Tumor Suppressor Gene PTEN Research Articles

ATP citrate lyase is an essential player in the metabolic rewiring induced by PTEN loss during T-ALL development.

Alterations inactivating the tumor suppressor gene PTEN drive the development of solid and hematological cancers, such as T-cell acute lymphoblastic leukemia (T-ALL), whereby PTEN loss defines poor-prognosis patients. We investigated the metabolic rewiring induced by PTEN loss in T-ALL, aiming at identifying novel metabolic vulnerabilities. We showed that the enzyme ATP citrate lyase (ACLY) is strictly required for the transformation of thymic immature progenitors and for the growth of human T-ALL, which remain dependent on ACLY activity even upon transformation. Whereas PTEN mutant mice all died within 17 weeks, the concomitant ACLY deletion prevented disease initiation in 70% of the animals. In these animals, ACLY promoted BCL-2 epigenetic upregulation and prevented the apoptosis of pre-malignant DP thymocytes. Transcriptomic and metabolic analysis of primary T-ALL cells next translated our findings to the human pathology, showing that PTEN-altered T-ALL cells activate ACLY and are sensitive to its genetic targeting. ACLY activation thus represents a metabolic vulnerability with therapeutic potential for high-risk T-ALL patients.

Tumor suppressors RBL1 and PTEN are epigenetically silenced in IPF mesenchymal progenitor cells by a CD44/Brg1/PRMT5 regulatory complex.

The idiopathic pulmonary fibrosis (IPF) lung contains mesenchymal progenitor cells (MPCs) that display durable activation of oncogenic signaling and cell-autonomous fibrogenicity in vivo. Prior work identified a CD44/Brg1/PRMT5 nuclear regulatory module in IPF MPCs that increased the expression of genes positively regulating pluripotency and self-renewal. Left unanswered is how IPF MPCs evade negative regulation of self-renewal. Here we sought to identify mechanisms disabling negative regulation of self-renewal in IPF MPCs. We demonstrate that expression of the tumor suppressor genes rbl1 and pten is decreased in IPF MPCs. The mechanism involves the CD44-facilitated association of the chromatin remodeler Brg1 with the histone-modifying methyltransferase PRMT5. Brg1 enhances chromatin accessibility leading to PRMT5-mediated methylation of H3R8 and H4R3 on the rbl1 and pten genes, repressing their expression. Genetic knockdown or pharmacological inhibition of either Brg1 or PRMT5 restored RBL1 and PTEN expression reduced IPF MPC self-renewal in vitro and inhibited IPF MPC-mediated pulmonary fibrosis in vivo. Our studies indicate that the CD44/Brg1/PRMT5 regulatory module not only functions to activate positive regulators of pluripotency and self-renewal but also functions to repress tumor suppressor genes rbl1 and pten. This confers IPF MPCs with the cancer-like property of cell-autonomous self-renewal providing a molecular mechanism for relentless fibrosis progression in IPF.NEW & NOTEWORTHY Here we demonstrate that a CD44/Brg1/PRMT5 epigenetic regulatory module represses the tumor suppressor genes RBL1 and PTEN in IPF mesenchymal progenitor cells, thereby promoting their self-renewal and maintenance of a critical pool of fibrogenic mesenchymal progenitor cells.

Novel gene therapy for drug-resistant melanoma: Synergistic combination of PTEN plasmid and BRD4 PROTAC-loaded lipid nanocarriers

Patients suffering from BRAF mutant melanoma have tumor recurrence within merely 7 months of treatment with a potent BRAF inhibitor (BRAFi) like vemurafenib (VEM). It has been proven that diverse molecular pathways driving BRAFi resistance converge to activation of c-Myc in melanoma. Therefore, we identified a novel combinatorial therapeutic strategy by targeting loss of PTEN tumor suppressor gene and upregulated BRD4 oncoprotein as Myc-dependent vulnerabilities of drug-resistant melanoma. Being promising therapeutic targets, we decided to concomitantly deliver PTEN plasmid and BRD4 targeted PROteolysis-TArgeting Chimera (ARV) to drug the “undruggable” c-Myc in BRAFi-resistant melanoma. Since PTEN plasmid and ARV are distinct in their physicochemical properties, we fabricated PTEN-plasmid loaded LNPs (PL-NANO) and ARV-825 loaded nanoliposomes (AL-NANO) to yield mean particle size of <100 nm and >99% encapsulation efficiency for each therapeutic payload. Combination of PL-NANO and AL-NANO displayed synergistic tumor growth inhibition and substantial apoptosis in in vitro 2D and 3D models. Importantly, simultaneous delivery of PL-NANO and AL-NANO achieved significant upregulation of PTEN expression levels and degradation of BRD4 protein to ultimately downregulate c-Myc levels in BRAFi-resistant melanoma cells. Altogether, lipid nanocarriers delivering this novel lethal cocktail stands as “one-of-a-kind” gene therapy to target “undruggable” c-Myc oncogene in BRAFi-resistant melanoma.

WTAP/IGF2BP3 mediated m6A modification of the EGR1/PTEN axis regulates the malignant phenotypes of endometrial cancer stem cells

Endometrial cancer (EC) stem cells (ECSCs) are pivotal in the oncogenesis, metastasis, immune escape, chemoresistance, and recurrence of EC. However, the specific mechanism of stem cell maintenance in EC cells (ECCs) has not been clarified. We found that WTAP and m6A levels decreased in both EC and ECSCs, and that knocking down WTAP promoted ECCs and ECSCs properties, including proliferation, invasion, migration, cisplatin resistance, and self-renewal. The downregulation of WTAP leads to a decrease in the m6A modification of EGR1 mRNA, and it is difficult for IGF2BP3, as an m6A reader, to recognize and bind to EGR1 mRNA that has lost m6A modification, resulting in a decrease in the stability of EGR1 mRNA. A decrease in the EGR1 level led to a decrease of in the expression tumor suppressor gene PTEN, resulting in deregulation and loss of cellular homeostasis and thereby fostering EC stem cell traits. Notably, the enforced overexpression of WTAP, EGR1, and PTEN inhibited the oncogenic effects of ECCs and ECSCs in vivo, and the combined overexpression of WTAP + EGR1 and EGR1 + PTEN further diminished the tumorigenic potential of these cells. Our findings revealed that the WTAP/EGR1/PTEN pathway is important regulator of EC stem cell maintenance, chemotherapeutic resistance, and tumorigenesis, suggesting a novel and promising therapeutic avenue for treating EC.

A black phosphorus nanosheet-based RNA delivery system for prostate cancer therapy by increasing the expression level of tumor suppressor gene PTEN via CeRNA mechanism

BackgroundProstate cancer (PCa) has a high incidence in men worldwide, and almost all PCa patients progress to the androgen-independent stage which lacks effective treatment measures. PTENP1, a long non-coding RNA, has been shown to suppress tumor growth through the rescuing of PTEN expression via a competitive endogenous RNA (ceRNA) mechanism. However, PTENP1 was limited to be applied in the treatment of PCa for the reason of rapid enzymatic degradation, poor intracellular uptake, and excessively long base sequence to be synthesized. Considering the unique advantages of artificial nanomaterials in drug loading and transport, black phosphorus (BP) nanosheet was employed as a gene-drug carrier in this study.ResultsThe sequence of PTENP1 was adopted as a template which was randomly divided into four segments with a length of about 1000 nucleotide bases to synthesize four different RNA fragments as gene drugs, and loaded onto polyethyleneimine (PEI)-modified BP nanosheets to construct BP-PEI@RNA delivery platforms. The RNAs could be effectively delivered into PC3 cells by BP-PEI nanosheets and elevating PTEN expression by competitive binding microRNAs (miRNAs) which target PTEN mRNA, ultimately exerting anti-tumor effects.ConclusionsTherefore, this study demonstrated that BP-PEI@RNAs is a promising gene therapeutic platform for PCa treatment.Graphical

DNA Mutational and Copy Number Variation Profiling of Primary Craniofacial Osteosarcomas by Next-Generation Sequencing.

Craniofacial osteosarcomas (CFOS) are uncommon malignant neoplasms of the head and neck with different clinical presentation, biological behavior and prognosis from conventional osteosarcomas of long bones. Very limited genetic data have been published on CFOS. In the current study, we performed comprehensive genomic studies in 15 cases of high-grade CFOS by SNP array and targeted next generation sequencing. Our study shows high-grade CFOS demonstrate highly complex and heterogenous genomic alterations and harbor frequently mutated tumor suppressor genes TP53, CDKN2A/B, and PTEN, similar to conventional osteosarcomas. Potentially actionable gene amplifications involving CCNE1, AKT2, MET, NTRK1, PDGFRA, KDR, KIT, MAP3K14, FGFR1, and AURKA were seen in 43% of cases. GNAS hotspot activating mutations were also identified in a subset of CFOS cases, with one case representing malignant transformation from fibrous dysplasia, suggesting a role for GNAS mutation in the development of CFOS. High-grade CFOS demonstrate highly complex and heterogenous genomic alterations, with amplification involving receptor tyrosine kinase genes, and frequent mutations involving tumor suppressor genes.

HOLOTHURIA SCABRA METHANOL EXTRACT INHIBITS CANCER GROWTH THROUGH TGF-β/PI3K/PTEN SIGNALING PATHWAY IN BREAST CANCER MICE MODEL.

Molecules and cytokines can be targeted in cancer therapy. Transforming growth factor-beta (TGF-β) is a cytokine that acts on protein kinase receptors in the plasma membrane. The signaling pathway of TGF-β can trigger the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway, a signal transduction pathway important in cancer growth and development. However, this PI3K/AKT cascade can be inhibited by phosphatase and tensin homolog (PTEN) tumor suppressor genes. To determine the inhibitory effect of Holothuria scabra methanol extract (HSE) on breast cancer growth through the TGF-β/PI3K pathways and PTEN tumor suppressor gene on a breast cancer (BC) mice model. Female C57BL6 mice were subcutaneously injected with carcinogen DMBA 1 mg/kg body weight (BW) and fed a high-fat diet (HFD). Mice were randomly divided into five groups (n = 6): negative control (NC) administered with a standard diet, positive control (PC) administered with DMBA and HFD, and three treatment groups (T1, T2, and T3) treated with HSE doses of 0.33, 0.66, and 0.99 g/kg BW for 12 weeks. TGF-β concentration in the blood serum of mice was assessed by ELISA and the PIK3CA and PTEN gene expression by qRT-PCR. The treatment with HSE resulted in a significant decrease in TGF-β concentrations in the blood sera of treatment groups T1 (35.31 ± 17.33), T2 (43.31 ± 17.42), and T3 (48.67 ± 20.94) pg/mL compared to the PC group (162.09 ± 11.60) pg/mL (p < 0.001). However, only HSE at a dose of 0.99 g/kg BW decreased the PIK3CA gene expression (p = 0.026), and at a dose of 0.66 g/kg BW increased the PTEN expression up to 4.93-fold. HSE is capable of inhibiting the TGF-β/PIK3CA pathway and increasing the PTEN gene expression.

Genetic Links between Endometriosis and Endometriosis-Associated Ovarian Cancer-A Narrative Review (Endometriosis-Associated Cancer).

Endometriosis is a frequent, estrogen-dependent, chronic disease, characterized by the presence of endometrial glands and stroma outside of the uterine cavity. Although it is not considered a precursor of cancer, endometriosis is associated with ovarian cancer. In this review, we summarized the evidence that clear-cell and endometrioid ovarian carcinomas (endometriosis-associated ovarian carcinoma-EAOC) may arise in endometriosis. The most frequent genomic alterations in these carcinomas are mutations in the AT-rich interaction domain containing protein 1A (ARID1A) gene, a subunit of the SWI/SNF chromatin remodeling complex, and alterations in phosphatidylinositol 3-kinase (PI3K) which frequently coexist. Recent studies have also suggested the simultaneous role of the PTEN tumor-suppressor gene in the early malignant transformation of endometriosis and the contribution of deficient MMR (mismatch repair) protein status in the pathogenesis of EAOC. In addition to activating and inactivating mutations in cancer driver genes, the complex pathogenesis of EAOC involves multiple other mechanisms such as the modulation of cancer driver genes via the transcriptional and post-translational (miRNA) modulation of cancer driver genes and the interplay with the inflammatory tissue microenvironment. This knowledge is being translated into the clinical management of endometriosis and EAOC. This includes the identification of the new biomarkers predictive of the risk of endometriosis and cancer, and it will shape the precision oncology treatment of EAOC.

Exploring the Prevalence of Oral Features for Early Detection of PTEN Hamartoma Tumour Syndrome

AimsPatients with PTEN hamartoma tumour syndrome (PHTS) have an increased risk of developing cancer due to a pathogenic germline variant in the PTEN tumour suppressor gene. Early recognition of PHTS facilitates initiation of cancer surveillance which is highly effective in preventing the development of advanced malignancies. PHTS is rare and due to its varied phenotype, even within families, oral abnormalities may be a valuable tool in the identification of these patients at an early stage before cancer development. Materials and methodsBetween 1997 and 2020, phenotypic characteristics were evaluated in 81 paediatric (median age: 9 years) and 86 adult (median age: 40 years) PHTS patients by one of 2 medical experts during yearly surveillance visits at a Dutch PHTS expertise centre. Oral features evaluated included gingival hypertrophy, oral papillomas, and high palate (in adults). ResultsWithin adults, gingival hypertrophy was present in 94%, oral papillomas in 88%, and a high palate in 89%. All adult patients had at least one of these oral features, and 99% showed at least 2 oral features. Oral features were less common in paediatric patients, especially under 11 years of age. Gingival hypertrophy was observed in 44% and oral papillomas in 54% of paediatric patients. ConclusionsThe presence of 2 or 3 oral features may indicate PHTS in adults or adolescents, especially if macrocephaly is present. Dental professionals are well-positioned to recognise these oral manifestations could be related to PHTS. They can initiate an overall clinical assessment of the patient by alerting the patient's medical practitioner of the findings and the possible need for genetic testing. This could significantly improve outcomes, including life expectancy, for patients and possibly for their relatives. Clinical relevanceDental professionals are ideally placed to recognise oral features and initiate early assessment of PHTS which could significantly improve patient outcomes.

Genomic Correlates of Prostate-Specific Membrane Antigen Expression and Response to 177Lu-PSMA-617: A Retrospective Multicenter Cohort Study.

While 177Lu-PSMA-617 (LuPSMA) is an effective therapy for many patients with metastatic castration-resistant prostate cancer (mCRPC), biomarkers associated with outcomes are not well defined. We hypothesized that prostate cancer mutational profile may associate with clinical activity of LuPSMA. We devised a study to evaluate associations between mCRPC mutational profile with LuPSMA clinical outcomes. This was a multicenter retrospective analysis of patients with mCRPC with next-generation sequencing (NGS) who received LuPSMA. PSA50 response (ie, ≥50% decline in prostate-specific antigen [PSA]) rate, PSA progression free survival (PSA PFS), and overall survival (OS) were compared between genetically defined subgroups. One hundred twenty-six patients with NGS results who received at least one cycle of LuPSMA were identified. The median age was 73 (IQR, 68-78) years, 124 (98.4%) received ≥1 prior androgen receptor-signaling inhibitor, and 121 (96%) received ≥1 taxane-based chemotherapy regimen. Fifty-eight (46%) patients with a DNA damage repair gene mutation (DNA damage response group) and 59 (46.8%) with a mutation in TP53, RB1, or PTEN tumor suppressor genes (TSG group) were identified. After adjusting for relevant confounders, the presence of ≥1 TSG mutation was associated with shorter PSA PFS (hazard ratio [HR], 1.93 [95% CI, 1.05 to 3.54]; P = .034) and OS (HR, 2.65 [95% CI, 1.15 to 6.11]; P = .023). There was improved OS favoring the DNA damage response group (HR, 0.37 [95% CI, 0.14 to 0.97]; P = .044) on multivariable analysis. Univariate analysis of patients with ATM mutations had significantly higher rates of PSA50 response, PSA PFS, and OS. Outcomes on LuPSMA varied on the basis of mutational profile. Prospective studies to define the clinical activity of LuPSMA in predefined genomic subgroups are justified.

Abstract 7271: Proteasome inhibitor FHND6091: A potent therapeutic candidate forPTEN-deficient cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA), characterized by high genetic heterogeneity, poses a significant challenge due to limited therapeutic options. Notably, the tumor suppressor gene PTEN is frequently altered in more than 50% of CCA clinical specimens. PTEN deficiency enhances proteasome subunit expression and proteasome proteolytic activity, and facilitates the potential therapeutic efficacy of proteasome inhibitors in CCA. The proteasome inhibitor FHND6091, a novel drug developed by Jiangsu Chia Tai Fenghai Pharmaceutical Co., Ltd., has obtained IND approval from NMPA and is currently undergoing a first-in-human phase 1 trial for multiple myeloma (MM) and mantle cell lymphoma (MCL). Method: This study employed a comprehensive approach, encompassing both in vitro and in vivo pharmacology studies. The investigation involved purified human proteasome activity assays, cell viability assays, and in vivo anti-tumor activity studies utilizing a cholangiocarcinoma patient-derived xenograft (CCA PDX) model with PTEN deficiency. Results: FHND6091 exhibited potent inhibitory effects on the β5 subunit of the 20S proteasome, with IC50 value of 2.22 nM. In cell viability assessments, FHND6091 demonstrated selective efficacy in PTEN-deficient CCA cell lines, with IC50 value of 5.87 nM in HCCC-9810 (PTEN deletion, homozygous loss of exon 6 to 9) and 10.08 nM in RBE (PTEN intact expression). In a PDX xenograft model with PTEN deficiency, gavage administration of FHND6091 BIW at 1.0 mg/kg (first week)/1.5 mg/kg (subsequent two weeks) exhibited advanced anti-tumor activity (TGI = 52.32%) compared to intraperitoneal administration of Bortezomib at 0.5 mg/kg BIW (TGI = 31.88%). Tissue distribution studies in rats revealed the preference of FHND6091 for liver accumulation, with an exposure (AUC0-t) approximately 44 times that of plasma after a single dose. These data corroborated that FHND6091 could achieve a specific level of exposure in the target organ liver. Conclusion: FHND6091, characterized by its irreversibility, high potency and oral bioavailability, emerges as a promising proteasome inhibitor for the treatment of both hematologic and solid tumors. These findings strongly support FHND6091 as a potential candidate for the treatment of CCA in clinical settings. Furthermore, the regulating of sensitivity to proteasome inhibitors by the PTEN status extends beyond cholangiocarcinoma to encompass various human cancers, such as ovarian, prostate, and digestive system malignancies. This broader implication establishes a theoretical foundation supporting the potential efficacy of FHND6091 as a therapeutic agent for solid tumors. Citation Format: Yongqiang Zhu, Jingmiao Shi, Chenhui Li. Proteasome inhibitor FHND6091: A potent therapeutic candidate forPTEN-deficient cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7271.

Abstract 2975: Vitamin D receptor signaling limits DNA damage at early stage of prostate tumorigenesis and prevents tumor aggressiveness

Abstract Prostate cancer (PCa) is the most common visceral neoplasm and the second leading cause of cancer-related deaths in male of western societies. Low circulating vitamin D levels and reduced expression of its receptor (VDR) correlate with PCa risk and severity, but the clinical use of vitamin D is limited by its pro-calcemic activities. To characterize the role of VDR in PCa, PTEN(i)pe-/− mice, that recapitulate the human disease and in which the tumor suppressor gene PTEN is selectively inactivated in prostatic epithelial cells (PECs) at adulthood, were compared to PTEN/VDR(i)pe-/− mice in which PECs are PTEN and VDR-deficient. Histological analysis showed that prostatic intraepithelial neoplasia (PIN) are formed between 1-3 months after gene inactivation (AGI). Importantly, VDR-deficiency increased by 2-fold the PECs proliferation rate as early as 1-month AGI. At this stage, RNA-seq and immunohistochemistry analysis revealed the presence of oxidative DNA damage selectively in VDR-deficient PINs. Moreover, a 4-week supplementation with the antioxidant N-acetyl cysteine reduced oxidative DNA damage in PTEN/VDR(i)pe-/- prostates, and normalized the number of Ki-67+ cells to the levels of PTEN(i)pe-/- ones. After 3 months AGI, both PTEN(i)pe-/- and PTEN/VDR(i)pe-/- tumors entered in a latency phase characterized by cellular senescence. However, the prostate weight in PTEN/VDR(i)pe-/− mice was 2-fold higher than in PTEN(i)pe-/− mice 12 months AGI. In addition, tumors of PTEN(i)pe-/− were mostly composed of non-invasive PIN-like adenocarcinoma, whereas invasive adenocarcinoma, sarcomatoid and squamous-cell carcinoma were observed in those of PTEN/VDR(i)pe-/- mice. Using single cell and spatial transcriptomics, we unraveled the presence of epithelial cells with epithelial-mesenchymal transition (EMT) hallmarks in PTEN/VDR(i)pe-/− prostate 10 months AGI. In addition, immunophenotyping revealed a higher infiltration of neutrophils, also termed as myeloid-derived suppressive cells and implicated in EMT and metastasis dissemination, in PTEN/VDR(i)pe-/− prostates compared to PTEN(i)pe-/− ones. Moreover, pan-cytokeratin-positive micro-metastasis were detected in lymph nodes and livers of PTEN/VDR(i)pe-/− mice, but not in those of PTEN(i)pe-/- mice, demonstrating that VDR-deficiency in PECs promotes aggressive PCa. Thus, VDR signaling exerts a protective role on PTEN-deficient PECs by limiting the oxidative-stress and proliferation during PINs initiation, and prevents PCa progression. Citation Format: Kateryna Len, Justine Gantzer, Nicolas Le May, Christelle Thibault-Carpentier, Daniel Metzger, Gilles Laverny. Vitamin D receptor signaling limits DNA damage at early stage of prostate tumorigenesis and prevents tumor aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2975.

Abstract 1740: Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion

Abstract Metastatic potential of basal-like breast cancers typically is initiated by genetic alterations that lead to a process known as epithelial-mesenchymal transition (EMT). However, much is currently not understood regarding the role of epigenetic modifications that lead to invasive characteristics and EMT phenotype of metastatic breast cancers. Based on the previous notion connecting epigenetic changes to breast cancer metastasis, we performed DIA-based mass spectrometry of isolated histones from an isogenic panel of MCF10A breast cell lines where the tumor suppressor genes TP53 and PTEN were silenced and induced EMT. With this approach, we determined which histone modifications were differentially enriched in the non-EMT and EMT-induced MCF10A cell lines. From approximately 72 histone modifications identified and annotated from our mass spectrometry results, we were able to identify 5 histone events that were differentially enriched in our MCF10A cell line panel. Two events of note were histone H3 lysine-14 acetylation (H3K14ac) significantly increasing and histone H4 arginine 55 dimethylation (H4R55me2) significantly decreasing in our EMT-transformed MCF10A p53-/PTEN- cell lines when compared to the parental, non-tumorigenic MCF10A cell line, showing these events are differentially affected during the EMT process in breast cancer cells. Furthermore, significant arginine demethylation of H4R55me2 &amp; H3.1R83me in the EMT-transformed MCF10A p53-/PTEN- cell lines corresponded with JMJD6, an established histone arginine demethylase, being overexpressed in basal-like breast cancer cell lines as well as basal-like breast cancer patients from The Cancer Genome Atlas (TCGA) and METABRIC datasets. Based on histone proteomic profiling of our isogenic cell line model, the loss of specific histone arginine methylation events corresponding with JMJD6 overexpression could highlight the potential for a targetable epigenetic mechanism in breast cancer metastasis. Citation Format: Charlotte B. McGuinness, Megan A. Wilson, Margaret V. Leonard, Maria Ouzounova, Hasan Korkaya, Austin Y. Shull. Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1740.

Abstract 164: Genetic elimination of β-catenin using a doxycycline-regulated GEM model fails to restore checkpoint blockade efficacy due to persistent M2-like macrophages

Abstract Response to checkpoint blockade immunotherapy (CBI) is not universal, partly due to a wide variation in baseline immune cell infiltration in the tumor microenvironment (TME). One mechanism contributing to this variability is tumor cell-intrinsic activation of the β-catenin signaling pathway, which has previously been shown to result in a non-T cell-inflamed TME. Constitutive β-catenin signaling resulted in reduced Batf3-lineage dendritic cell (cDC1) recruitment, leading to reduced T cell activation and infiltration and loss of anti-CTLA-4+anti-PD-L1 therapeutic efficacy. Efforts to target the Wnt/β-catenin pathway to enhance immune responses in β-catenin-active tumors have seen limited success. A genetic experiment eliminating β-catenin expression after establishment of a non-T cell-inflamed TME might illustrate the maximal biologic effect that could be expected with blockade of the pathway. We developed a genetically engineered mouse (GEM) model that allows for dynamic regulation of β-catenin on and off. This model employs tyrosinase-driven tamoxifen-regulated Cre-recombinase to activate the BRAF V600E oncogene and delete the tumor suppressor gene PTEN, along with a transactivator for doxycycline-regulated expression of a non-degradable β-catenin. Topical application of 4-hydroxytamoxifen resulted in melanoma formation, and administration of doxycycline resulted in robust nuclear expression of melanocyte-specific β-catenin compared to non-treated controls. This was accompanied by a significant reduction in CD3+ T cell infiltration within the TME, confirming the link between active β-catenin signaling and T cell exclusion. Discontinuing doxycycline treatment led to complete reduction of nuclear β-catenin expression in the tumor cells within seven days and a substantial return of CD3+ T cells into the TME. However, despite the re-infiltration of CD3+ T cells, the tumors previously expressing β-catenin (ex-β-catenin tumors) did not regain therapeutic responsiveness to anti-CTLA-4+anti-PD-L1. Single cell RNA sequencing of the ex-β-catenin tumors indicated the presence of an immunosuppressive-like macrophage population, characterized by the expression of Ccl8, Gas6 and Cd163. This finding suggests that the prior presence of β-catenin may induce long-lasting changes in the TME that suppress the immune response even after β-catenin levels are reduced. These data corroborate previous findings on the role of β-catenin in modulating the immunological landscape of melanoma and extend it by demonstrating the long-term persistence of immune evasion by suppressive myeloid cells even after β-catenin signaling is eliminated. These insights have implications for clinical strategies aiming to block Wnt/β-catenin signaling, which may additionally require myeloid cell modulation to restore immunotherapy efficacy. Citation Format: Alexandra Cabanov, Ruxandra Tonea, Jason Shapiro, Anna Martinez, Yuanyuan Zha, Thomas F. Gajewski. Genetic elimination of β-catenin using a doxycycline-regulated GEM model fails to restore checkpoint blockade efficacy due to persistent M2-like macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 164.

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

Abstract B028: Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia

Abstract Somatic mutations in the phosphatidylinositol 3-kinase (PI3K) pathway occur in 84% of endometrial carcinomas (EC) and up to 68% of endometrial hyperplasia (EH). Regulation of PI3K/Akt/mTOR signaling is integral for normal endometrial function and is governed by tumor suppressor genes PTEN and PIK3R1 and proto-oncogene PIK3CA. While the actions of PTEN and PIK3CA are in direct opposition as a phosphatase and kinase, respectively, PIK3R1 regulates PI3K/Akt/mTOR signaling in two distinct ways. As the regulatory subunit of the PI3K enzyme, PIK3R1 controls the catalytic activity of PIK3CA and augments PTEN activity. Mutations or alterations in PIK3R1 are present in 33% of EC and 9% of EH, the majority of which lead to loss of PIK3R1 protein (p85a). Both loss of PTEN and PIK3R1 lead to increased proliferation through PI3K/Akt/mTOR signaling, but the structural, functional, and mechanistic implications of PIK3R1 loss in the endometrial epithelium (EE) in vivo are largely unexplored. To further interrogate the changes in the endometrium resulting from PIK3R1 loss, we have created a murine model of endometrial epithelial-specific PIK3R1 loss driven by Pax8-Cre. Mice were estrus cycle staged and sacrificed in the diestrus phase to reduce confounding variables when assessing phenotypes between genotypes. PIK3R1 loss was confirmed by immunohistochemistry (IHC). Histology and immunostaining of control and PIK3R1 knockout (KO) mice were assessed by IHC and immunofluorescence. We found that PIK3R1 loss in the EE leads to EH distinct from what is observed with PTEN loss. In contrast to the EH with atypia seen In PTEN KO mice, the EH resulting from PIKR1 loss more closely resembles EH without atypia. The endometrium of PIK3R1 KO mice is characterized by thickened luminal epithelia with jagged edges and irregularities, mild glandular crowding, and occasional nuclear atypia. PIK3R1 KO mice also showed an increase in proliferation (Ki-67) and downstream PI3K signaling (phospho-S6) in the EE. Gene expression changes occurring within the EE from PIK3R1 KO and Cre-negative control mice were also assessed by RNA-seq. In vivo genomic analyses were compared to in vitro knockdown of PIK3R1 and publicly available patient data. We found that PIK3R1 loss leads to an increase in signaling through the PI3K/Akt/mTOR and MAPK pathways. Our data show that the effect of PIK3R1 loss is sufficient to cause EH and may have shared and distinct actions from PTEN. Citation Format: Shannon K. Harkins, Hilary J. Skalski, Amelia Arendt, Laura Pavliscak, Mike R. Wilson, Ronald L. Chandler. Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B028.

Real-World Overall Survival and Treatment Patterns by PTEN Status in Metastatic Castration-Resistant Prostate Cancer.

It is estimated that the PTEN tumor suppressor gene is functionally lost in 40%-50% of patients with metastatic castration-resistant prostate cancer (mCRPC). There is limited information on the prognostic significance of PTEN status identified with genomic testing. This real-world cohort study assessed PTEN as a genetic biomarker using data from US-based oncology practices. This retrospective real-world cohort study used a deidentified US-based metastatic prostate cancer clinicogenomic database linked to longitudinal clinical data derived from electronic health records. Patients were aged 18 years and older and diagnosed with mCRPC between January 1, 2018, and June 30, 2021. Comprehensive genomic profiling (CGP) of tumor specimens was performed using next-generation sequencing. First-line (1L) and second-line (2L) treatment patterns were assessed and stratified by PTEN status. Kaplan-Meier methods and a multivariable Cox model were used to compare the real-world overall survival by PTEN status among patients who received 1L novel hormone therapy or taxanes. In patients with mCRPC who underwent CGP, PTEN loss of function (LOF) was associated with decreased survival compared with intact PTEN (hazard ratio, 1.61 [95% CI, 1.07 to 2.42]; P = .024). The results were not influenced by 1L treatment type. 1L treatment patterns were similar between intact PTEN and PTEN LOF subgroups, with abiraterone and enzalutamide being the two most common treatments in both groups. Patients with PTEN LOF were less likely to receive 2L treatments than patients with intact PTEN. PTEN LOF, identified with genomic testing, was associated with decreased survival and negative prognoses in patients with mCRPC.

The Role of the PTEN Tumor Suppressor Gene and Its Anti-Angiogenic Activity in Melanoma and Other Cancers.

Human malignant melanoma and other solid cancers are largely driven by the inactivation of tumor suppressor genes and angiogenesis. Conventional treatments for cancer (surgery, radiation therapy, and chemotherapy) are employed as first-line treatments for solid cancers but are often ineffective as monotherapies due to resistance and toxicity. Thus, targeted therapies, such as bevacizumab, which targets vascular endothelial growth factor, have been approved by the US Food and Drug Administration (FDA) as angiogenesis inhibitors. The downregulation of the tumor suppressor, phosphatase tensin homolog (PTEN), occurs in 30-40% of human malignant melanomas, thereby elucidating the importance of the upregulation of PTEN activity. Phosphatase tensin homolog (PTEN) is modulated at the transcriptional, translational, and post-translational levels and regulates key signaling pathways such as the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways, which also drive angiogenesis. This review discusses the inhibition of angiogenesis through the upregulation of PTEN and the inhibition of hypoxia-inducible factor 1 alpha (HIF-1-α) in human malignant melanoma, as no targeted therapies have been approved by the FDA for the inhibition of angiogenesis in human malignant melanoma. The emergence of nanocarrier formulations to enhance the pharmacokinetic profile of phytochemicals that upregulate PTEN activity and improve the upregulation of PTEN has also been discussed.

CHIP suppresses the proliferation and migration of A549 cells by mediating the ubiquitination of eIF2α and upregulation of tumor suppressor RBM5

The PERK-eIF2α pathway plays an essential role in endoplasmic reticulum (ER) stress. When the PERK-eIF2α pathway is activated, PERK phosphorylates eIF2α (p-eIF2α) at Ser51 and quenches global protein synthesis. In this study, we verified eIF2α as a bona fide substrate of the E3 ubiquitin ligase CHIP both in vitro and in cells. CHIP mediated the ubiquitination and degradation of nonphosphorylated eIF2α in a chaperone-independent manner and promoted the upregulation of the transcription factor ATF4 under ER stress conditions. ATF4 induced the transcriptional enhancement of the tumor suppressor genes PTEN and RBM5. Although transcription was enhanced, the PTEN protein was subsequently degraded by CHIP, but the expression of the RBM5 protein was upregulated, thereby suppressing the proliferation and migration of A549 cells. Overall, our study established a new mechanism that deepened the understanding of the PERK-eIF2α pathway through the ubiquitination and degradation of eIF2α. The crosstalk between the phosphorylation and ubiquitination of eIF2α shed light on a new perspective for tumor progression.

Genetically engineered mouse model of pleomorphic liposarcoma: Immunophenotyping and histologic characterization

IntroductionPleomorphic liposarcoma is a rare and aggressive subset of soft-tissue sarcomas with a high mortality burden. Local treatment largely consists of radiation therapy and wide surgical resection, but options for systemic therapy in the setting of metastatic disease are limited and largely ineffective, prompting exploration of novel therapeutic strategies and experimental models. As with other cancers, sarcoma cell lines and patient-derived xenograft models have been developed and used to characterize these tumors and identify therapeutic targets, but these models have inherent limitations. The establishment of genetically engineered mouse models represents a more realistic framework for reproducing clinically relevant conditions for studying pleomorphic liposarcoma. MethodsTrp53fl/fl/Rb1fl/fl/Ptenfl/fl (RPP) mice were used to reliably generate an immunocompetent model of mouse pleomorphic liposarcoma through Cre-mediated conditional silencing of the Trp53, Rb1, and Pten tumor suppressor genes. Evaluation of tumor-infiltrating lymphocytes was assessed with immunostaining for CD4, CD8, and PD-L1, and flow cytometry with analysis of CD45, CD3, CD4, CD8, CD19, F4/80, CD11b, and NKp46 sub-populations. ResultsMice reliably produced noticeable soft-tissue tumors in approximately 6 weeks with rapid tumor growth between 100 and 150 days of life, after which mice reached euthanasia criteria. Histologic features were consistent with pleomorphic liposarcoma, including widespread pleomorphic lipoblasts. Immunoprofiling and assessment of tumor-infiltrating lymphocytes was consistent with other soft-tissue sarcomas. ConclusionGenetically engineered RPP mice reliably produced soft-tissue tumors consistent with pleomorphic liposarcoma, which immunological findings similar to other soft-tissue sarcomas. This model may demonstrate utility in testing treatments for this rare disease, including immunomodulatory therapies.