily for 6 weeks. Primary end points were: basal and pentagastrin-stimulated gastric acid and 24 h circulating gastrin and chromogranin A (CgA) at baseline, start and end of treatment, gastric biopsies at baseline and end of treatment and basal and pentagastrin-stimulated gastric acid and dyspepsia questionnaire after treatment withdrawal. RESULTS: All treatments similarly inhibited pentagastrin-stimulated gastric acid secretion. All treatments increased serum gastrin, but the combination and rabeprazole did so more than netazepide alone. The combination also reduced basal acid secretion. Rabeprazole increased plasma CgA, whereas netazepide and the combination reduced it. None of the biopsies showed enterochromaffin-like (ECL) cell hyperplasia. Withdrawal of treatments led neither to rebound hyperacidity nor dyspepsia. CONCLUSIONS: Netazepide suppressed pentagastrin-stimulated gastric acid secretion as effectively as did rabeprazole. The reduction in basal acid secretion and greater increase in serum gastrin by the combination is consistent with more effective acid suppression. Despite our failure to show rabeprazole-induced ECL cell hyperplasia and rebound hyperacidity, the increase in plasma CgA after rabeprazole is consistent with a trophic effect on ECL cells, which netazepide prevented. Thus, netazepide is a potential treatment for the trophic effects of hypergastrinaemia and, with or without a PPI, is a potential treatment for acid-related conditions.

Combined CDKN1A/TP53 mutation in bladder cancer is a therapeutic target.

Invasive bladder cancer has high morbidity and nearly uniform mortality when metastatic, with no therapeutic improvement in many years. Although chemotherapy combined with Chk1 inhibition has been investigated in several cancer types in which TP53 mutation is seen, this combination treatment approach has not been studied in bladder cancer. Recently, cancer genome sequencing efforts have identified CDKN1A (p21) mutations at 14% frequency in invasive bladder cancer, co-occurring half the time with TP53 mutations. We hypothesized that combined CDKN1A-TP53 loss would make bladder cancer sensitive to combined treatment with gemcitabine and Chk1 inhibitor. Here, we show that TP53-CDKN1A double-mutant bladder cancer cell lines, 647V and RT-112, have a remarkable increase in p-Chk1 levels and G2-M arrest in response to gemcitabine treatment, with a heightened sensitivity to combination treatment with gemcitabine and either Chk1 inhibitor PF477736 or AZD7762, in comparison with other bladder cancer cell lines (either TP53 or p21 deficient). In addition, CDKN1A restoration in p21-deficient bladder cancer cells significantly reduced their sensitivity to combined treatment by protecting them from DNA damage and apoptosis. Furthermore, xenograft studies using RT-112 showed a significant synergistic effect of combined gemcitabine-PF477736 treatment on tumor growth. Our findings suggest that TP53/CDKN1A double-mutant bladder cancer cells have a unique dependence on Chk1 activity for the G2-M cell-cycle checkpoint in response to chemotherapy-induced DNA damage. This combination or others involving genotoxic agents and Chk kinase inhibitors is a promising therapeutic approach for bladder cancer with these mutations.

ERK5 pathway regulates transcription factors important for monocytic differentiation of human myeloid leukemia cells.

Mitogen-activated protein kinases (MAPKs) are important transducers of external signals for cell growth, survival, and other cellular responses including cell differentiation. Several MAPK cascades are known with the MEK1/2-ERK1/2, JNK, and p38MAPKs receiving most attention, but the role of MEK5-ERK5 in intracellular signaling deserves more scrutiny, as this pathway transmits signals that can complement ERK/2 signaling. We hypothesized that the ERK5 pathway plays a role in the control of monocytic differentiation, which is disturbed in myeloid leukemia. We therefore examined the cellular phenotype and key molecular events which occur when human myeloid leukemia cells, acute (AML) or chronic (CML), are forced to differentiate by vitamin D derivatives (VDDs). This study was performed using established cell lines HL60 and U937, and primary cultures of blasts from 10 patients with ML. We found that ERK5 and its direct downstream target transcription factor MEF2C are upregulated by 1,25D in parallel with monocytic differentiation. Further, inhibition of ERK5 activity by specific pharmacological agents BIX02189 and XMD8-92 alters the phenotype of these cells by reducing the abundance of the VDD-induced surface monocytic marker CD14, and concomitantly increasing surface expression of the general myeloid marker CD11b. Similar results were obtained when the expression of ERK5 was reduced by siRNA or short hairpin (sh) RNA. ERK5 inhibition resulted in an expected decrease in MEF2C activation. We also found that in AML cells the transcription factor C/EBPß is positively regulated, while C/EBPα is negatively regulated by ERK5. These findings provide new understanding of dysregulated differentiation in human myeloid leukemia.