Murine Double Minute 2 Inhibitor Research Articles

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Impacts of Nutlin-3a and exercise on murine double minute 2-enriched glioma treatment.

JOURNAL/nrgr/04.03/01300535-202504000-00029/figure1/v/2024-07-06T104127Z/r/image-tiff Recent research has demonstrated the impact of physical activity on the prognosis of glioma patients, with evidence suggesting exercise may reduce mortality risks and aid neural regeneration. The role of the small ubiquitin-like modifier (SUMO) protein, especially post-exercise, in cancer progression, is gaining attention, as are the potential anti-cancer effects of SUMOylation. We used machine learning to create the exercise and SUMO-related gene signature (ESLRS). This signature shows how physical activity might help improve the outlook for low-grade glioma and other cancers. We demonstrated the prognostic and immunotherapeutic significance of ESLRS markers, specifically highlighting how murine double minute 2 (MDM2), a component of the ESLRS, can be targeted by nutlin-3. This underscores the intricate relationship between natural compounds such as nutlin-3 and immune regulation. Using comprehensive CRISPR screening, we validated the effects of specific ESLRS genes on low-grade glioma progression. We also revealed insights into the effectiveness of Nutlin-3a as a potent MDM2 inhibitor through molecular docking and dynamic simulation. Nutlin-3a inhibited glioma cell proliferation and activated the p53 pathway. Its efficacy decreased with MDM2 overexpression, and this was reversed by Nutlin-3a or exercise. Experiments using a low-grade glioma mouse model highlighted the effect of physical activity on oxidative stress and molecular pathway regulation. Notably, both physical exercise and Nutlin-3a administration improved physical function in mice bearing tumors derived from MDM2-overexpressing cells. These results suggest the potential for Nutlin-3a, an MDM2 inhibitor, with physical exercise as a therapeutic approach for glioma management. Our research also supports the use of natural products for therapy and sheds light on the interaction of exercise, natural products, and immune regulation in cancer treatment.

Navtemadlin, a Novel MDM2 Inhibitor, Potentiated Venetoclax-Induced Antitumor Efficacy in TP53 Wild-Type Acute Myeloid Leukemia (AML)

Introduction: The tumor suppressor p53 ( TP53) maintains genomic integrity, and nearly 50% of cancers are characterized by a loss or mutation in the TP53 gene. Murine double minute 2 (MDM2) negatively regulates p53 expression and promotes oncogenesis in TP53 wild-type ( TP53 WT) tumors. Navtemadlin (nvtm) is a potent, selective, orally available MDM2 inhibitor that overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis in TP53 WT tumors. AML is a heterogeneous hematological malignancy resulting from the transformation of stem cells and is characterized by poor therapy response. Thus, combination therapies are required to avoid tumor escape mechanisms, such as alterations in apoptotic machinery and metabolism. Venetoclax (ven), a BCL-2 inhibitor, is a promising therapy in AML. Overexpression of anti-apoptotic members of the BCL-2 family, including myeloid cell leukemia-1 (MCL-1) limits ven response, thus requiring MCL-1 inhibitors to further improve sensitivity. A proof-of-concept study with idasanutlin (idasa), a first-generation MDM2 inhibitor and ven demonstrated manageable safety and encouraging efficacy in relapsed/refractory AML patients (Daver et al. Blood 2023). Here, we present a preclinical evaluation of nvtm alone and in combination with ven in the setting of TP53 WT AML, with a focus on metabolic pathways, pro-survival signaling, and bone marrow (BM) niche. Methods: AML-derived cell line, MOLM-13 ( TP53 WT, FLT3-ITD) was cultured alone, with HS5 AML stromal cells or with cytokines. Cultures were treated with DMSO, nvtm (0.1-1.5 μM), or ven (0.5 μM) for short-term (4-8 hrs) or long-term (48-72 hrs) and analyzed for changes in cell cycle progression, viability, clonogenicity potential, and metabolism (glycolysis and mitochondrial oxidative phosphorylation by analyzing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) respectively). Primary AML BM mononuclear cells (MNCs) were cultured in cytokine support media. Results: In MOLM-13 cells, short-termnvtm (0.5 µM) treatment rapidly restored p53 expression and phosphorylation, with greater efficacy than idasa. Long-term treatment significantly induced apoptosis in a dose-dependent manner from 0.25 μM (p<0.01) with 80% cytotoxicity at 0.75 μM (p<0.001). Cell cycle analysis at 48 hr showed that nvtm significantly arrested cells in the sub-G0-G1 phase. Clonogenicity potential was significantly lowered in nvtm-treated cells. Metabolic alterations in the Mito Stress and Glycostress tests showed nvtm significantly lowered EACR, OCR, and mitochondrial membrane potential, as compared to idasa. We next evaluated the effects of nvtm in ven-treated MOLM-13 cells. Combination treatment significantly lowered the metabolism, cell viability, and clonogenicity potential of MOLM-13 cells, compared to nvtm or ven treatment. Immunoblotting analysis revealed a reduction in key anti-apoptosis proteins (BCL-2, MCL-1, PARP). Strikingly, nvtm downregulated ven-induced MCL-1 expression in a dose-dependent manner. Culturing MOLM-13 cells in HS5-conditioned media, transwells overlying HS5 cells, or with HS5 cells increased MOLM-13 cell resistance to nvtm. However, in HS5 co-cultures, a combination of nvtm and ven strongly re-sensitized MOLM-13 cells for apoptosis. Nvtm also significantly blocked the cytoprotective effects of AML-relevant cytokines (IL-6, SCF, FLT3 ligand, TPO, and GM-SCF). In cultures of primary AML BM-MNCs (n=11), nvtm alone at clinically relevant concentrations reduced viability (14-40%), and a combination nvtm and ven significantly enhanced AML cytotoxicity (30-52%). Conclusions: Nvtm, a potent second-generation MDM2 inhibitor, rapidly restored p53 function, induced apoptosis in p53 WT AML MOLM-13 by impeding cell cycle, viability, clonogenicity potential, and reducing both glycolysis and oxidative phosphorylation. Nvtm combined with ven significantly enhanced these effects. Importantly, this combination blocked stromal cell-mediated (contact and soluble factors) cytoprotection. These encouraging results provide a mechanistic basis for the evaluation of this combination in vivo and as a therapeutic strategy to overcome documented ven resistance mechanisms in TP53 WT AML patients.

Population Pharmacokinetic and Pharmacodynamic Analysis of Navtemadlin in Patients with Relapsed and Refractory (R/R) Myelofibrosis (MF) and Other Myeloid or Solid Tumor Malignancies

Background:Murine double minute 2 (MDM2), a key negative regulator of the tumor suppressor protein p53, is overexpressed in malignancies such as MF and Merkel cell carcinoma (MCC). Navtemadlin is a potent, selective, orally available MDM2 inhibitor that overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis of TP53WT tumors. Navtemadlin has been tested in 21- or 28-day treatment cycles, with 5 or 7 consecutive days of dosing per cycle. Navtemadlin elicits plasma concentration-dependent increases in serum macrophage inhibitory cytokine-1 (MIC-1). An increase in MIC-1 relative to baseline MIC-1 serum concentration is a key pharmacodynamic (PD) marker of response to navtemadlin treatment (Zhang et al. Xenobiotica 2022). A population pharmacokinetic (PK) model was developed, based on patients with R/R MF (N=113, NCT03662126); MCC (N=35, NCT03787602); solid tumors and multiple myeloma (ST/MM N=105, Gluck et al. Invest New Drugs 2019); R/R acute myeloid leukemia (AML, N=35, Erba et al. Blood Adv 2019); and healthy volunteers (HV, N=30, Wong et al. Clin Pharmacol Drug Dev 2022).The objectives wereto characterize navtemadlin PK and covariate effects and compare steady-state (SS) PK and PD across cancer types. Methods:A two-compartment PK model with first-order absorption and elimination was estimated using NONMEM ® (first-order conditional estimation with interaction). Four transit compartments captured absorption. Inter-subject variability was modeled as lognormal. A stepwise covariate search used forward addition (p<0.05) followed by backward elimination (p<0.01). Log-transformed SS MIC-1 ratio to baseline (fold ratio) was tested in a fixed-effects linear regression model with respect to SS exposures, with baseline MIC-1 as the covariate. The final population PK and MIC-1 models were evaluated by inspection of parameter tables, by overlaying model predictions with observed data, and by standard residual diagnostics. Results:The PK estimation used 5,282 concentrations from 318 subjects, including 1,415 concentrations in 113 patients with R/R MF. Tumor type affected absorption rate (median, 0.635/hr in MCC; slower vs other types), apparent clearance (median, 8.90 L/hr in MCC; lower vs other types), and apparent central volume (median, 64.4 L in MCC; similar to AML, but lower vs other types) (Table 1). Accordingly, simulated SS area under the curve (AUCss) differed across populations in the order MCC>AML>MF>ST/MM = HV (Figure 1). Covariate analysis indicated median AUC increased with the inflammation marker C-reactive protein, by 21% at 90 th percentile CRP, and with age, by 5% at 90 th percentile age of 75 y, compared to the median, 66 y. Men had 24% lower relative bioavailability than women. Taking navtemadlin with food slowed absorption by 10% but had no impact on AUCss. Other tested covariates, including body weight, creatinine clearance, aspartate transaminase, alanine transaminase, bilirubin, albumin, alpha 1-acid glycoprotein, race, UGT1A1 genotype, recent ruxolitinib treatment in MF, and CYP3A4 inducer/inhibitor concomitant medications, were not significant. Median MIC-1 fold ratio increased with navtemadlin AUCss. Higher baseline MIC-1 resulted in lower SS MIC-1 fold ratio. Median baseline MIC-1 differed by tumor type. In R/R MF, baseline MIC-1 was 2,297 ng/L and SS fold ratio of MIC-1 was 7.79 (N=91). In MCC, baseline MIC-1 was 1,718 ng/L and SS fold ratio of MIC-1 was 16.7 (N=29). In R/R MF, higher baseline spleen volume was also associated with lower SS MIC-1 fold ratio. Exposure-MIC-1 relationships in R/R MF and MCC were generally similar to other tumor types and HV populations (Allard et al. Hemasphere 2019; Zhang et al. Xenobiotica 2022). Conclusions:Tumor type influenced navtemadlin exposure with simulated exposure in R/R MF patients at the selected Phase 3 dose of 240 mg comparable to exposures in MCC patients at 180 mg, the selected Phase 2 dose for that indication. Cancer-associated inflammation and/or treatment history, as well as age and female sex, may increase navtemadlin exposure. The PD marker MIC-1 responds to navtemadlin in a concentration-dependent fashion. The overall SS fold change of MIC-1 PD responses was lower in disease states or subjects with higher baseline MIC-1. These studies demonstrate potent, dose-dependent PK/PD responses following p53 activation with the novel MDM2 inhibitor, navtemadlin, across diverse tumor types.

Identification of MDM2 as a prognostic and immunotherapeutic biomarker in a comprehensive pan-cancer analysis: A promising target for breast cancer, bladder cancer and ovarian cancer immunotherapy

BackgroundThe murine double minute 2 (MDM2) gene is a crucial factor in the development and progression of various cancer types. Multiple rigorous scientific studies have consistently shown its involvement in tumorigenesis and cancer progression in a wide range of cancer types. However, a comprehensive analysis of the role of MDM2 in human cancer has yet to be conducted. MethodsWe used various databases, including TIMER2.0, TCGA, GTEx and STRING, to analyze MDM2 expression and its correlation with clinical outcomes, interacting genes and immune cell infiltration. We also investigated the association of MDM2 with immune checkpoints and performed gene enrichment analysis using DAVID tools. ResultsThe pan-cancer MDM2 analysis found that MDM2 expression and mutation status were observably different in 25 types of cancer tissue compared with healthy tissues, and prognosis analysis showed that there was a significant correlation between MDM2 expression and patient prognosis. Furthermore, correlation analysis showed that MDM2 expression was correlated with tumor mutational burden, microsatellite instability and drug sensitivity in certain cancer types. We found that there was an association between MDM2 expression and immune cell infiltration across cancer types, and MDM2 inhibitors might enhance the effect of immunotherapy on breast cancer, bladder cancer and ovarian cancer. ConclusionsThe first systematic pan-cancer analysis of MDM2 was conducted, and it demonstrated that MDM2 was a reliable prognostic biomarker and was closely related to cancer immunity, providing a potential immunotherapeutic target for breast cancer, bladder cancer and ovarian cancer.

Exploratory novel biomarker and resistance mechanism of milademetan, an MDM2 inhibitor, in amplified MDM2 intimal sarcoma from an open-label phase 1b/2 trial (NCCH1806/MK004).

11544 Background: Amplified Murine double minute 2 ( MDM2) is found in > 70% of intimal sarcoma, known as one of the ultra-rare sarcomas. Milademetan (DS3032, RAIN-32) is a novel, specific, small-molecule MDM2 inhibitor that disrupts MDM2 and the tumor suppressor protein p53 interactions in tumor cells. We conducted a phase 1b/2 trial (Trial registration No: JMA-IIA00402) in patients with amplified MDM2 wild-type TP53 intimal sarcoma as a sub-study under the nationwide large registry for rare cancers in Japan (MASTERKEY Project). Eleven patients were enrolled, and ten were included in the efficacy analysis. Two (20%) patients had durable responses for > 15 months. Milademetan provided clinical benefits in patients with amplified MDM2 intimal sarcoma. Predictive biomarkers other than amplified MDM2 and acquired resistance mechanisms for milademetan are unknown. Methods: Whole-exome and RNA sequencing analyses of pre-treatment tissue samples were conducted to identify determinants of response. Genomic alterations were analyzed for 10 patients, and gene expression was analyzed for 9 patients using their pre-treatment tissue samples. Targeted sequencing of cell-free DNA (cfDNA) samples (liquid biopsy) was also conducted sequentially at three points [before treatment with milademetan (baseline), at Cycle 2 Day1, and at the time of disease progression] to identify determinants of response and resistance. Results: From whole-exome and RNA sequencing analyses of pre-treatment tissue samples, we could not find any molecular pathways associated with the anti-tumor activity of milademetan. Focusing on 8 genes ( CDK4, CDKN2A, CDKN2B, EGFR, ERBB3, MDM2, PDGFRA, TP53) known to be frequently affected in intimal sarcoma and 10 genes ( AKT1, ATM, BBC3, CDKN1A, CDKN1C, CHEK2, MDM4, PMAIP1, PPM1D, TWIST1) reported to be associated with MDM2 inhibitor responses; we found that anti-tumor activity correlated with amplified TWIST1 ( p-value = 0.028) and negatively with CDKN2A loss ( p-value = 0.071). Eight of the 10 patients had their cfDNA collected sequentially at baseline, at Cycle 2 Day1 and disease progression; however, one did not consent to the exploratory analysis study using cfDNA, and one had cfDNA collected at baseline but not at disease progression due to ongoing treatment. Of the eight patients, TP53 mutations in cfDNA were detected in one and five patients at baseline and disease progression, respectively. The cfDNA allele frequency of TP53 mutations increased with disease progression. Conclusions: CDKN2A loss and amplified TWIST1 could be associated with the anti-tumor activity of milademetan in patients with amplified MDM2 intimal sarcoma. Acquired TP53 mutations were detected in sequential liquid biopsies as loss-of-function mutations, and these TP53 mutations might compromise the anti-tumor activity. Clinical trial information: JMA-IIA00402 .

A phase 0/Ia study of BI 907828 concentrations in brain tissue and a nonrandomized, open-label, dose escalation study of BI 907828 in combination with radiotherapy in patients with newly diagnosed glioblastoma (GBM).

TPS2081 Background: Murine double minute 2 (MDM2) directly regulates the stability of the tumor suppressor p53, and dysregulation of this pathway can critically disrupt cellular response to genomic stress and lead to cell death. BI 907828 is a highly potent inhibitor of the MDM2-p53 interface that triggers accumulation of p53 and subsequent apoptosis in multiple GBM patient-derived xenografts (PDXs) with wild-type p53. Moreover, the combination of BI 907828 and radiotherapy provides significant survival extension in multiple orthotopic GBM PDXs. Methods: An integrated Phase 0/1 trial design is being used to evaluate the potential for BI 907828 to achieve a therapeutic concentration in brain tumor tissue and to test the safety of the combination with radiation. The co-primary endpoints for the Phase 0 study are i) measured total concentration of BI 907828 and ii) calculated unbound concentration of BI 907828 in contrast and non-contrast enhancing regions of tumor. Key inclusion criteria for the Phase 0 study are patients at least 18 years of age, ECOG performance status of 0 or 1, histologic or radiologic new diagnosis of GBM, and planned surgical resection. The first approximately 6 patients enrolled in the Phase 0 will be treated at the starting dose of 30 mg, and the future patients enrolled will be dosed at higher levels (45 mg or 60 mg) provided there are no safety concerns. If dose-limiting toxicities (DLTs) are encountered, then a Bayesian Logistic Regression Model (BLRM) with overdose control will be used to guide the dose level decisions for the additional patients. Multiple, image-registered tumor samples and intra-operative plasma samples will be analyzed for BI 907828 by LC-MS/MS. Intra-tumoral variation in BI 907828 will be correlated with pharmacodynamic effects of MDM2 inhibition (elevation of p53, p21, MDM2, GDF15, PUMA). Those patients completing the Phase 0 study and who meet additional eligibility criteria can enroll on the Phase 1a portion of the study. The pathologic inclusion criteria are TP53 wild-type, IDH1/2 wild-type, MGMT unmethylated GBM. Co-primary endpoints for the Phase 1a trial are i) occurrence of DLTs during and within 21 days after completion of radiation therapy, and ii) occurrence of adverse events throughout the treatment period. Tolerability of inter-patient dose-escalation of BI 907828 (30, 45, 60 mg) combined with radiation therapy (60 Gy in 30 fractions) will be evaluated with dose-escalation guided by a two-parameter BLRM with overdose control. Patients will remain on BI 907828 monotherapy following completion of radiotherapy until progression. To date, two patients have enrolled on the Phase 0 study, and one patient went on to enroll on the Phase 1a portion of the study. Clinical trial information: NCT05376800 .

Synthesis and Antineoplastic Activity of a Dimer, Spiroindolinone Pyrrolidinecarboxamide.

The mutation or function loss of tumour suppressor p53 plays an important role in abnormal cell proliferation and cancer generation. Murine Double Minute 2 (MDM2) is one of the key negative regulators of p53. p53 reactivation by inhibiting MDM2-p53 interaction represents a promising therapeutic option in cancer treatment. Here, to develop more effective MDM2 inhibitors with lower off-target toxicities, we synthesized a dimer, spiroindolinone pyrrolidinecarboxamide XR-4, with potent MDM2-p53 inhibition activity. Western blotting and qRT-PCR were performed to detect the impact of XR-4 on MDM2 and p53 protein levels and p53 downstream target gene levels in different cancers. Cancer cell proliferation inhibition and clonogenic activity were also investigated via the CCK8 assay and colony formation assay. A subcutaneous 22Rv1-derived xenografts mice model was used to investigate the in vivo anti-tumour activity of XR-4. The results reveal that XR-4 can induce wild-type p53 accumulation in cancer cells, upregulate the levels of the p53 target genes p21 and PUMA levels, and then inhibit cancer cell proliferation and induce cell apoptosis. XR-4 can also act as a homo-PROTAC that induces MDM2 protein degradation. Meanwhile, the in vivo study results show that XR-4 possesses potent antitumour efficacy and a favourable safety property. In summary, XR-4 is an interesting spiroindolinone pyrrolidinecarboxamide-derivative dimer with effective p53 activation activity and a cancer inhibition ability.

Abstract P6-10-10: Genetic alterations in breast cancer associated with MDM2 dependency and sensitivity to the MDM2 inhibitor milademetan

Abstract Background: Murine double minute 2 (MDM2), a potent negative regulator of p53, promotes tumorigenesis if dysregulated. MDM2 dysregulation occurs via different mechanisms, including MDM2 gene amplification, MDM2 overexpression, and loss of cyclin-dependent kinase inhibitor 2A (CDKN2A), which encodes the MDM2 regulator p14ARF. Combined inactivation of MDM2 and GATA3 in hormone receptor-positive (HR+) breast cancer is lethal to the cell. Pharmacologic inhibition of MDM2 is a rational therapeutic strategy for MDM2-dependent, TP53 wildtype (WT) tumors, including tumors with MDM2 amplification or CDKN2A loss, and GATA3-mutant HR+ breast cancers. We determined the frequency and associated characteristics of genetic alterations of MDM2-dependent breast cancers, and evaluated sensitivity of these tumors to the small-molecule MDM2 inhibitor milademetan (RAIN-32). Methods: Genomic data were obtained from three datasets: METABRIC; TCGA PanCancer Atlas, GDC v23.0 (April 2020); AACR Genie, v11. Among TP53 WT breast cancer samples from each dataset, the frequency of GATA3 frameshift mutations, MDM2 amplification (copy number [CN] ≥12), and CDKN2A homozygous loss was determined individually and as co-alterations. The antitumoral activity of milademetan was evaluated in a GATA3-mutant, TP53 WT HR+ breast cancer cell line (MCF7 GATA3 G336fs*17), a breast xenograft model (MCF7 GATA3 G335fs), and ex vivo in MDM2-amplified patient-derived breast cancer organoids (CTG-2810, ER+/PR+/HER2–, MDM2 CN 8). Results: Genetic alteration frequencies in TP53 WT breast cancers by dataset are shown in the Table. GATA3 frameshift mutations (7.3–11.7%), MDM2 amplification (0.3–1.1%), and CDKN2A loss (0.2–1.2%) occurred across breast tumors, but were found with highest frequencies in HR+ tumors. Co-alteration frequencies in TP53 WT breast cancers across the aforementioned datasets were < 1%: GATA3 mutations/MDM2 CN ≥12 (0.2–0.3%); GATA3 mutations/CDKN2A loss (0.1–0.2%); MDM2 CN ≥12/CDKN2A loss (0%). Mean MDM2 expression (log2 (TPM+1)) in HR+ breast cancers (TCGA) were: GATA3 mutations, 5.12; CDKN2A loss, 5.88; MDM2 CN ≥12, 8.13, TP53 WT without these alterations, 4.78; mutant TP53, 4.35. A GATA3-mutant ER+ breast cancer cell line was sensitive to milademetan in vitro (IC50 126 nM). Milademetan 100 mg/kg displayed antitumor activity in GATA3-mutant HR+ breast xenograft and PDX models (p< 0.05 vs. vehicle). Milademetan also displayed activity in MDM2-amplified HR+ breast cancer organoids (IC50 0.2 μM). In a phase I study (NCT01877382), a patient with heavily pretreated MDM2-amplified breast cancer (MDM2 CN 16.8) had tumor shrinkage (18.2%) and PFS of 7.3 months with milademetan (orally 260 mg 3/14 days). Conclusions: The frequency of genetic alterations potentially targetable by MDM2 inhibition among TP53 WT breast cancers (i.e., GATA3 mutations, MDM2 amplification, and CDKN2A loss) is greatest in the HR+ subset, and these genetic biomarkers are associated with higher MDM2 expression. Preclinical data show that the MDM2 inhibitor milademetan has antitumor activity in GATA3-mutant and MDM2-amplified HR+ breast cancers, and support the clinical evaluation of milademetan in these tumors. Two clinical trials of milademetan – MANTRA-2 (Phase 2 basket study in solid tumors with TP53 WT and MDM2 CN ≥12; NCT05012397) and MANTRA-4 (Phase 1 study of milademetan + atezolizumab in solid tumors with CDKN2A loss) – are enrolling patients or in planning. Table. Citation Format: Francois-Clement Bidard, Diana Bello Roufai, Arielle J. Medford, Vijaya Tirunagaru, Robert C. Doebele, Aditya Bardia. Genetic alterations in breast cancer associated with MDM2 dependency and sensitivity to the MDM2 inhibitor milademetan [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-10.

Synthesis, Docking Study, and Biological Evaluation of 2‐Phenylchroman‐4‐one Derivatives as Murine Double Minute 2 (MDM2) Inhibitors

AbstractInhibiting the interaction between the p53 tumor suppressor and its negative regulator murine double minute 2 (MDM2) is a promising therapeutic opportunity in cancer drug research. Herein, we describe the design, synthesis, and in vitro screening of phenyl chroman‐based derivatives 7 a–l as MDM2 inhibitors. Among target compounds, 7 e, 7 h, and 7 j had considerable cytotoxicity activity at concentrations of 50 μM and 100 μM against MCF7 and HCT‐116 cell lines. Designed compounds that demonstrated good toxicity, were selected for enzyme inhibition assay. The most potent MDM2 inhibitor in this series is compound 7 h, which showed a Ki value of 13.66 μM. Moreover, compound 7 h with acceptable safety profiles in normal cells was found the least effective compound against human umbilical vein endothelial cells (HUVECs). In addition, cell cycle analysis was also investigated for representative compound 7 h. Also, the docking study was done to predict the possible interaction between the synthesized compound and both MDM2 and murine double minute x (MDMX) proteins. Interestingly, the docking results were in good agreement with the experimental assay.

Reduced murine double minute 2 and 4 protein, but not messenger RNA, expression is associated with more severe disease in myelodysplastic syndromes and acute myeloblastic leukaemia.

The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34+ /CD45low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.

BOREAS: aglobal, phase III study of the MDM2 inhibitor navtemadlin (KRT-232) in relapsed/refractory myelofibrosis.

Patients with myelofibrosis (MF) who discontinue ruxolitinib due to progression/resistance have poor prognoses. JAK inhibitors control symptoms and reduce spleen volumes with limited impact on underlying disease pathophysiology. Murine double minute 2 (MDM2), a negative regulator of p53, is overexpressed in circulating malignant CD34+ MF cells. The oral MDM2 inhibitor navtemadlin (KRT-232) restores p53 activity to drive apoptosis of wild-type TP53 tumor cells by inducing expression of pro-apoptotic Bcl-2 family proteins. Navtemadlin demonstrated promising clinical and disease-modifying activity and acceptable safety in a phase II study in patients with relapsed/refractory MF. The randomized phase III BOREAS study compares the efficacy and safety of navtemadlin to best available therapy in patients with MF that is relapsed/refractory to JAK inhibitor treatment.

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

Development of KT-253, a Highly Potent and Selective Heterobifunctional MDM2 Degrader for the Treatment of Acute Myeloid Leukemia

The murine double minute 2 (MDM2) oncoprotein is a key E3 ubiquitin ligase that degrades and thereby inactivates the tumor-suppressor p53. Targeting of the MDM2/p53 interaction with reversible small molecule inhibitors (SMI) to stabilize p53 and induce apoptosis in wildtype (WT) p53 tumors has been an emerging therapeutic approach in AML and in other WT p53 hematologic and solid tumor malignancies. However, recent clinical trials with MDM2 inhibitors, especially in R/R AML, have resulted in suboptimal clinical activity, highlighting the need for novel therapeutic approaches. KT-253 is a novel, highly potent heterobifunctional MDM2 degrader that suppresses p53-dependent MDM2 protein upregulation that is known to be triggered by the MDM2 SMIs and thereby limits their clinical activity. Previously, we have shown that KT-253 has superior activity compared to MDM2 SMIs, demonstrating >200-fold improvements in both in vitro cell growth inhibition and apoptosis. Because of its superior pharmacological profile, a single dose of KT-253 was sufficient to induce rapid apoptosis and sustained tumor regression in the MV4;11 AML and RS4;11 ALL cell line-derived (CDX) mouse xenograft models, supporting an intermittent dosing schedule of KT-253. Because of the initial promising clinical activity of SMIs in AML, and the potential for KT-253 to provide superior activity, we assessed the activity of KT-253 in AML patient-derived xenograft (PDX) models. In vivo, KT-253 administered once every 3 weeks at 1 mg/kg led to tumor regression in a variety of these models, including CTG-2227 (Figures 1A and B). In addition, in an AML CDX model resistant to venetoclax such as MOLM13, KT-253 administered in combination with venetoclax once every three weeks showed more substantial activity than the single agents alone and led to sustained tumor regression. In summary, intermittent dosing of the clinical candidate KT-253 achieved tumor regression in a variety of AML PDX and CDX models. Combination of KT-253 with the AML standard of care treatment venetoclax achieved durable tumor regression in additional AML xenograft models that are resistant to standard of care, suggesting potential benefit to an expanded patient population. In addition, we have identified hematological and solid tumor indications that respond acutely to MDM2 degradation, we plan to share more details at upcoming meetings. 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

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).

Abstract CT123: A phase 2 study of the MDM2 inhibitor milademetan in patients with TP53-wild type and MDM2-amplified advanced or metastatic solid tumors (MANTRA-2)

Abstract Background: Murine double minute 2 (MDM2) is a potent negative regulator of the tumor suppressor p53. MDM2 induces degradation of p53 and promotes tumorigenesis in solid tumors, and preclinical models have shown that inhibition of MDM2 can restore p53 tumor suppressor activity in TP53-wild type (WT), MDM2-amplified tumors. We performed a mutual exclusivity analysis of patients with solid tumors (n=42,125; AACR Project GENIE) and found that the frequency of co-occurring TP53 mutations decreased with increasing MDM2 copy number. An MDM2 copy number of 12 was chosen as the threshold. An estimated 1.1% of solid tumors meet this molecular criteria, excluding glioblastomas, de-differentiated liposarcomas, and intimal sarcomas where this signature is enriched. Milademetan (RAIN-32), an oral, selective MDM2 inhibitor, inhibits growth of TP53-WT/MDM2-amplified cell lines and patient-derived xenograft models from varying tumor types. Furthermore, tumor regression was observed in 3/3 non-liposarcoma patients with MDM2 copy number &amp;gt;12 in a phase 1 trial of milademetan. MANTRA-2 (RAIN-3202) is a phase 2, multicenter, single-arm, open-label, basket trial designed to evaluate the efficacy or clinical benefit of milademetan in TP53-WT solid tumors with MDM2 amplification (copy number ≥12). Methods: Eligible patients must be ≥18 years of age with histologically and/or cytologically confirmed locally advanced, incurable or metastatic solid tumors refractory to standard therapy. Local testing demonstrating TP53 WT and MDM2 amplification is required, defined as a MDM2 copy number ≥12 or 6-fold increase. Patients with well-differentiated/de-differentiated liposarcomas, intimal sarcomas, or primary central nervous system tumors are excluded. Prior treatment with an MDM2 inhibitor is not permitted. Patients receive milademetan 260 mg orally once daily on Days 1-3 and 15-17 of a 28-day cycle. Tumor response is evaluated by RECIST v1.1 at Weeks 8, 16, 24, and 32, and then every 12 weeks. Primary endpoint: objective response rate. Secondary endpoints include: duration of response; progression-free survival; growth modulation index; disease control rate; overall survival; safety; health-related quality of life scores. Exploratory endpoints include: biomarkers in blood and/or tumor tissue; pharmacodynamics; pharmacokinetics. Enrollment of 65 patients is planned to ensure that 57 patients have centrally confirmed TP53 WT and MDM2 copy number ≥12. The trial opened in November 2021 and is actively enrolling patients. ClinicalTrials.gov: NCT05012397. Citation Format: Ecaterina I. Dumbrava, Glenn J. Hanna, Gregory M. Cote, Thomas E. Stinchcombe, Melissa L. Johnson, Christopher T. Chen, Siddhartha H. Devarakonda, Naisargee Shah, Feng Xu, Robert C. Doebele, Mrinal M. Gounder. A phase 2 study of the MDM2 inhibitor milademetan in patients with TP53-wild type and MDM2-amplified advanced or metastatic solid tumors (MANTRA-2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT123.

Abstract CT235: MANTRA: A randomized, multicenter, phase 3 study of the MDM2 inhibitor milademetan versus trabectedin in patients with de-differentiated liposarcomas

Abstract Background: Murine double minute 2 (MDM2) is a negative regulator of tumor suppressor protein p53. MDM2 induces degradation of p53 and promotes tumorigenesis. MDM2 amplification occurs in many cancers but is documented in up to 100% of well-differentiated or de-differentiated liposarcomas (WD/DDLPS) [Cancer Genome Atlas Research Network. Cell 2017]. Inhibition of the MDM2-p53 interaction is a promising therapeutic approach to restore p53 tumor suppressor activity in WD/DDLPS. Milademetan (RAIN-32) is a small-molecule MDM2 inhibitor that inhibits the MDM2-p53 interaction and restores p53 function at nanomolar concentrations. In a phase 1 study, milademetan showed promising efficacy in 53 patients with WD/DDLPS when administered on an intermittent schedule (260 mg QD on Days 1-3 and 15-17 on a 28-day cycle), with a median progression-free survival (PFS) of 7.4 months [Gounder et al. AACR-NCI-EORTC 2020]. WD/DDLS are relatively resistant to chemotherapy, and systemic treatment options for patients with advanced disease are limited. MANTRA (RAIN-3201) is a randomized, multicenter, open-label, phase 3 registration study designed to evaluate the efficacy and safety of milademetan versus trabectedin in patients with unresectable or metastatic DDLPS with disease progression on ≥1 prior systemic therapies. Methods: Eligible patients are ≥18 years of age with histologically confirmed unresectable and/or metastatic DDLPS, with or without a WD component, who have received ≥1 prior systemic therapies, including ≥1 anthracycline-based regimen, with radiographic evidence of progression by RECIST v1.1 within 6 months before study entry. Prior treatment with trabectedin or an MDM2 inhibitor is not permitted. Patients will be randomly assigned (1:1) to receive milademetan (260 mg once daily orally Days 1-3 and 15-17 on a 28-day cycle) or trabectedin (1.5 mg/m2 as a 24-hour intravenous infusion every 3 weeks). Randomization is stratified by Eastern Cooperative Oncology Group performance status (0 or 1) and number of prior treatments for WD/DDLPS (≤2 or &amp;gt;2). Tumor response will be evaluated by RECIST v1.1 at Weeks 8, 16, 24, and 32, and then every 12 weeks. Primary endpoint: PFS by blinded independent central review. Secondary endpoints: overall survival; disease control rate; objective response rate; duration of response; PFS by investigator assessment; safety; health-related quality of life. Exploratory endpoints: molecular markers in peripheral blood and/or tumor tissue; milademetan pharmacokinetics. To demonstrate a 3-month increase in PFS (from 3 to 6 months) corresponding to a hazard ratio of 0.5, approximately 160 patients will be required to observe 105 events with 93.9% power and 2-sided significance level of 5%. ClinicalTrials.gov: NCT04979442. Citation Format: Mrinal M. Gounder, Gary K. Schwartz, Robin L. Jones, Sant P. Chawla, Victoria S. Chua-Alcala, Silvia Stacchiotti, Andrew J. Wagner, Gregory M. Cote, Robert G. Maki, Hanna Kosela-Paterczyk, Dale R. Shepard, Naisargee Shah, Richard Bryce, Robert C. Doebele, Shreyaskumar Patel. MANTRA: A randomized, multicenter, phase 3 study of the MDM2 inhibitor milademetan versus trabectedin in patients with de-differentiated liposarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT235.

A phase 2 study of the MDM2 inhibitor milademetan in patients with TP53-wild type and MDM2-amplified advanced or metastatic solid tumors (MANTRA-2).

TPS3165 Background: Murine double minute 2 (MDM2) is a potent negative regulator of the tumor suppressor p53. MDM2 induces degradation of p53 and promotes tumorigenesis in solid tumors, and preclinical models have shown that inhibition of MDM2 can restore p53 tumor suppressor activity in TP53-wild type (WT), MDM2-amplified tumors. We performed a mutual exclusivity analysis of patients with solid tumors (n = 42,125; AACR Project GENIE) and found that the frequency of co-occurring TP53 mutations decreased with increasing MDM2 copy number. An MDM2 copy number of 12 was chosen as the threshold. An estimated 1.1% of solid tumors meet this molecular criteria, excluding glioblastomas, dedifferentiated liposarcomas, and intimal sarcomas where this signature is enriched. Milademetan (RAIN-32), an oral, selective MDM2 inhibitor, inhibits growth of TP53-WT/ MDM2-amplified cell lines and patient-derived xenograft models from varying tumor types. Furthermore, tumor regression was observed in 3/3 non-liposarcoma patients with MDM2 copy number &gt; 12 in a phase 1 trial of milademetan. MANTRA-2 (RAIN-3202) is a phase 2, multicenter, single-arm, open-label, basket trial designed to evaluate the efficacy or clinical benefit of milademetan in TP53-WT solid tumors with MDM2 amplification (copy number ≥ 12). Methods: Eligible patients must be ≥ 18 years of age with histologically and/or cytologically confirmed locally advanced, incurable or metastatic solid tumors refractory to standard therapy. Local testing demonstrating TP53 WT and MDM2 amplification is required, defined as a MDM2 copy number ≥ 12 or 6-fold increase. Patients with well-differentiated/de-differentiated liposarcomas, intimal sarcomas, or primary central nervous system tumors are excluded. Prior treatment with an MDM2 inhibitor is not permitted. Patients receive milademetan 260 mg orally once daily on Days 1–3 and 15–17 of a 28-day cycle. Tumor response is evaluated by RECIST v1.1 at Weeks 8, 16, 24, and 32, and then every 12 weeks. Primary endpoint: objective response rate. Secondary endpoints include: duration of response; progression-free survival; growth modulation index; disease control rate; overall survival; safety; health-related quality of life scores. Exploratory endpoints include: biomarkers in blood and/or tumor tissue; pharmacodynamics; pharmacokinetics. Enrollment of 65 patients is planned to ensure that 57 patients have centrally confirmed TP53 WT and MDM2 copy number ≥ 12. The trial opened in November 2021 and is actively enrolling patients. Clinical trial information: NCT05012397.

MANTRA: A randomized, multicenter, phase 3 study of the MDM2 inhibitor milademetan versus trabectedin in patients with de-differentiated liposarcomas.

TPS11589 Background: Murine double minute 2 (MDM2) is a negative regulator of tumor suppressor protein p53. MDM2 induces degradation of p53 and promotes tumorigenesis. MDM2 amplification occurs in many cancers but is documented in up to 100% of well-differentiated or dedifferentiated liposarcomas (WD/DDLPS) [Cancer Genome Atlas Research Network. Cell 2017]. Inhibition of the MDM2-p53 interaction is a promising therapeutic approach to restore p53 tumor suppressor activity in WD/DDLPS. Milademetan (RAIN-32) is a small-molecule MDM2 inhibitor that inhibits the MDM2-p53 interaction and restores p53 function at nanomolar concentrations. In a phase 1 study, milademetan showed promising efficacy in 53 patients with WD/DDLPS when administered on an intermittent schedule (260 mg QD on Days 1–3 and 15–17 on a 28-day cycle), with a median progression-free survival (PFS) of 7.4 months [Gounder et al. AACR-NCI-EORTC 2020]. WD/DDLS are relatively resistant to chemotherapy, and systemic treatment options for patients with advanced disease are limited. MANTRA (RAIN-3201) is a randomized, multicenter, open-label, phase 3 registration study designed to evaluate the efficacy and safety of milademetan versus trabectedin in patients with unresectable or metastatic DDLPS with disease progression on ≥ 1 prior systemic therapies. Methods: Eligible patients are ≥ 18 years of age with histologically confirmed unresectable and/or metastatic DDLPS, with or without a WD component, who have received ≥ 1 prior systemic therapies, including ≥ 1 anthracycline-based regimen, with radiographic evidence of progression by RECIST v1.1 within 6 months before study entry. Prior treatment with trabectedin or an MDM2 inhibitor is not permitted. Patients will be randomly assigned (1:1) to receive milademetan (260 mg once daily orally Days 1–3 and 15–17 on a 28-day cycle) or trabectedin (1.5 mg/m2 as a 24-hour intravenous infusion every 3 weeks). Randomization is stratified by Eastern Cooperative Oncology Group performance status (0 or 1) and number of prior treatments for WD/DDLPS (≤ 2 or &gt; 2). Tumor response will be evaluated by RECIST v1.1 at Weeks 8, 16, 24, and 32, and then every 12 weeks. Primary endpoint: PFS by blinded independent central review. Secondary endpoints: overall survival; disease control rate; objective response rate; duration of response; PFS by investigator assessment; safety; health-related quality of life. Exploratory endpoints: molecular markers in peripheral blood and/or tumor tissue; milademetan pharmacokinetics. To demonstrate a 3-month increase in PFS (from 3 to 6 months) corresponding to a hazard ratio of 0.5, approximately 160 patients will be required to observe 105 events with 93.9% power and 2-sided significance level of 5%. Clinical trial information: NCT04979442.