MDM2 Inhibitor Research Articles

Icariin improves cognitive impairment by inhibiting ferroptosis of nerve cells.

We investigated the effect and mechanism of Icariin (ICA) on improving neurobehavioral ability of mice with Alzheimer's disease (AD). We selected 10-month-old APP/PS1 mice (AD) and wild-type C57BL/6J mice (Normal). After intragastric administration of ICA, Morris water maze was employed to detect neurobehavioral improvements, and to assay key ferroptosis indicators and oxidative stress levels. The common target of ICA for resisting ferroptosis and AD was predicted by network pharmacology. ICA could improve the neurobehavioral, memory and motor abilities of AD mice. It could lower the ferroptosis level and enhance the resistance to oxidative stress. After inhibition of MDM2, ICA could no longer improve the cognitive ability of AD mice, nor could it further inhibit ferroptosis. Network pharmacological analysis revealed that MDM2 might be the target of ICA action. We found that ICA can inhibit ferroptosis of nerve cells, thereby ameliorating neural damage in mice with AD.

Idasanutlin and navitoclax induce synergistic apoptotic cell death in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in which activating mutations in the Notch pathway are thought to contribute to transformation, in part, by activating c-Myc. Increased c-Myc expression induces oncogenic stress that can trigger apoptosis through the MDM2-p53 tumor suppressor pathway. Since the great majority of T-ALL cases carry inactivating mutations upstream in this pathway but maintain wildtype MDM2 and TP53, we hypothesized that T-ALL would be selectively sensitive to MDM2 inhibition. Treatment with idasanutlin, an MDM2 inhibitor, induced only modest apoptosis in T-ALL cells but upregulated the pro-apoptotic BH3 domain genes BAX and BBC3, prompting us to evaluate the combination of idasanutlin with BH3 mimetics. Combination treatment with idasanutlin and navitoclax, a potent Bcl-2/Bcl-xL inhibitor, induces more consistent and potent synergistic killing of T-ALL PDX lines in vitro than venetoclax, a Bcl-2 specific inhibitor. Moreover, a marked synergic response to combination treatment with idasanutlin and navitoclax was seen in vivo in all four T-ALL xenografts tested, with a significant increase in overall survival in the combination treatment group. Collectively, these preclinical data show that the combination of idasanutlin and navitoclax is highly active in T-ALL and may merit consideration in the clinical setting.

Mdm2 Targeting Reassembly Peptide (Trap) Nanoparticles For P53 Based Cancer Therapy.

Gene mutations and functional inhibition are the major obstacles for p53-mediated oncotherapy. For p53-wild type tumors, the underlying mechanisms of functional inhibition of p53 during oncogenesis are unknown. Our results revealed that the expression of the MDM2 inhibitor ARF was inhibited in p53-wild type tumors, indicating that the restoration of ARF could be a potential oncotherapy strategy for p53-wild type tumors. Therefore, we developed ARF-mimetic MDM2-targeting reassembly peptide nanoparticles (MtrapNPs) for p53-based tumor therapy. Our results elucidated that the MtrapNPs responded to and formed a nanofiber structure with MDM2. By trapping MDM2, the MtrapNPs stabilized and activated p53 for the inhibition of p53-wild type tumors. In most cases, reactivated mutant p53 is inhibited and degraded by MDM2. In the present study, MtrapNPs were used to load and deliver arsenic trioxide, a p53 mutation rescuer, for p53-mutated tumor treatment in both orthotopic and metastatic models, and they exhibited significant therapeutic effects. Therefore, our study provides evidence supporting a link between decreased ARF expression and tumor development in patients with p53-wild type tumors. Thus, the novel MDM2-trap strategy, which addresses both the inhibition and mutations of p53, is an efficient strategy for the treatment of p53-mutated tumors. This article is protected by copyright. All rights reserved.

Susceptibility of pediatric acute lymphoblastic leukemia to STAT3 inhibition depends on p53 induction.

Advances in the clinical management of pediatric B-cell acute lymphoblastic leukemia (B-ALL) have dramatically improved outcomes for this disease. However, relapsed and high-risk disease still contribute to significant numbers of treatment failures. Development of new, broad range therapies is urgently needed for these cases. We previously reported the susceptibility of ETV6-RUNX1+ pediatric B-ALL to inhibition of signal transducer and activator of transcription 3 (STAT3) activity. In the present study, we demonstrate that pharmacological or genetic inhibition of STAT3 results in p53 induction and that CRISPR-mediated TP53 knockout substantially reverses susceptibility to STAT3 inhibition. Furthermore, we demonstrate that sensitivity to STAT3 inhibition in patient-derived xenograft (PDX) B-ALL samples is not restricted to any particular disease subtype, but rather depends on TP53 status, the only resistant samples being TP53 mutant. Induction of p53 following STAT3 inhibition is not directly dependent on MDM2 but correlates with degradation of MDM4. As such, STAT3 inhibition exhibits synergistic in vitro and in vivo anti-leukemia activity when combined with MDM2 inhibition. Taken together with the relatively low frequency of TP53 mutations in this disease, these data support the future development of combined STAT3/ MDM2 inhibition in the therapy of refractory and relapsed pediatric B-ALL.

Integrative analyses reveal outcome-associated and targetable molecular partnerships between TP53, BRD4, TNFRSF10B, and CDKN1A in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a common, genomically heterogenous disease that presents a clinical challenge despite the success of frontline regimens and second-line chimeric antigen receptor T-cell (CAR-T) therapy. Recently, genomic alterations and tumor microenvironment features associated with poor CAR-T response have been identified, namely those to the TP53 tumor suppressor gene. This retrospective analysis aimed to integrate various data to identify genomic partnerships capable of providing further clarity and actionable treatment targets within this population. Publicly available data were analyzed for differential expression based on TP53 and 24-month event-free survival (EFS24) status, revealing enrichments of the BRD4 bromodomain oncogene (p < 0.0001, p = 0.001). High-BRD4 and TP53 alterations were significantly associated with lower CDKN1A (p21) and TNFRSF10B (TRAIL-R2), a key tumor suppressor and CAR-T modulator, respectively. Significant loss of CD8 T-cell presence within low-TNFRSF0B (p = 0.0042) and altered-TP53 (p = 0.0424) patients showcased relevant outcome-associated tumor microenvironment features. Furthermore, reduced expression of CDKN1A was associated with low TNFRSF10B (FDR < 0.0001) and increased BRD4 interactant genes (FDR < 0.0001). Promisingly, in vitro MDM2 inhibition with Idasnutlin and TP53 reactivation via Eprenetapopt was able to renew TNFRSF10B protein expression. Additionally, applying the BRD4-degrading PROTAC ARV-825 and the CDK4/6 inhibitor Abemaciclib as single-agents and in synergistic combination significantly reduced TP53-altered DLBCL cell line viability. Our analysis presents key associations within a genomic network of actionable targets capable of providing clarity within the evolving precision CAR-T treatment landscape.

Recent Advancements in the Discovery of MDM2/MDM2-p53 Interaction Inhibitors for the Treatment of Cancer.

Discovery of MDM2 and MDM2-p53 interaction inhibitors changed the direction of anticancer research as it is involved in about 50% of cancer cases globally. Not only the inhibition of MDM2 but also its interaction with p53 proved to be an effective strategy in anticancer drug design and development. Various molecules of natural as well as synthetic origin have been reported to possess excellent MDM2 inhibitory potential. The present review discusses the pathophysiology of the MDM2-p53 interaction loop and MDM2/MDM2-p53 interaction inhibitors from literature covering recent patents. Focus has also been put on characteristic features of the active site of the target and its desired interactions with the currently FDA-approved inhibitor. The designing approach of previously reported MDM2/MDM2-p53 interaction inhibitors, their SAR studies, in silico studies, and the biological efficacy of various inhibitors from natural as well as synthetic origins are also elaborated. An attempt is made to cover recently patented MDM2/MDM2- p53 interaction inhibitors.

674P Response of thrombopoietin receptor agonists in MDM2 inhibitor induced thrombocytopenia

674P Response of thrombopoietin receptor agonists in MDM2 inhibitor induced thrombocytopenia

LBA89 Efficacy and safety findings from MANTRA: A global, randomized, multicenter, phase III study of the MDM2 inhibitor milademetan vs trabectedin in patients with dedifferentiated liposarcomas

LBA89 Efficacy and safety findings from MANTRA: A global, randomized, multicenter, phase III study of the MDM2 inhibitor milademetan vs trabectedin in patients with dedifferentiated liposarcomas

USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

Antagonizing MDM2 Overexpression Induced by MDM4 Inhibitor CEP-1347 Effectively Reactivates Wild-Type p53 in Malignant Brain Tumor Cells.

The development of MDM4 inhibitors as an approach to reactivating p53 in human cancer is attracting increasing attention; however, whether they affect the function of MDM2 and how they interact with MDM2 inhibitors remain unknown. We addressed this question in the present study using CEP-1347, an inhibitor of MDM4 protein expression. The effects of CEP-1347, the genetic and/or pharmacological inhibition of MDM2, and their combination on the p53 pathway in malignant brain tumor cell lines expressing wild-type p53 were investigated by RT-PCR and Western blot analyses. The growth inhibitory effects of CEP-1347 alone or in combination with MDM2 on inhibition were examined by dye exclusion and/or colony formation assays. The treatment of malignant brain tumor cell lines with CEP-1347 markedly increased MDM2 protein expression, while blocking CEP-1347-induced MDM2 overexpression by genetic knockdown augmented the effects of CEP-1347 on the p53 pathway and cell growth. Blocking the MDM2-p53 interaction using the small molecule MDM2 inhibitor RG7112, but not MDM2 knockdown, reduced MDM4 expression. Consequently, RG7112 effectively cooperated with CEP-1347 to reduce MDM4 expression, activate the p53 pathway, and inhibit cell growth. The present results suggest the combination of CEP-1347-induced MDM2 overexpression with the selective inhibition of MDM2's interaction with p53, while preserving its ability to inhibit MDM4 expression, as a novel and rational strategy to effectively reactivate p53 in wild-type p53 cancer cells.

SF3B1 Mutations Are Associated with Resistance to Non-Genotoxic MDM2 Inhibition in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a genetically and clinically heterogeneous malignancy affecting older individuals. There are a number of current treatment options for CLL, including monoclonal antibodies, targeted drugs, chemotherapy, and different combinations of these. However, for those patients who are intrinsically treatment resistant, or relapse following initial responses, novel targeted therapies are still needed. Targeting the mouse double-minute-2 human homolog (MDM2), a primary negative regulator of p53, is an appealing therapeutic strategy for non-genotoxic reactivation of p53, since the TP53 gene is in its wild-type state at diagnosis in approximately 90% of patients. Mutated SF3B1 and TP53 are both associated with more aggressive disease, resistance to therapies and poorer overall survival for CLL. In this study, we performed a screen for SF3B1 and TP53 mutations and tested RG7388 (idasanutlin), a second-generation MDM2 inhibitor, in a cohort of CLL primary patient samples. SF3B1 mutations were detected in 24 of 195 cases (12.3%) and found associated with poor overall survival (hazard ratio [HR] 2.12, p = 0.032) and high CD38 expression (median CD38 (%) 32 vs. 5; p = 0.0087). The novel striking finding of this study was an independent link between SF3B1 mutational status and poor response to RG7388. Overall, SF3B1 mutations in CLL patient samples were associated with resistance to treatment with RG7388 ex vivo, and patients with the wild type for both SF3B1 and TP53 are more likely to benefit from treatment with MDM2 inhibitors.

MDM2 Inhibition in the Treatment of Glioblastoma: From Concept to Clinical Investigation.

Inhibition of the interaction between MDM2 and p53 has emerged as a promising strategy for combating cancer, including the treatment of glioblastoma (GBM). Numerous MDM2 inhibitors have been developed and are currently undergoing rigorous testing for their potential in GBM therapy. Encouraging results from studies conducted in cell culture and animal models suggest that MDM2 inhibitors could effectively treat a specific subset of GBM patients with wild-type TP53 or functional p53. Combination therapy with clinically established treatment modalities such as radiation and chemotherapy offers the potential to achieve a more profound therapeutic response. Furthermore, an increasing array of other molecularly targeted therapies are being explored in combination with MDM2 inhibitors to increase the effects of individual treatments. While some MDM2 inhibitors have progressed to early phase clinical trials in GBM, their efficacy, alone and in combination, is yet to be confirmed. In this article, we present an overview of MDM2 inhibitors currently under preclinical and clinical investigation, with a specific focus on the drugs being assessed in ongoing clinical trials for GBM patients.

A Phase I study of Milademetan (DS3032b) in combination with low dose cytarabine with or without venetoclax in acute myeloid leukemia: Clinical safety, efficacy, and correlative analysis

In TP53 wild-type acute myeloid leukemia (AML), inhibition of MDM2 can enhance p53 protein expression and potentiate leukemic cell apoptosis. MDM2 inhibitor (MDM2i) monotherapy in AML has shown modest responses in clinical trials but combining options of MDM2i with other potent AML-directed agents like cytarabine and venetoclax could improve its efficacy. We conducted a phase I clinical trial (NCT03634228) to study the safety and efficacy of milademetan (an MDM2i) with low-dose cytarabine (LDAC)±venetoclax in adult patients with relapsed refractory (R/R) or newly diagnosed (ND; unfit) TP53 wild-type AML and performed comprehensive CyTOF analyses to interrogate multiple signaling pathways, the p53-MDM2 axis and the interplay between pro/anti-apoptotic molecules to identify factors that determine response and resistance to therapy. Sixteen patients (14 R/R, 2 N/D treated secondary AML) at a median age of 70 years (range, 23–80 years) were treated in this trial. Two patients (13%) achieved an overall response (complete remission with incomplete hematological recovery). Median cycles on trial were 1 (range 1–7) and at a median follow-up of 11 months, no patients remained on active therapy. Gastrointestinal toxicity was significant and dose-limiting (50% of patients ≥ grade 3). Single-cell proteomic analysis of the leukemia compartment revealed therapy-induced proteomic alterations and potential mechanisms of adaptive response to the MDM2i combination. The response was associated with immune cell abundance and induced the proteomic profiles of leukemia cells to disrupt survival pathways and significantly reduced MCL1 and YTHDF2 to potentiate leukemic cell death. The combination of milademetan, LDAC±venetoclax led to only modest responses with recognizable gastrointestinal toxicity. Treatment-induced reduction of MCL1 and YTHDF2 in an immune-rich milieu correlate with treatment response.

Clinical activity and exploratory resistance mechanism of milademetan, an MDM2 inhibitor, in intimal sarcoma with MDM2 amplification: an open-label phase 1b/2 study.

Intimal sarcoma is an extremely rare life-threatening malignant neoplasm. Murine double minute 2 (MDM2) amplification is observed in > 70% of intimal sarcomas. Milademetan, an MDM2 inhibitor, may provide a clinical benefit in this patient population. We conducted a phase 1b/2 study in patients with MDM2-amplified wild-type TP53 intimal sarcoma as a sub-study of a large nationwide registry for rare cancers in Japan. Milademetan (260 mg) was administered orally once daily for three days every 14 days, twice in a 28-day cycle. Of 11 patients enrolled, 10 were included in the efficacy analysis. Two patients (20%) showed durable responses for >15 months. Anti-tumor activity correlated with TWIST1 amplification (P = 0.028) and negatively with CDKN2A loss (P = 0.071). Acquired TP53 mutations were detected in sequential liquid biopsies as a novel exploratory resistance mechanism to milademetan. These results suggest that milademetan could be a potential therapeutic strategy for intimal sarcoma.

Epichaperome inhibition targets TP53-mutant AML and AML stem/progenitor cells

AbstractTP53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53–wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.

MDB-23. ELP1 GERMLINE DEFICIENCY SENSITIZES THE GRANULE NEURON LINEAGE TO SHH MEDULLOBLASTOMA AND EXPOSES NOVEL THERAPEUTIC VULNERABILITIES

Abstract Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events in childhood medulloblastoma (MB). ELP1 germline carriers develop SHH-MBs that exhibit coincident somatic PTCH1 mutations and universal loss-of-heterozygosity of the remaining ELP1 allele through chromosome 9q deletion. The molecular, biochemical, and cellular mechanisms by which germline ELP1/Elongator deficiency contribute to SHH-MB tumorigenesis remain largely unknown. Herein, we report that mice engineered to mimic germline Elp1 LOF (i.e., Elp1HET) seen in SHH-MB patients exhibit hallmark features of premalignancy events in cycling cerebellar granule neuron progenitors (GNPs), the lineage-of-origin for SHH-MB. Compared to wild-type counterparts, Elp1HET GNPs exhibit increased replication stress-associated DNA damage, homologous recombination-associated genomic instability, accelerated cell cycle kinetics, reduced p53-dependent apoptosis in response to genotoxic stress, and slowed differentiation. Orthotopic transplantation of Elp1HET GNPs harboring somatic Ptch1 inactivation into the cerebella of immunocompromised mice promotes onset of SHH-MB tumors with incomplete penetrance that exhibit reduced p53 transcriptional activity through a currently unknown mechanism(s). Concomitant Elp1 and Ptch1 gene targeting in p53-null GNPs reproduces highly penetrant cerebellar tumors recapitulating the molecular and phenotypic features of ELP1-associated SHH-MB. Finally, reactivation of the p53 pathway through preclinical treatment with an MDM2 inhibitor promotes cell death and prolongs the survival of patient-derived xenograft tumor (PDX) models harboring deleterious ELP1 mutations. Together, our findings reveal that germline Elp1 LOF heightens genomic instability and malignant transformation in cycling GNPs, providing a mechanistic model for the subgroup-restricted pattern of predisposition associated with pathogenic ELP1 germline carriers. These results provide essential mechanistic insight into the molecular and cellular basis of SHH-MB predisposition driven by ELP1 LOF and nominate therapies that overcome p53 pathway inhibition as a rational treatment option for affected children.

The current state and future of neoadjuvants in resectable melanoma: Review of phase II/III studies conducted between 2018-2022.

e21568 Background: Adjuvant therapy with single agent anti-PD1 and BRAF/MEK-inhibitors (BRAF/MEKi) is approved for patients diagnosed with high-risk stage III melanoma. The goal of adjuvant therapy is to prevent the growth of the occult metastasis to distant organs. The premise of neoadjuvant therapy in clinical stage III melanoma is to allow for a better antigen exposure when checkpoint inhibitors are administered. We aimed to get an overview of the current state of neoadjuvant therapy in resectable melanoma. Methods: A systematic literature search was conducted in November 2022, using PubMed, American Society of Clinical Oncology (ASCO) Journal of Clinical Oncology and European Society for Medical Oncology (ESMO) abstract databases. Eligible studies included Phase 2 and 3 randomized controlled trials (RCT), single-arm trials, pooled analyses, and planned future trials published between 2018-2022 looking at the efficacy of neoadjuvants in resectable melanoma. Two individuals (CL and MM) screened all the studies, and 3 individuals (CM, MM, MW) went over the list of Abstracts. Primary outcomes and secondary outcomes, as well as median duration of follow up and Grade 3 or above adverse effects (AEs) were recorded. Abstracts with more recent findings or final publications were excluded in the final analysis. Results: A total of 14 published studies (4 RTCs, 5 single-arm, 2 OpACIN-neo and 3 related sub-analysis studies) and 16 abstracts (5 ESMO, 11 ASCO) were identified, representing 34 unique Neoadjuvant arms and a total of 1,496 patients. Thirteen of 27 trials making 71% of patients (n = 1061) tested immune checkpoint inhibitors (ICIs). Four of 27 trials of total n = 112 tested BRAF/MEKi. All ongoing or future studies (7 trials) tested combination of ICI with BRAF/MEKi or other molecules such as MDM2 inhibitors, interferon-α, or oncolytic viruses. Most of studies (54% of ICI studies, 50% of BRAF/MEK inhibitor trials, and 80% of other studies) used pathological response as their primary outcome. Other primary end points included event-free survival (EFS) and relapse-free survival (RFS), and 13/14 published studies and 13/16 abstracts met the primary end point. A pooled analysis of complete pathologic response (pCR) revealed an estimate of 38.3% ± 19.7 (mean ± SD) from 16 unique ICI neoadjuvant arms, 51.9% ± 5.3 for BRAF/MEKi (from 3 data points), and 21.0% ± 7.2 for other agents/combinations (from 3 data points). Rate of ≥ Grade 3 AEs was 28.7% ± 31.0 (from 11 data points) for studies testing ICI, 32.5% ± 14.1 (from 6 data points) for BRAF/MEKi, and 23.6% ± 20.2 (from 3 data points) from studies testing other agents/combinations. Conclusions: Recent studies have shown that neoadjuvant in resectable melanoma can offer increased therapeutic efficacy. While past studies have focused on PD-1 and CTLA-4 inhibitors, ongoing and future studies are beginning to test newer agents and novel molecular targets.

CCT6A and triple-negative breast cancer metastasis: Activating the AKT/MDM2/P53 pathway.

e13087 Background: Triple-negative breast cancer (TNBC) patients are prone to distant recurrence and poorer prognosis. The key molecules and underlying mechanisms of TNBC's aggressive phenotype remain to be further clarified. Methods: First, using the Kaplan-Meier Plotter ( https://kmplot.com/analysis ) and analyzing our collected clinical samples, we established the expression pattern and clinical value of the chaperonin containing TCP1 subunit 6A (CCT6A) in TNBC. We carried out the Cell Counting Kit-8 (CCK8), Transwell, and flow cytometry assay in the TNBC cell lines including MDA-MB-231 and BT549 to assess the effects of knockdown and overexpress of CCT6A. The mechanism of CCT6A in TNBC was identified by using the RNA-seq, KEGG analysis and Co-Immunoprecipitation Mass Spectrometry. Rescue assays verified the role of the AKT Serine/Threonine Kinase 1 (AKT) / Mouse double minute 2 homolog (MDM2)/P53 pathway on CCT6A. Then, the effect of CCT6A on tumor progression and metastasis in vivo was evaluated by the mouse models of tumorigenesis in situ and caudal artery lung metastasis. APG115, a pharmacological p53 activator and immune stimulatory MDM2 inhibitor, combined with anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody Ipilimumab was found to prevent TNBC metastasis. Finally, we did a profiling of the tumor immune environment in mice. Results: CCT6A was highly expressed in TNBC ( p&lt;0.0001), and patients with high expression of CCT6A had poor prognosis ( p=0.0053, HR=2.36 [1.27-4.39]). Of note, CCT6A was significantly associated with lymph node metastasis ( p&lt;0.0001). CCT6A promoted cell proliferation, migration, and invasion while inhibiting cell apoptosis and achieving G1/S stage arrest through gain- and loss-of-function approaches. In terms of mechanism, Co-IP and Immunofluorescence assay verified that CCT6A interacted with Keratin 10 (KRT10) which can promote tumor metastasis. Compared with the control group, RNA-seq, and KEGG analysis suggested that AKT/MDM2/P53 signaling was inhibited after knockdown of CCT6A in MDA-MB-231. Furthermore, we found that the tumor volume and the number of lung metastasis sites were significantly reduced in the APG115 combined with Ipilimumab treatment group. Flow cytometry and multiple Immunofluorescence assay showed that TGF-β level from regulatory T cells decreased, while IFN-γ amount from CD8+T cells significantly increased with the combination (both p&lt;0.05). Conclusions: Our research demonstrated that CCT6A can enhance metastasis of triple-negative breast cancer via up-regulating the AKT/MDM2/P53 pathway. These results provide rationales for MDM2 inhibitor combined with anti-CTLA-4 antibody for preventing TNBC metastasis.

Genomic characterization of the GATA3 mutational landscape in breast cancer.

1039 Background: GATA3 expression is broadly used as a biomarker for diagnosing cancer of breast origin and its expression is strongly associated with estrogen receptor (ER)-positive luminal phenotype. Although GATA3 mutations are observed in 12-18% of breast cancers (BC), they are poorly characterized. Recently, the pharmacological inhibition of MDM2 has been shown to significantly impair tumor growth in GATA3-deficient models in vitro, in vivo, and patient-derived xenograft harboring GATA3 somatic mutations. Therefore, given the potential targetability of GATA3-mutated BC cells, it is important to better understand and characterize the mutational landscape of GATA3 to help guide future prospective clinical studies. Methods: We accessed a cohort of BC samples profiled genomically and transcriptomically from two open-source datasets: TCGA (n=961) and METABRIC (n=1866). We used the chi-squared test to analyze the frequency of GATA3 mutations across PAM50 subtypes (Basal, HER2, Luminal A, Luminal B, Normal-like) and histological BC subtypes (invasive ductal carcinoma [IDC] and invasive lobular carcinoma [ILC]). Mutations affecting other genes in the GATA3-mutated samples and the GATA3 wild-type (WT) samples were compared using the chi-squared test. We analyzed the mutational landscape of the GATA3 gene and correlated different sites of mutations with GATA3 expression, MDM2 amplification, other co-occurring mutations, and clinical behavior. GATA3 expression levels were compared using the Mann-Whitney test. Results: Of the analyzed 2,827 BC samples, GATA3 mutations were unevenly distributed in the five PAM50 subtypes, as Luminal A and B subtypes had the highest frequency (16% and 17%, respectively) and the basal subtype had no observed GATA3 mutations (p &lt;0.0001). Further analysis showed that GATA3 mutations were more common in luminal IDC than ILC (17% vs 8.7%; p &lt;0.0001). While mutations in CBFB, AKT1, TBX3 and ARID1A were more frequently co-occurring with GATA3 mutations, mutations in TP53, CDH1 and PIK3CA were mutually exclusive with GATA3 mutations (adjusted p &lt;0.0001 for all genes). Analysis of the GATA3 mutational landscape identified two types of mutations in GATA3: a group of truncating mutations occurring at the end of the GATA3 gene (after the 307 position including the splice site at 308) and a group occurring before the 307 position. The first group was associated with increased GATA3 expression, MDM2 amplification, and was mutually exclusive with TP53 mutations. The second group had little to no effect on GATA3 expression and behaved similarly to GATA3 WT. Conclusions: A subtype of GATA3 mutations are mutually exclusive with TP53 mutations and are associated with increased MDM2 amplification, making them an ideal target for clinical trials involving MDM2 inhibitors.

A phase 2 study of alrizomadlin (APG-115) in combination with pembrolizumab in patients with unresectable or metastatic cutaneous melanoma that has failed immuno-oncologic (IO) drugs.

9559 Background: Alrizomadlin is a novel, orally active small-molecule MDM2 inhibitor that activates p53-mediated apoptosis in tumor cells, also functions as a host immunomodulator, and may restore antitumor activity in patients with cancer that has progressed on PD-1/PD-L1 inhibitors. Alrizomadlin is currently being investigated for treatment of patients with unresectable or metastatic cutaneous melanoma that progressed on prior immunotherapy. Methods: This multicenter clinical trial was conducted in the United States and Australia in patients with unresectable or metastatic cutaneous melanoma that had progressed on PD-1/PD-L1 immunotherapy. Eligible patients had an ECOG performance status of 0-2. Alrizomadlin 150 mg was administered orally every other day for 2 consecutive weeks, with 1 week off, and pembrolizumab 200 mg intravenously for 30 minutes on Day 1 of a 21-day cycle. Results: As of December 12, 2022, preliminary and interim results are reported for 31 patients with cutaneous melanoma, of whom 21 were male and 10 female, with a median (range) age of 65 (27-84) years. Treatment-related adverse events (TRAE) of any grade ( &gt; 10%) for either alrizomadlin or pembrolizumab were reported in 30/31 (96.8%) patients. These AEs included nausea (71%), vomiting (38.7%), fatigue (35.5%), thrombocytopenia (32.3%), diarrhea (25.8%), neutropenia (19.4%), decreased appetite (16.1%), and decreased leukocyte count (12.9%). A total of 4/31 (12.9%) patients reported treatment-related serious AEs, including anemia, thrombocytopenia, deep vein thrombosis, joint effusion, pulmonary embolism, and vomiting. In 26 efficacy-evaluable patients, the confirmed overall response rate (ORR; partial response [PR] + complete response [CR]) was 23.1%, including 2 patients reporting CRs and 4 reporting PRs based on RECIST v.1.1. The initial analysis indicated that the ORR observed in patients whose disease had failed IO treatment was primarily due to alrizomadlin combined with pembrolizumab, not the delayed effect of prior immunotherapy. Conclusions: Alrizomadlin combined with pembrolizumab is well tolerated and demonstrates clinical efficacy in these patients with highly refractory unresectable or metastatic cutaneous melanoma that had progressed on PD-1/PD-L1 immunotherapy. Internal study identifier: APG-115-US002. Clinical trial information: NCT03611868 .