Yang Yang-Hartwich

Preclinical Efficacy of the Estrogen Receptor Degrader Fulvestrant in Combination with RAF/MEK Clamp Avutometinib and FAK Inhibitor in a Low-Grade Serous Ovarian Cancer Animal Model with Acquired Resistance to Chemotherapy and Aromatase Inhibitor

Low-grade-serous ovarian carcinomas (LGSOC) are rare tumors characterized by a high recurrence rate and limited treatment options. Most LGSOC are estrogen receptor (ER)-positive and demonstrate alterations in the RAS/MAPK pathway. Avutometinib is a dual RAF/MEK clamp, whereas defactinib and VS-4718 are focal adhesion kinase (FAK) inhibitors. Fulvestrant is an ER antagonist/degrader. We assessed the preclinical efficacy of fulvestrant, avutometinib + VS-4718 (FAKi), and the triple combination in a chemotherapy/aromatase inhibitor-resistant LGSOC patient-derived tumor xenograft (PDX) model. Tissue obtained from a LGSOC patient wild-type for KRAS/NRAS/BRAF mutations in progression after chemotherapy/anastrozole was transplanted into female CB17/lcrHsd-Prkdc/SCID mice (PDX-OVA(K)250). The animals were treated with either saline/control, fulvestrant, avutometinib/FAKi, or the triple combination of avutometinib/FAKi/fulvestrant. Avutometinib and FAKi were given five-days on and two-days off through oral gavage. Fulvestrant was administered subcutaneously weekly. Mechanistic studies were performed ex vivo using Western blot assays. Animals treated with the triple combination demonstrated stronger tumor growth inhibition compared to all the other experimental groups including control/saline (p < 0.001), single-agent fulvestrant (p = 0.04 from day eight and onwards), and avutometinib/FAKi (p = 0.02 from day 18). Median survival for mice treated with saline/control was 29 days while mice in all other experimental groups were alive at day 60 (p < 0.0001). Treatment was well tolerated across all experimental treatments. By Western blot, exposure of OVA(K)250 to the triple combination demonstrated a decrease in phosphorylated MEK (p-MEK) and p-ERK levels. The addition of fulvestrant to avutometinib/FAKi is well tolerated in vivo and enhances the antitumor activity of avutometinib/FAKi in a LGSOC-PDX model with acquired resistance to chemotherapy/aromatase inhibitors. These results support the clinical evaluation of avutometinib/defactinib in combination with fulvestrant or an aromatase inhibitor in patients with recurrent LGSOC.

Preclinical Activity of Datopotamab Deruxtecan, an Antibody–Drug Conjugate Targeting Trophoblast Cell-Surface Antigen 2, in Uterine Serous Carcinoma

Abstract Uterine serous carcinoma (USC) is a rare subset of endometrial cancer with a poor prognosis and high recurrence rate. Datopotamab deruxtecan (Dato-DXd) is a novel antibody–drug conjugate (ADC). The objective of this study was to evaluate the preclinical activity of Dato-DXd in USC in vitro against primary USC cell lines with various trophoblast cell-surface antigen 2 (TROP2) expression and in vivo in TROP2-overexpressing cell line–derived mice xenografts. USC primary tumor cell lines were treated with Dato-DXd and a control ADC (CTL ADC) to evaluate cell viability following exposure. Antibody-dependent cell-mediated cytotoxicity against TROP2-overexpressing and -nonexpressing cell lines was evaluated using a 4-hour chromium release assay. USC xenografts in mice were treated with Dato-DXd, CTL ADC, datopotamab, and vehicle to assess the in vivo effects via retro-orbital Dato-DXd administration. We found USC cell lines with TROP2 overexpression to be significantly more sensitive to killing induced by Dato-DXd compared with CTL ADC in vitro (e.g., IC50: 0.11 µmol/L vs. 30.07 µmol/L, P = 0.0074 and 0.11 µmol/L vs. 48.95 µmol/L, P = 0.0127, respectively). Dato-DXd induced antibody-dependent cell-mediated cytotoxicity in the presence of peripheral blood lymphocytes from healthy donors. TROP2-nonexpressing cell lines demonstrated minimal killing by Dato-DXd; however, when admixed with TROP2-overexpressing cells, a significant bystander effect was appreciated. In vivo, mice xenografts overexpressing TROP2 treated with Dato-DXd demonstrated tumor growth suppression and longer overall survival compared with CTL ADC–treated xenografts. These data demonstrate Dato-DXd to be highly active against TROP2-overexpressing USC in vitro and in vivo. Our preclinical activity results warrant future clinical trials for patients with advanced or recurrent USC. Significance: Targeted treatment of USC using the biomarker TROP2 represents a significant opportunity for further treatment options for patients already resistant to other lines of treatment. In this study, we present data showing preclinical evidence of effectiveness of this biomarker-targeted therapy in USC.

Identification of miPEP133 as a novel tumor-suppressor microprotein encoded by miR-34a pri-miRNA

Abstract Background Very few proteins encoded by the presumed non-coding RNA transcripts have been identified. Their cellular functions remain largely unknown. This study identifies the tumor-suppressor function of a novel microprotein encoded by the precursor of miR-34a. It consists of 133 amino acid residues, thereby named as miPEP133 (pri-microRNA encoded peptide 133). Methods We overexpressed miPEP133 in nasopharyngeal carcinoma (NPC), ovarian cancer and cervical cancer cell lines to determine its effects on cell growth, apoptosis, migration, or invasion. Its impact on tumor growth was evaluated in a xenograft NPC model. Its prognostic value was analyzed using NPC clinical samples. We also conducted western blot, immunoprecipitation, mass spectrometry, confocal microscopy and flow cytometry to determine the underlying mechanisms of miPEP133 function and regulation. Results miPEP133 was expressed in normal human colon, stomach, ovary, uterus and pharynx. It was downregulated in cancer cell lines and tumors. miPEP133 overexpression induced apoptosis in cancer cells and inhibited their migration and invasion. miPEP133 inhibited tumor growth in vivo. Low miPEP133 expression was an unfavorable prognostic marker associated with advanced metastatic NPC. Wild-type p53 but not mutant p53 induced miPEP133 expression. miPEP133 enhanced p53 transcriptional activation and miR-34a expression. miPEP133 localized in the mitochondria to interact with mitochondrial heat shock protein 70kD (HSPA9) and prevent HSPA9 from interacting with its binding partners, leading to the decrease of mitochondrial membrane potential and mitochondrial mass. Conclusion miPEP133 is a tumor suppressor localized in the mitochondria. It is a potential prognostic marker and therapeutic target for multiple types of cancers.