Murine Double Minute Research Articles

Elucidating the Mechanism of Action (MOA) of Navtemadlin, an MDM2 Inhibitor, and Its Synergy with Gilteritinib in Myeloid Malignancies

Introduction: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent, selective, orally available MDM2 inhibitor, is being evaluated in the phase 3 BOREAS trial of patients with relapsed/refractory myelofibrosis (MF; NCT03662126) and in phase 1b/2 trials in various tumor types. In preclinical studies, navtemadlin has overcome MDM2 dysregulation and has restored p53 activity to drive apoptosis in TP53WT malignancies. The primary determinant of tumor cell sensitivity to navtemadlin is TP53 mutation status. Because MDM2 is often overexpressed and p53 mutations are less common in treatment-naïve acute myeloid leukemia (AML) and MF, navtemadlin represents a rational therapeutic candidate for these malignancies (Prokocimer et al. Blood. 2017). The present study aims to elucidate the effects of navtemadlin on proteins downstream of p53 and synergy of navtemadlin with the FLT3 inhibitor gilteritinib in myeloid malignancies. Methods: Cell viability was evaluated in three TP53WT myeloid cell lines, MOLM-13 (FLT3 AML), MV-4-11, and UKE-1, and one TP53MUT myeloid cell line, HEL-92, treated with 1-10 µM navtemadlin for 72 h. Growth inhibition was determined using the ATP-based Cell Titer Glo assay. The treatment effect on cell cycle progression, BCL-2 family proteins, and apoptosis was determined by flow cytometry. The effects of navtemadlin (0.5-1 µM) + gilteritinib (0.5-1 µM) were assessed in MOLM-13 and MV-4-11 (TP53WT and TP53MUT clones) and visualized using Combenefit. A MOLM-13 TP53WT mouse xenograft model was used to assess in vivo activity. Engrafted mice were randomly assigned to one of five treatment groups (n=10 each): vehicle control, 30 mg/kg gilteritinib, 50 mg/kg navtemadlin, 50 mg/kg navtemadlin + 30 mg/kg gilteritinib, or 100 mg/kg navtemadlin. Mice were treated orally once a day until the mice met early removal criteria or the study was terminated. Survival was measured by the percentage of mice in each group that remained alive at the end of the study. Results: Navtemadlin exposure led to dose-dependent growth inhibition in the TP53WT myeloid cell lines MOLM13, MV-4-11, and UKE-1 with an EC90 of 2-6 µM. Conversely, no growth inhibition was observed in the TP53MUT myeloid cell line HEL-92, up to 10 µM of navtemadlin. Assessment of cell cycle progression following navtemadlin exposure showed the induction of threshold-dependent apoptosis in the TP53WT myeloid cell lines, but not in the TP53MUT myeloid cell line. The threshold at which a switch from p21-mediated cell-cycle arrest to p53-directed apoptosis occurs is illustrated in Figure 1a. Navtemadlin-induced apoptosis was indicated by a release of cytochrome c and increase in processed caspase-3. Clinically, a steady-state Cmax of 2-6 µM is achieved at therapeutically active and relevant doses of navtemadlin ≥180 mg given once a day on a 5-day or 7-day schedule in clinical trials, leading to maximum tumor cell death. To gain better insight into the contribution of BCL-2 family members alterations as an MOA for navtemadlin-induced apoptosis, expression levels of BCL-2 family members were evaluated in TP53WT myeloid cell lines. Navtemadlin increased levels of the effector pro-apoptotic proteins Bak, Bax, and Bim in leukemia cells. Synergy was observed in vitro for navtemadlin + gilteritinib in TP53WT, but not TP53MUT myeloid cell lines. Treatment with 100 mg/kg navtemadlin monotherapy and 50 mg/kg navtemadlin + gilteritinib combination led to prolonged survival in mice harboring MOLM-13 TP53WT xenografts, with all mice still alive at day 113 when the study was terminated (Figure 1b). In contrast, median survival times for the vehicle, 50 mg/kg navtemadlin monotherapy, and gilteritinib monotherapy groups were 18, 53, and 65 days, respectively. Conclusion: Navtemadlin induces potent, threshold-dependent apoptosis in cell culture models of TP53WT myeloid malignancies at clinically relevant concentrations. Navtemadlin-induced p53 activity initiates apoptosis by activating the caspase cascade via cytochrome c release. Navtemadlin was shown to induce the expression of pro-apoptotic BCL-2 family proteins, key mediators in driving cell death. These pro-apoptotic effects translate to robust antitumor activity in xenograft models of TP53WT myeloid malignancies, with the navtemadlin + gilteritinib combination demonstrating synergy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Siremadlin in Combination with Venetoclax (VEN) Plus Azacitidine (AZA) in Adult Patients with Acute Myeloid Leukemia (AML) Who Are Ineligible for Intensive Chemotherapy: A Phase Ib/II Trial

Background: Patients with AML who are ineligible for intensive chemotherapy have limited treatment options. Adding VEN to standard-of-care hypomethylating agents (HMAs) improves remission rates and survival for these patients. However, relapse rates with VEN+HMA are high, and many patients do not achieve a complete remission (CR)/CR with incomplete hematological recovery (CRi), or do so and remain measurable residual disease-positive (DiNardo CD, N Engl J Med, 2020), which is associated with poor outcomes. Murine double minute 2 (MDM2) is a key negative regulator of tumor protein 53 (p53; encoded by the TP53 gene). Inhibition of the p53-MDM2 interaction upregulates the tumor suppressor effects of p53. Siremadlin (HDM201) is a selective inhibitor of the p53-MDM2 interaction. In combination with VEN, siremadlin had durable antitumor responses in p53-wild-type AML patient-derived xenograft models (Wang Y, Cancer Res, 2019). Siremadlin was tolerable and demonstrated preliminary single-agent antitumor activity in patients with relapsed/refractory AML in a first-in-human Phase I trial (Stein EM, Clin Cancer Res, 2021; NCT02143635). In combination with VEN, siremadlin also demonstrated promising preliminary antileukemic activity in a Phase Ib trial in patients with relapsed/refractory AML (Wei AH, ASH 2021; NCT03940352). Study design: This international, multicenter, Phase Ib/II, open-label, dose-confirmation, proof-of-concept trial will provide a dosing recommendation and assess the preliminary efficacy of siremadlin in combination with VEN+AZA in adults (≥18 years) with AML who are ineligible for intensive chemotherapy (NCT05155709). For this Phase Ib/II trial, patients must have an Eastern Cooperative Oncology Group performance status of 0-2 (if age ≥75 years) or 0-3 (if age ≥18-74 years) with at least 1 predefined comorbidity rendering the patient ineligible for intensive chemotherapy. Patients with TP53 mutations, chromosomal del17p, or prior MDM2-inhibitor or FLT3-inhibitor treatment are ineligible. Patients will be enrolled into 2 arms: (Arm 1) patients who have received 2-4 cycles of first-line VEN+AZA without achieving a best response of CR, CR with partial hematological recovery (CRh), CRi, or morphologic leukemia-free state, and (Arm 2) newly diagnosed patients with adverse genetic risk as defined in Figure 1. The trial has 2 parts: (Part 1) a safety run-in to assess the safety and confirm the recommended Phase II dose (RP2D) of siremadlin+VEN+AZA and (Part 2) an expansion phase to evaluate the preliminary efficacy of siremadlin in combination with VEN+AZA. Treatment will be administered in 28-day cycles until disease progression, relapse, or unacceptable toxicity. In Part 1, 3-6 patients per arm will receive the starting dose of siremadlin (20 mg; once-daily oral capsule) from Day 1 to Day 5 in each 28-day treatment cycle in combination with VEN+AZA. For Arm 1, VEN and AZA will be administered at doses determined prior to trial enrollment. For Arm 2, VEN will be administered once daily at 400 mg on each day of the cycle, following a 3-day ramp-up, and AZA will be administered intravenously or subcutaneously at 75 mg/m2 either on Days 1-7, or Days 1-5 and Days 8-9 (according to standard local clinical practice). If the starting dose of siremadlin is deemed safe, an additional ~6-9 patients will receive dose level +1 (30 mg) of siremadlin to determine the RP2D for Part 2. Approximately 26 patients will receive siremadlin at the RP2D in combination with VEN+AZA in Part 2. Two primary outcomes will be assessed: in Arms 1 and 2, the proportion of patients with dose-limiting toxicities reported during the first cycle (28 days); and in Arm 1 only, the proportion of patients at the expansion dose who achieve CR. Secondary outcomes include, in Arm 2 only, proportion of patients who achieve CR, and in Arms 1 and 2, duration of CR; proportion of patients who achieve CR, or CRh, or CRi; duration of CR/CRh and CR/CRi; overall survival; pharmacokinetics; proportion of CR-MRD negative overall and MRD negativity in patients achieving a CR, CR/CRh, and CR/CRi. Conclusions: This Phase Ib/II trial investigates siremadlin in combination with VEN+AZA in patients with AML who are ineligible for intensive chemotherapy and will aid in the understanding of the effectiveness of an MDM2 inhibitor with venetoclax combination in patients with AML. The trial is in progress and expected to enroll approximately 55 patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RANKL down-regulates the mast cell proliferation through inducing senescence.

An increase in the number of mast cells could contribute to inflammatory diseases and pathologic conditions. A receptor activator of NF-κB ligand (RANKL)/RANK system is one of the key signaling pathways accelerating mast cell-mediated allergic inflammatory reactions. However, the biological functions of RANKL in mast cell proliferation remains to be clarified. The aim of the present study is to clarify the role of RANKL in mast cell proliferation. Surprisingly, RANKL remarkably reduced the proliferation of human mast cell line, HMC-1 cells through the inhibition of murine double minute 2 (MDM2) and Ki-67 mRNA expressions in a dose-dependent manner. RANKL significantly reduced cell viability, whereas it increased cellular senescence via increasing levels of p53, phosphorylated(p)-p53, p21, and p16 and decreasing levels of retinoblastoma protein (pRb) and p-pRb in HMC-1 cells. Even in rat peritoneal mast cells, RANKL induced cellular senescence by increasing filamentous-actin polymerization. In addition, RANKL remarkably reduced thymic stromal lymphopoietin (TSLP)-induced mast cell proliferation via the downregulation of MDM2 and Ki-67. RANKL decreased levels of p-signal transducer and activator of transcription 6 in TSLP-stimulated HMC-1 cells. The mast cell growth factor, interleukin-13 was remarkably down-regulated by treatment with RANKL in TSLP-stimulated HMC-1 cells. Furthermore, RANKL increased the number of senescence-associated β-galactosidase-stained cells and protein levels of p53, p-p53, and p21 in TSLP-stimulated HMC-1 cells. These data suggest that RANKL down-regulates mast cell proliferation by inducing senescence.

Phase I dose-escalation study of milademetan in patients with relapsed or refractory acute myeloid leukemia

Long-term survival in patients with acute myeloid leukemia (AML) remains low, and current treatment modalities are inadequate. Milademetan (DS-3032, RAIN-32), a small-molecule specific murine double minute 2 inhibitor, has shown a p53 status-dependent antitumor effect in vitro studies. This is the first phase I study report of milademetan monotherapy in relapsed/refractory (R/R) AML patients evaluating the safety, tolerability, pharmacokinetics, and preliminary tumor response for further clinical development. Fourteen patients received 90 (starting dose, n = 4), 120 (n = 6), or 160 mg (n = 4) of oral milademetan once daily in a 14/28 treatment cycle. The median total treatment duration was 1.5 cycles. Dose-limiting toxicity did not occur, and the maximum tolerated dose was not reached. Thus, the recommended dose was defined as 160 mg. The most common adverse events (AEs) were decreased appetite (64.3%), febrile neutropenia (50%), nausea (42.9%), and anemia (35.7%). No deaths or AEs leading to treatment discontinuation occurred. Five serious treatment-emergent AEs occurred in 4 patients. Plasma concentration increased linearly with milademetan dose. However, trends in the safety and efficacy of oral milademetan in patients with R/R AML warrant further clinical investigation. This study can inform future milademetan studies in hematologic malignancies.

Disulfiram enhances chemotherapeutic effects of doxorubicin liposomes against human hepatocellular carcinoma via activating ROS-induced cell stress response pathways.

Increasing evidences have revealed the anti-cancer effect of disulfiram. Current disulfiram-based cancer therapies still have limitations, such as poor tumor-targeting ability and insufficient studies on anti-tumor mechanisms. In the present study, tumor-targeting liposomes were prepared as drug carriers to increase retention of disulfiram in tumor cells. Then, anti-tumor efficacy of liposomes and the underlying mechanisms were investigated in in vitro, in vivo, and transcriptomic level. The results showed that disulfiram enhanced sensitivity of human hepatocellular carcinoma cells to doxorubicin by 15-27-fold, and increased reactive oxygen species (ROS) production as well as caspase-dependent apoptosis. Inhibition of tumor migration and invasion by doxorubicin were further enhanced by disulfiram. In vivo study showed that disulfiram additive doxorubicin liposomes had better performance in suppressing tumor growth than single doxorubicin liposomes. Gene expression profiling found that cellular components destruction, cell stress, check point regulation, and immunoregulation were the main anti-tumor mechanisms of disulfiram. More importantly, disulfiram possessed a great potential to be a protein ubiquitination and murine double minute 4 (MDM4) targeting compound. Due to its low price and good safety, it is worth to repurposing disulfiram as a chemotherapeutic drug. Furthermore, MDM4 may act as a biomarker for observation the clinical effect of disulfiram-based treatment.

Rapeseed peptide inhibits HepG2 cell proliferation by regulating the mitochondrial and P53 signaling pathways.

Rapeseed peptide, extracted from rapeseed protein, is known to have a variety of biological activities. In this study, the anti-proliferation effect and molecular mechanism of rapeseed peptide on HepG2 cells were investigated. In vitro anticancer experiments showed that the rapeseed peptide NDGNQPL could inhibit HepG2 cell proliferation in a concentration-dependent manner [half maximal inhibitory concentration (IC50 ), 1.56 mmol L-1 ). HepG2 cells were induced by NDGNQPL at a 0.5 mmol L-1 concentration and exhibited a 28.39 ± 0.80% apoptosis rate and a cell cycle arrest in the G0/G1 phase. Meanwhile, rapeseed peptide induced a decrease in mitochondrial membrane potential, an increase in reactive oxygen species (ROS) release, and changes in the nuclear morphology of HepG2 cells, indicating that rapeseed peptide could induce cell apoptosis through the mitochondrial pathway. In addition, rapeseed peptide activated the proliferation-related P53 signaling pathway, in which the expression levels of P53, P21, and cleaved-caspase3 were up-regulated, while the expression levels of murine double minute 2 (MDM2) were down-regulated. In molecular docking simulations, NDGNQPL exhibited a good affinity for the MDM2 molecule, which supported the notion that the rapeseed peptide is able to inhibit MDM2, a negative regulator of P53. The current results indicate that the rapeseed-derived NDGNQPL peptide has the potential to inhibit the proliferation of HepG2 cells and promote human health. © 2022 Society of Chemical Industry.

Insights into the role of P2X7R/DUSP6/ERK1/2 and SIRT2/MDM2 signaling in the nephroprotective effect of berberine against cisplatin-induced renal fibrosis in rats

AimsSeveral signaling events have been identified for mediating cisplatin-induced chronic inflammation and progressive renal fibrosis, but the majority of them have not yet been established as therapeutic targets. This study investigated the modulatory effects of berberine on purinergic 2X7 receptors (P2X7R) and some potential intracellular profibrogenic signaling as molecular mechanisms that could hinder renal fibrosis associated with cisplatin administration in rats. Main methodsFor induction of kidney injury, rats were injected with cisplatin (1 mg/kg, i.p.) daily for two weeks. Concurrently, the rats were treated with berberine (100 or 200 mg/kg, p.o). The gene expressions of P2X7R, dual-specificity phosphatase 6 (DUSP6), and murine double-minute 2 (MDM2) were determined. The expressions of alpha smooth-muscle actin and tumor necrosis factor alpha (TNF-α) were assessed by immunohistochemical staining. Phosphorylated extracellular signal-regulated kinase 1/2, (p-ERK1/2) was evaluated by western blotting. Sirtuin 2 (SIRT2), kidney injury molecule-1, and galectin-3 were measured by enzyme-linked immunosorbent assay. The degree of renal fibrosis was assessed by microscopic examination and picrosirius red staining. Key findingsBerberine effectively inhibited cisplatin-induced renal histopathological changes, enhanced renal function, and markedly mitigated inflammatory and fibrotic alterations as well as TNF-α protein expression. Additionally, P2X7R, p-ERK1/2, MDM2, and SIRT2 were suppressed and DUSP6 was upregulated by berberine. SignificanceThe nephroprotective effects of berberine were mediated in part by downregulating P2X7R and modulating DUSP6-mediated inactivation of ERK1/2 as well as by suppressing SIRT2/MDM2-triggered renal fibrosis.

POTENTIAL CARDIOPROTECTIVE EFFECT OF GENIPIN VIA CYCLOOXIDASE 2 SUPPRESSION AND P53 SIGNAL PATHWAY ATTENUATION IN INDUCED MYOCARDIAL INFARCTION IN RATS.

Background and aims: Genipin, an iridoid derived from geniposide by β-glucosidase hydrolysis, has shown potential benefit in the treatment of heart function insufficiency despite its unclear therapeutic mechanism. This study aimed to investigate the primary cardioprotective mechanism of genipin. We hypothesized that genipin demonstrated the antiapoptosis and anti-inflammation for cardiac protection by inhibiting the cyclooxidase 2 (COX2)-prostaglandin D2 (PGD2) and murine double minute 2 (MDM2)-p53 pathways. Methods: The normal Sprague-Dawley rats were made into myocardial infarction models by conventional methods. Animals were treated with genipin for 5 weeks after myocardial infarction (MI). Morphometric and hemodynamic measurements were performed 5 weeks post-MI. Biological and molecular experiments were performed after the termination. Results: Both morphometry and hemodynamics in systole and diastole were significantly impaired in the model group but restored close to basal level after treatment with genipin. Genipin also restored the post-MI upregulated expressions of cytochrome c, p53, COX2, and PGD2 and downregulated expression of MDM2 to the approximate baseline. Genipin inhibited apoptotic and inflammatory pathways to prevent post-MI structure-function remodeling. Conclusions: This study showed the cardioprotective mechanism of genipin and implied its potential clinical application for the treatment of ischemic heart failure.

232 (PB112) - Low SKP2 expression is predictive of sensitivity to an MDM2 antagonist in p53 wild-type AML

Background: Non-mutational aberrations of p53 function are a common event in AML. Using fragment-based drug discovery we have identified a Murine double minute 2 (MDM2) antagonist that potently activates p53 pathway. Here, we present the discovery of a novel patient selection strategy for MDM2 antagonist-induced apoptosis in wild-type p53 AML. Material and Methods: Cell panel screen and bioinformatics analyses of p53 wild-type AML cell lines that undergo apoptosis following Compound 1 (MDM2 antagonist) treatment versus those that do not were employed to identify novel patient selection strategy. Correlation between low levels of SKP2 (S-Phase Kinase Associated Protein 2) and induction of apoptosis following Compound 1 treatment was tested in vitro using Western Blot and flow cytometry analysis. Genetic manipulation of SKP2 levels using knockdown and over-expression approaches in wild-type p53 AML cells were used to validate the role of SKP2 expression in in vitro and in vivo experiments. Finally, bone marrow-derived primary AML blasts were tested to confirm the novel patient selection strategy. Results: Cell panel screen of p53 wild-type cell lines showed a wide range of sensitivity to Compound 1 suggesting that additional biomarkers are required to ensure patient stratification. Further bioinformatics analysis of apoptotic and non-apoptotic AML cancer cell lines following treatment with Compound 1 demonstrated differential gene expression levels and identified several potential predictive biomarkers. Western blot and flow cytometry analyses demonstrated that basal SKP2 expression level is lower in apoptotic AML cell lines than in non-apoptotic cells. Over-expression of SKP2 levels in MV4-11 cells reduced MDM2 antagonist-dependent cell death in previously apoptotic cell line. Similarly, SKP2 knockdown in OCI-AML3 cells increased MDM2 antagonist-induced cytotoxicity. Studies performed in mouse systemic AML model in vivo also demonstrated that high levels of SKP2 reduced p53 response to Compound 1. Finally, the correlation of SKP2 expression and Compound 1 sensitivity was confirmed in primary AML blasts isolated from patients. Conclusions: This work demonstrates in vitro and in vivo activity of MDM2 antagonist in AML models with low basal SKP2 levels. These pre-clinical data validate low levels of SKP2 as a novel patient selection strategy in AML and support the clinical investigation of MDM2 antagonist as therapeutic strategy for the treatment of SKP2low p53wt AML. No conflict of interest.

61P Biomarkers EGFR, p53, IDH1 and MDM2 as prognostic indicators for overall survival of glioblastoma patients

Glioblastoma (GBM) is one of the deadliest cranial tumors occurring in adults. Various biomarkers have been tested for their significance in diagnosis, prognosis, and treatment of GBM. Some well-studied markers in GBM are Isocitrate dehydrogenase1 (IDH1), Murine double minute2 (MDM2), Epidermal Growth Factor Receptor (EGFR) and p53. The aim of this study was to investigate the protein expression of these markers in GBM patients of Pakistan.

AML-143 Trial in Progress: Phase Ib/II Study of Siremadlin in Combination With Venetoclax + Azacitidine in Patients With Acute Myeloid Leukemia (AML) Who Are Ineligible for Intensive Chemotherapy (IC)

Treatments for AML patients unfit for IC are limited. Venetoclax+hypomethylating agents (HMAs) demonstrated improved survival; however, relapse rates are high, and most patients remain measurable residual disease (MRD) positive. Siremadlin is a selective inhibitor of the tumor protein 53 (p53)-murine double minute-2 (MDM2) interaction. AML patients treated with siremadlin experienced a tolerable safety profile and preliminary antitumor activity in a Phase I study (NCT02143635). Initial results from a Phase lb study exploring siremadlin+venetoclax showed promising antileukemic activity in patients with relapsed/refractory AML (NCT03940352). To describe the study design of a Phase lb/II study assessing the safety and efficacy of siremadlin+venetoclax+azacitidine in AML patients unfit for IC (NCT05155709). The study will consist of a safety run-in (part 1) and an expansion phase (part 2). Adult AML patients unfit for IC due to age (≥75 years) or comorbidities are eligible. Patients with TP53 mutation or del17p are excluded. Two subpopulations will be evaluated: patients with prior suboptimal response (no CR, CRh, CRi, or morphologic leukemia-free state) after 2-4 cycles of first-line venetoclax+azacitidine (Arm 1) and patients with newly diagnosed AML with high-risk features that confer a low likelihood of response to venetoclax+HMAs (adverse genetic risk stratification per European Leukemia Network, 2017; Arm 2). Safety run-in/part 1: siremadlin 20 or 30 mg from Day (D)1 to D5 in each 28-day cycle with venetoclax+azacitidine. Arm 1: patients will continue receiving their tolerated venetoclax+azacitidine doses prior to study enrollment. Arm 2: venetoclax will be increased over 3 days to a target daily dose of 400 mg and azacitidine (75 mg/m2) from D1-D7 or from D1-D5, and D8+D9. Upon confirmation of the recommended dose for expansion (RDE) for each arm, enrollment for the expansion phase/part 2 will open; patients will be treated with siremadlin at the RDE in combination with venetoclax+azacitidine. Primary endpoints: incidence of dose-limiting toxicities (Arms 1 and 2 separately) and CR rate (Arm 1). Secondary endpoints: safety and tolerability; CR rate (Arm 2); CR/CRh and CR/CRi rates, duration of CR, CRi, and CRh; OS; early mortality; MRD; pharmacokinetics.

CEP-1347 Targets MDM4 Protein Expression to Activate p53 and Inhibit the Growth of Glioma Cells.

The development of pharmacological inhibitors targeting negative regulators of p53, such as murine double minute (MDM) 2 and, more recently, MDM4, has been actively pursued as a potential strategy to treat cancers with wild-type p53. We previously showed that CEP-1347, a small molecule kinase inhibitor originally developed for the treatment of Parkinson's disease, suppressed MDM4 expression and activated wild-type p53 in retinoblastoma cells. However, it remains unknown whether CEP-1347 acts as an MDM4 inhibitor and as such activates p53 in other types of human cancer cells. The effects of CEP-1347 and MDM4 knockdown on the mRNA and protein expression of components of the p53 pathway, including MDM4, in human glioma cell lines with and without p53 mutation were examined by RT-PCR and western blot analyses. Trypan blue dye exclusion was used to examine the effect of CEP-1347 on cell growth. CEP-1347 decreased the expression of MDM4, increase that of p53, and activated the p53 pathway in glioma cells with wild-type p53. Knockdown-mediated inhibition of MDM4 expression in a glioma cell line with wild-type p53 that overexpresses MDM4 resulted in increased p53 expression and activation of the p53 pathway. CEP-1347 preferentially inhibited the growth of glioma cells with wild-type p53 without showing toxicity to normal cells at clinically relevant concentrations. Our findings suggest CEP-1347 is a novel inhibitor of MDM4 protein expression and as such activates p53 to inhibit the growth of cancer cells with wild-type p53, including retinoblastoma and glioblastoma.

LncRNA SNHG1 Facilitates Tumor Proliferation and Represses Apoptosis by Regulating PPARγ Ubiquitination in Bladder Cancer.

Simple SummaryOur study elucidated that SNHG1 promotes MDM2 expression by binding to miR-9-3p to promote PPARγ ubiquitination and downregulate PPARγ expression and that SNHG1 plays an important role in bladder cancer and provides a potential therapeutic target for bladder cancer.Background: Long noncoding RNAs regulate various biological effects in the progression of cancers. We found that the expression of SNHG1 was significantly up-regulated in bladder cancer after analyzing data obtained from TCGA and GEO. However, the potential role of SNHG1 remains to be investigated in bladder cancer. It was validated that SNHG1 was overexpressed in bladder cancer tissues detected by qRT-PCR and FISH, which was also associated with poor clinical outcome. Additionally, SNHG1 was verified to facilitate tumor proliferation and repress apoptosis in vitro and in vivo. Results: SNHG1 could act as a competitive endogenous RNA and decrease the expression of murine double minute 2 (MDM2) by sponging microRNA-9-3p. Furthermore, MDM2 induced ubiquitination and degradation of PPARγ that contributed to the development of bladder cancer. Conclusions: the study elucidated that SNHG1 played an important role in bladder cancer and provided a potential therapeutic target for bladder cancer.

Navtemadlin (KRT-232) in combination with avelumab for patients with anti-PD-1/L1 treatment-naïve TP53WT Merkel cell carcinoma (MCC)

Background and aims: Murine double minute 2 (MDM2) is the key negative regulator of the tumor suppressor protein p53. Upregulation of MDM2 contributes to MCC oncogenesis by inhibiting p53, thus promoting tumor proliferation. MDM2 is, therefore, a rational target in TP53WT MCC. Navtemadlin, a potent, selective, orally available MDM2 inhibitor, restores p53 function and drives tumor cell apoptosis. Preliminary analysis of the navtemadlin monotherapy cohort of the KRT-232-103 trial documents an ORR of 33% and an acceptable safety profile in 11 patients with advanced MCC who were relapsed/refractory to anti-PD-1/PD-L1 immunotherapy (Wong 2020).

Quantitation of navtemadlin in human plasma and brain tissue by liquid chromatography-tandem mass spectrometry.

Navtemadlin is an orally bioavailable small molecule that blocks the protein-protein interaction between murine double minute 2 protein (MDM2) and the tumor suppressor protein p53, leading to p53-mediated cell cycle arrest and apoptosis. It is being evaluated in clinical trials for a variety of malignancies, both as a single agent and in combination regimens. A sensitive, robust LC-tandem mass spectrometry (LC-MS/MS) method was developed to quantitate navtemadlin in plasma, and this method was also validated using brain tissue homogenate. Sample preparation involved protein precipitation of plasma or brain tissue homogenate using acetonitrile. Navtemadlin, navtemadlin glucuronide, and the internal standard, D6 -navtemadlin, were separated from microsomal incubation extracts using gradient elution and a ZORBAX XDB C18 column. Analytes were detected using a SCIEX 5500 triple quadrupole mass spectrometer in positive electrospray ionization mode. The assay range of 1-1000 ng/mL was shown to be accurate (96.1-102.0% and 95.7-104%) and precise (coefficient of variation ≤ 10.6% and ≤ 6.6%) in plasma and brain tissue homogenate, respectively. An 8000 ng/mL navtemadlin sample diluted 1:10 (v/v) with plasma was also accurately quantitated. Navtemadlin has been stable in frozen plasma at -70°C for at least 20 months. This validated LC-MS/MS method was applied to determine navtemadlin concentrations in plasma and brain tissue samples from two separate patients receiving 120 mg/day navtemadlin on protocol ABTC1604.

MTBP enhances the activation of transcription factor ETS-1 and promotes the proliferation of hepatocellular carcinoma cells

Increasing evidence indicates that the oncoprotein murine double minute (MDM2) binding protein (MTBP) can be considered a pro-oncogene of human malignancies; however, its function and mechanisms in hepatocellular carcinoma (HCC) are still not clear. In the present work, our results demonstrate that MTBP could function as a co-activator of transcription factor E26 transformation-specific sequence (ETS-1), which plays an important role in HCC cell proliferation and/or metastasis and promotes proliferation of HCC cells. Using luciferase and real-time polymerase chain reaction (qPCR) assays, MTBP was found to enhance the transcription factor activation of ETS-1. The results from chromatin co-immunoprecipitation showed that MTBP enhanced the recruitment of ETS-1 to its downstream gene’s (mmp1’s) promoter region with ETS-1 binding sites. In cellular and nude mice models, overexpression of MTBP was shown to promote the proliferation of MHCC97-L cells with low endogenous MTBP levels, whereas the knockdown of MTBP led to inhibition of the proliferation of MHCC97-H cells that possessed high endogenous levels of MTBP. The effect of MTBP on ETS-1 was confirmed in the clinical specimens; the expression of MTBP was positively correlated with the downstream genes of ETS-1, mmp3, mmp9, and uPA. Therefore, by establishing the role of MTBP as a novel co-activator of ETS-1, this work expands our knowledge of MTBP or ETS-1 and helps to provide new ideas concerning HCC-related research.

Discovery of the oncogenic MDM2, a direct binding target of berberine and a potential therapeutic, in multiple myeloma.

Recent studies have suggested the potency of berberine (BBR) for multiple cancer treatments, including multiple myeloma (MM). However, the direct target and underlying mechanism of BBR remain largely understood in MM. Here, we demonstrated that BBR inhibited cell proliferation and acted synergistically with bortezomib in MM.1S cells. BBR treatment induced MM cell cycle arrest by downregulating several cell cycle-related proteins. Murine double minute 2 (MDM2) as a BBR-binding protein was identified by surface plasmon resonance image (SPRi) analysis and molecular docking. Overexpression of MDM2 is associated with MM progression and a poor prognosis. Knockdown MDM2 by siRNA transfection can repress MM malignant progression and attenuate the BBR sensitivity to MM.1S cells. BBR treatment induced the degradation of MDM2 through the ubiquitin-proteasome system and reactivated P53/P21 in MM cells. Overall, our data has illustrated that MDM2, as a binding protein of BBR for the first time, may serve as a potential therapeutic option for MM.

IHC expression of AKT in different grades of epithelial dysplasia: An in vitro study.

Akt, also known as protein kinase B, is a serine/threonine-specific protein-kinase which plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, transcription and cell migration. The activation of Akt is one of the most frequent alterations observed in human cancer and tumour cells. Akt regulates cellular survival and metabolism by binding and regulating many downstream effectors, e.g., Nuclear Factor-kB, murine double minute 2(MDM2). To evaluate and compare immunohistochemical expression of Akt in normal epithelium and different histological grades of epithelial dysplasias. Forty paraffin-embedded tissue sections were used for the immunohistochemical study of which 10 cases of normal epithelium, 10 cases of each mild, moderate and severe epithelial dysplasia which were diagnosed by haematoxylin and eosin procedures. The tissue sections were immunohistochemically analysed for expression of Akt in different grades of epithelial dysplasia by using anti-Akt-1 monoclonal antibody. Statistical analysis was carried out using statistical package for social science (SPSS, V 10.5). The data were analysed using Chi-square test and P < 0.05 was considered statistically significant. An overall significant difference was observed when normal tissues were compared with epithelial dysplasia with a Chi-square value of 21.04, but there was no statistical significance between the three grades of epithelial dysplasias. In conclusion, this study suggests that Akt-1 overexpression can be one of the useful diagnostic markers for predicting the potential behaviour of oral dysplasias transforming into oral squamous cell carcinoma (OSCC).

Inhibitory effects of LOXL2 knockdown on cellular functions of liver cancer stem cells.

BackgroundLysyl oxidase-like 2 (LOXL2) plays a role in tumor microenvironment formation and metastasis of hepatocellular carcinoma (HCC), which has a high mortality burden. Liver cancer stem cells (LCSCs) are related with the major malignant phenotypes of HCC. The function of LOXL2 in regulation of LCSCs remains unknown.MethodsCD133+HepG2 and CD133+Hep3B cells were sorted by fluorescence-activated cell sorting (FACS) from two human hepatoblastoma cell lines. Spheroid formation, apoptosis, cell cycle, as well as transwell assays were performed upon LOXL2 knockdown in CD133+HepG2 and CD133+Hep3B cells. Protein and mRNA levels were quantified by Western blotting, immunofluorescence and reverse transcription-PCR (RT-PCR).ResultsKnockdown of LOXL2 decreased spheroid formation, migration and invasion (P<0.05), also induced apoptosis (P<0.05) and cell cycle arrest (P<0.05) in CD133+HepG2 and CD133+Hep3B cells. Knockdown of LOXL2 effectively inhibited expression of the anti-apoptosis proteins baculoviral inhibitor of apoptosis protein (IAP) repeat-containing 3 (BIRC3) and murine double minute 2 (MDM2) (P<0.01), as well as autophagy marker microtubule-associated protein 1 light chain 3 B (LC3B) and autophagy gene ATG5 in CD133+HepG2 and CD133+Hep3B cells (P<0.01).ConclusionsThe results revealed that LOXL2 inhibition could reduce the proliferation and expansion of LCSCs, making LOXL2 inhibitors an attractive and novel therapeutic strategy of HCC.

PB1849: TRIAL IN PROGRESS: PHASE IB/II STUDY OF SIREMADLIN IN COMBINATION WITH VENETOCLAX + AZACITIDINE IN PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) WHO ARE INELIGIBLE FOR INTENSIVE CHEMOTHERAPY

Background: Treatment options for patients with AML who are not candidates for intensive chemotherapy (IC) are limited. The addition of venetoclax to hypomethylating agents demonstrated improved remission rates and survival; however, relapse rates are high, and most patients remain measurable residual disease (MRD) positive (DiNardo CD, N Engl J Med, 2020). Murine double minute-2 (MDM2) is a key negative regulator of tumor protein 53 (p53). Inhibition of the p53-MDM2 interaction results in up-regulation of the tumor-suppressive effect of p53. Siremadlin is a selective inhibitor of the p53-MDM2 interaction. Siremadlin + venetoclax has shown durable antitumor responses in p53-wild-type AML patient-derived xenograft models (Wang Y, Cancer Res, 2019). Findings from a first-in-human Phase I study with siremadlin demonstrated a tolerable safety profile and preliminary antitumor activity in patients with AML (Stein EM, Clin Cancer Res, 2021; NCT02143635). Preliminary results from the ongoing Phase Ib study exploring siremadlin in combination with venetoclax showed promising antileukemic activity in patients with relapsed/refractory AML (Wei AH, ASH 2021; NCT03940352). Aims: Here we describe the study design of a Phase Ib/II study to assess the safety and efficacy of the novel combination of siremadlin + venetoclax + azacitidine in patients with AML who are unfit for IC (NCT05155709). Methods: The study will enroll approximately 55 adult patients with AML who are unfit for IC due to older age (≥75 y) or presence of comorbidities (cardiac or pulmonary, Eastern Cooperative Oncology Group performance status of 2 or 3). Patients with prior myelodysplastic syndrome, myelofibrosis, essential thrombocythemia, polycythemia, or therapy-related AML may also be eligible. Patients with TP53 mutation or del17p are excluded. Two subpopulations will be evaluated in 2 arms: patients with prior suboptimal response (no complete remission [CR], CR with partial/incomplete hematologic recovery [CRh/CRi], or morphologic leukemia-free state) after 2-4 cycles of first-line venetoclax + azacitidine (Arm 1) and patients with newly diagnosed AML with high-risk clinical features that confer a low likelihood of response to venetoclax + hypomethylating agents (adverse genetic risk stratification per European Leukemia Network, 2017; Arm 2). The study will consist of a safety run-in (Part 1) and expansion phase (Part 2; Figure). The safety run-in will evaluate safety across 2 siremadlin dose levels (20 or 30 mg) from Day (D)1 to D5 in each 28-d treatment cycle in combination with venetoclax and azacitidine. In Arm 1, pts will continue receiving their venetoclax and azacitidine doses that were evaluated as tolerable prior to study enrollment. In Arm 2, venetoclax will be ramped up over 3 days to a target daily dose of 400 mg and azacitidine (75 mg/m2) will be administered from D1–D7 or from D1–D5, and D8 and D9). Upon confirmation of the recommended dose for expansion (RDE, determined for each arm separately), enrollment for the expansion phase (Part 2) will open; patients will be treated with siremadlin at the RDE in combination with venetoclax + azacitidine. The primary endpoints are incidence of dose-limiting toxicities (Arm 1 and Arm 2 separately) and CR rate (Arm 1 only). Secondary endpoints include safety and tolerability; CR rate (Arm 2 only); CR/CRh and CR/CRi rates; duration of CR, CRi, and CRh; overall survival; early mortality; MRD; and pharmacokinetics. Results: None, trial in progress. Image:Summary/Conclusion: None, trial in progress.