Tumor Growth In Xenograft Model Research Articles

Design, synthesis, and antitumor activity of benzimidazole derivatives as CDK4/6 inhibitors

A series of benzimidazole derivatives were synthesized and characterized using 1H NMR, 13C NMR, HR-MS, FTIR and UV Vis. The cytotoxicity of these compounds was evaluated against various cell lines, including 293T (kidney cell), HeLa (cervical cancer), A498 (kidney cancer), and MDA-MB-321 (breast cancer), using the MTT assay. It was observed that the majority of the benzimidazole derivatives exhibited superior anti-tumor activity compared to Abemaciclib. Among them, compound 9f demonstrated the most potent anti-proliferative activity on the MDA-MB-321 cell line, with an IC50 value of 0.028±0.0016 μM, significantly lower than that of Abemaciclib (IC50=10.36 ± 5.08 μM). Further investigation into the anti-tumor activity of compound 9f revealed its effectiveness in inhibiting not only tumor cell proliferation, migration, and adhesion of tumor cells but also in demonstrating significant inhibitory effects in experiments involving a cell xenograft tumor growth model using MDA-MB-231 cells. Additionally, molecular docking studies provided insights into the mechanism of interaction between compound 9f and the CDK4/6 protein. These findings suggest that compound 9f holds considerable potential for breast cancer treatment and serves as a promising CDK4/6 inhibitors for further research.

Abstract 7125: Inhibition of IDH1 impairs homologous recombination and induces sensitivity to PARP inhibitor in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related deaths in the United States. While Poly (ADP-ribose) polymerase (PARP) inhibitors hold promise in treating PDAC with Homologous Recombination Deficiency (HRD). However, HRD occurs in only 10% of cases, leaving 90% with intact Homologous Recombination Proficiency (HRP). Our research highlights the key role of wild-type isocitrate dehydrogenase 1 (IDH1) in maintaining cellular function. We found that inhibiting IDH1 increases DNA damage, suppressing the DNA repair pathway crucial for genomic integrity. This makes cancer cells susceptible to PARP inhibitors, offering a new PDAC treatment approach. Methods: The effect of IDH1 inhibition (Ivosidenib) combined with PARP inhibition (Olaparib) on in vitro growth inhibition were assessed through Trypan blue and PicoGreen in drug combination assays. Additionally, we investigated the molecular mechanism underlying the synergistic effects of drug combination. To further evaluate the therapeutic potential of IDH1 inhibition in combination with PARP inhibitors, we examined tumor growth in xenograft models of PDAC in athymic nude mice. Results: Our data report that, mechanistically, IDH1 inhibition decreased expression of HR DNA repair pathway molecules, such as BRCA1 and BRCA2, and leading to decreased HR repair efficiency in pancreatic cancer cells. Short-term cell viability data demonstrated that targeting IDH1 with ivosidenib when combined with DNA-damaging agent (Olaparib) had a synergistic effect in HRP cell lines (MiaPaCa-2 and Panc-1) with a positive synergy score and Bliss score greater than 1. Additionally, we assessed long-term cell survival using colony formation assays, which yielded a dramatic reduction in cell survival for both Panc1 and MiaPaCa-2 cells when olaparib was combined with ivosidenib, as compared to single-agent controls. Consequently, this reduction increased the levels of apoptosis induced by olaparib. Furthermore, we discovered that these two compounds complemented each other in DNA damage response, leading to an increase in the DNA damage biomarker γ-H2AX. In vivo studies using xenograft models demonstrated that the combination of ivosidenib and olaparib synergistically enhanced anti-tumor activity as compared to either single-agent used alone. Conclusion: Our data provides insights into the mechanisms underlying the susceptibility of PDAC tumor growth to a dual treatment approach involving an IDH1 inhibitor and a PARP inhibitor, particularly in the context of HRP cancer. This highlights the promise of precision medicine for pancreatic cancer treatment, though further pre-clinical studies are needed for validation and refinement. Citation Format: Mehrdad Zarei, Omid Hajihassani, Hallie J. Graor, Alexander W. Loftus, Soubhi Tahhan, Faith Nakazzi, Parnian Naji, Luke D. Rothermel, Jonothan R. Brody, Jordan M. Winter. Inhibition of IDH1 impairs homologous recombination and induces sensitivity to PARP inhibitor in pancreatic cancer [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 7125.

Abstract 677: Discovery of a first-in-class allosteric inhibitor of the small GTPase Rac1

Abstract The Rac1 signaling pathway is known to be hyperactivated in numerous solid tumors. We developed a Rac1 AlphaLISA assay that is amenable to high-throughput screening (HTS) to screen large libraries of small molecules. Based on molecular modeling of a putative allosteric binding site for several of the hits, we optimized a series of compounds that lead to RP-102124. RP-102124 engages with Rac1 in both biochemical and cellular target engagement assays. RP-102124 has greater potency than previously reported Rac1 inhibitors in biochemical assays, with potent inhibition of phenotypic characteristics of Rac signaling such as cell migration and cytoskeletal rearrangements. RP-102124 is a Racmulti inhibitor with activity against oncogenic Rac1b. RP-102124 is orally bioavailable and shows dose dependent inhibition of tumor growth in xenograft models at doses that are well-tolerated. RP-102124 is a first-in-class allosteric Racmulti inhibitor that shows promising therapeutic potential. Citation Format: Robert Silverman, Alan Cooper, Ronodip Kar, Mayuri V. Raman, Ramu Ravirala, Mubarak Hasan Shah, Sandeep Mahankali, Erik T. Goka. Discovery of a first-in-class allosteric inhibitor of the small GTPase Rac1 [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 677.

Abstract 6728: Preclinical development of QL325, a novel T cell engager targeting CLEC12A-positive AML

Abstract Despite advancement in targeted therapies, treatment options for patients with acute myeloid leukemia (AML) that provide a tolerable and durable response remain elusive. We have developed a CD3 bispecific antibody, QL325, targeting CLEC12A, a c-type lectin protein that is highly expressed on AML blasts. Normally, CLEC12A expression is mostly confined to the CD34+CD38- progenitor population and largely absent on normal and regenerating bone marrow stem cells. Our bispecific antibody is configured in a 2 + 1 format, with bivalent avidity binding to CLEC12A, monovalent low affinity binding to CD3, and an effector-null Fc for extending half-life. In our preclinical studies, QL325 induced CLEC12A -dependent activation and proliferation of T cells. Redirected T cell-dependent cytotoxicity mediated by QL325 was restricted to AML cancer cells expressing CLEC12A. QL325 demonstrated dose dependent suppression of tumor growth in xenograft models of AML, significantly more potent than a clinical benchmark antibody. In non-human primates, QL325 was tolerated up to 0.03 mg/kg for IV dosing and under 0.1 mg/kg for SC dosing, well above the predicted effective therapeutic dose. A phase 1 clinical trial is currently ongoing to determine the safety and early efficacy in patients with refractory or relapsed AML. Citation Format: Shenda Gu, Hieu V. Tran, Shihao Chen, Ke Liu, Xin Zhang, Linchao Jia, Ruixue Ma, Jiayu Fu. Preclinical development of QL325, a novel T cell engager targeting CLEC12A-positive AML [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 6728.

Parathyroid hormone-related protein as a potential prostate cancer biomarker: Promoting prostate cancer progression through upregulation of c-Met expression.

Parathyroid hormone-related protein (PTHrP) plays a significant role in various tumor types, including prostate cancer. However, its specific role and underlying mechanisms in prostate cancer remain unclear. This study investigates the role of PTHrP and its interaction with the c-Met in prostate cancer. PTHrP was overexpressed and knocked down in prostate cancer cell lines to determine its effect on cell functions. Xenograft tumor models were employed to assess the impact of PTHrP overexpression on tumor growth. To delve into the interaction between PTHrP and c-Met, rescue experiments were conducted. Clinical data and tissue samples from prostate cancer patients were gathered and analyzed for PTHrP and c-Met expression. PTHrP overexpression in prostate cancer cells upregulates c-Met expression and augments cell functions. In contrast, PTHrP-knockdown diminishes c-Met expression and inhibits cell functions. In vivo experiments further demonstrated that PTHrP overexpression promoted tumor growth in xenograft models.Moreover, modulating c-Met expression in rescue experiments led to concurrent alterations in prostate cancer cell functions. Immunohistochemical analysis of clinical samples displayed a significant positive correlation between PTHrP and c-Met expression. Additionally, PTHrP expression correlated with clinical parameters like prostate-specific antigen (PSA) levels, tumor stage, lymph node involvement, distant metastasis, and Gleason score. PTHrP plays a crucial role in prostate cancer progression by upregulating c-Met expression. These insights point to PTHrP as a promising potential biomarker for prostate cancer.

Decoding Serine Metabolism: Unveiling Novel Pathways for Evolving Cancer Therapies.

Serine metabolism plays a pivotal role in cancer, making it an appealing therapeutic target. Two recent studies published in Nature Metabolism and Science Translational Medicine uncovered novel players and therapeutic opportunities within this crucial metabolic pathway. Papalazarou and colleagues employed genetic tools coupled with metabolomics and high-throughput imaging to identify and characterize membrane transporters involved in serine uptake and mitochondrial import in colorectal cancer. Notably, they showed that dual inhibition of these transporters in combination with impaired serine biosynthesis reduced tumor growth in xenograft models. In a parallel study, Zhang and colleagues identified isocitrate dehydrogenase I (IDH1) as a novel regulator of serine biosynthesis in non-small cell lung cancer. Through extensive mechanistic studies, they demonstrated that IDH1 enhances the expression of the key enzymes phosphoglycerate dehydrogenase and phosphoserine aminotransferase 1 via a noncanonical function independent of its enzymatic activity. Strikingly, pharmacologic disruption of this novel function of IDH1 not only diminished tumor growth but also enhanced the anticancer efficacy of dietary serine restriction in mouse models of lung cancer. Together, these studies advance our mechanistic understanding of how cancer cells fulfill their serine requirements and reveal innovative therapeutic avenues to deprive tumors of this vital nutrient.

Corosolic acid enhances oxidative stress-induced apoptosis and senescence in pancreatic cancer cells by inhibiting the JAK2/STAT3 pathway.

Pancreatic cancer (PC) is a fatal human malignancy with a poor prognosis. Corosolic acid (CRA) is a triterpenoid, has been reported to have inhibitory effects on tumor growth. However, the role of CRA on PC has not been explored. Here, we aimed to uncover the molecular mechanisms of CRA in PC progression. Cell viability, lactate dehydrogenase (LDH) release, cell apoptosis and senescence were detected by cell counting kit-8 (CCK-8), LDH, flow cytometry and senescence associated-β-galactosidase (SA-β-gal) assay. Levels of relevant proteins and oxidative stress (OS) markers were evaluated by Western blot and enzyme-linked immunosorbent assay (ELISA). A xenograft tumor model was established to explore the in vivo effects of CRA on PC. We found that CRA inhibited PC cell viability and promoted LDH release in a dose-dependent manner, but had no significant effect on human normal pancreatic ductal epithelial cells HPDE6C7. CRA increased OS-induced cell apoptosis and senescence in HAPC and SW1990 cells. And CRA decreased the levels of anti-apoptotic protein Bcl-2, and elevated the expression of pro-apoptotic protein Bax and senescence-associated proteins P21 and P53. Besides, CRA decreased tumor growth in xenograft models. Furthermore, CRA inactivated the Janus kinase-2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway in HAPC and SW1990 cells. Functional experiments demonstrated that activation of the JAK2/STAT3 pathway by the JAK2 activator coumermycin A1 (C-A1) or the STAT3 activator colivelin (col) reduced the contribution effect of OS, apoptosis and senescence by CRA. Taken together, our findings indicated that CRA exerted anti-cancer effects in PC by inhibiting the JAK2/STAT3 pathway.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer–promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer–promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

ATF5 promotes malignant T cell survival through the PI3K/AKT/mTOR pathway in cutaneous T cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma characterized by skin infiltration of malignant T cells. The biological overlap between malignant T cells and their normal counterparts has brought obstacles in identifying tumor-specific features and mechanisms, limiting current knowledge of CTCL pathogenesis. Transcriptional dysregulation leading to abnormal gene expression profiles contributes to the initiation, progression and drug resistance of cancer. Therefore, we aimed to identify tumor-specific transcription factor underlying CTCL pathology. We analyzed and validated the differentially expressed genes (DEGs) in malignant T cells based on single-cell sequencing data. Clinical relevance was evaluated based on progression-free survival and time to next treatment. To determine the functional importance, lentivirus-mediated gene knockdown was conducted in two CTCL cell lines Myla and H9. Cell survival was assessed by examining cell viability, colony-forming ability, in-vivo tumor growth in xenograft models, apoptosis rate and cell-cycle distribution. RNA sequencing was employed to investigate the underlying mechanisms. Activating transcription factor 5 (ATF5) was overexpressed in malignant T cells and positively correlated with poor treatment responses in CTCL patients. Mechanistically, ATF5 promoted the survival of malignant T cells partially through the PI3K/AKT/mTOR pathway, and imparted resistance to endoplasmic reticulum (ER) stress-induced apoptosis. These findings revealed the tumor-specific overexpression of the transcription factor ATF5 with its underlying mechanisms in promoting tumor survival in CTCL, providing new insight into the understanding of CTCL's pathology.

Synergistic approach to combat triple-negative breast cancer: Ti3C2/TiO2-based photothermal therapy combined with sonodynamic effect for tumor ablation

The incidence rate of female breast cancer in China ranks first in the world, where the five-year survival rate after chemotherapy for triple-negative breast cancer (TNBC) is less than 12%, and there is an urgent need to explore more novel combination therapies to augment efficacy of traditional chemotherapeutic modalities. In this study, a nanodrug was prepared by loading doxorubicin (DOX) and encrusting with calcium carbonate (CaCO3) on two-dimensional Ti3C2/TiO2 nanosheets (TDC). This study aims to investigate their anti-tumor effect of TDC in combination with photothermal and sonodynamic therapy. Uptake of TDC nanoparticles into TNBC cells and a pH-dependent release pattern of Ca2+ were observed. Critically, cell viability was remarkably inhibited by TDC-mediated combinatory photothermal and sonodynamic therapies. A subcutaneous tumor model of triple-negative breast cancer in nude mice was established to evaluate the efficacy of TDC in vivo photoacoustic imaging and anti-TNBC. The results show that TDC elicited photoacoustic imaging of tumor and suppressed tumor growth in xenograft models. Immunohistochemistry and immunofluorescence staining of tumor tissues revealed that TDC-based combinatory photothermal and sonodynamic therapies induced elevated expression of apoptosis relevant proteins. Furthermore, by establishing the mouse model of bilateral TNBC, we discovered that the immune response was activated in tumor microenvironment after combination therapy. Collectively, our study revealed that combinatory strategy of photothermal and sonodynamic therapies based on TDC suppressed proliferation and induced apoptotic cell death of breast cancer cells. Our findings provide new insights into exploiting novel therapies based on TDC in order to provide a possible safe and effective method for the treatment of TNBC.

The Clinical Relevance and Functional Implications of Thymosin Beta-10 in Glioma.

Glioma is a highly aggressive form of brain cancer characterized by limited treatment options and poor patient prognosis. In this study, we aimed to elucidate the oncogenic role of thymosin beta-10 (TMSB10) in glioma through comprehensive analyses of patient data from the TCGA and GTEx databases. Our investigation encompassed several key aspects, including the analysis of patients' clinical characteristics, survival analysis, in vitro and in vivo functional experiments, and the exploration of correlations between TMSB10 expression and immune cell infiltration. Our findings revealed a significant upregulation of TMSB10 expression in glioma tissues compared to normal brain tissues, with higher expression levels observed in tumors of advanced histological grades. Moreover, we observed positive correlations between TMSB10 expression and patient age, while no significant association with gender was detected. Additionally, TMSB10 exhibited marked elevation in gliomas with wild-type IDH and noncodeletion of 1p/19q. Survival analysis indicated that high TMSB10 expression was significantly associated with worse overall survival, disease-specific survival, and progression-free survival in glioma patients. Functionally, knockdown of TMSB10 in glioma cells resulted in reduced cellular growth rates and impaired tumor growth in xenograft models. Furthermore, our study revealed intriguing correlations between TMSB10 expression and immune cell infiltration within the tumor microenvironment. Specifically, TMSB10 showed negative associations with plasmacytoid dendritic cells (pDC) and γδ T cells (Tgd), while displaying positive correlations with neutrophils and macrophages. These findings collectively provide valuable insights into the oncogenic properties of TMSB10 in glioma, suggesting its potential as a therapeutic target and a biomarker for patient stratification.

(E)-SIS3 suppressed osteosarcoma progression via promoting cell apoptosis, arresting cell cycle, and regulating the tumor immune microenvironment.

Osteosarcoma is a prevalent malignant bone tumor with a poor prognosis. Mothers against decapentaplegic homolog 3 (Smad3) present as a therapeutic target in antitumor treatment, whereas its functions in the osteosarcoma have not been well explored. In the current study, we aimed to investigate the effects of Smad3 in the progression of osteosarcoma. The tumor immune single-cell hub 2 website was used for graph-based visualization of Smad3 status in osteosarcoma single-cell database. Western Blot was applied to detect the expression of Smad3 protein in cell lines. Colony formation and cell counting kit-8 assays were used to evaluate cell proliferation. Transwell and wound healing assays were used to detect the migration and invasion abilities of cells. Cell apoptosis rates and cell cycle changes were explored by using flow cytometry analysis. The xenograft tumor growth model was applied to explore the effect in tumor growth after Smad3 blockage in vivo. Moreover, to confirm the potential mechanism of Smad3's effects on osteosarcoma, bioinformatics analysis was performed in TARGET-Osteosarcoma and GSE19276 databases. Our study found that the Smad3 protein is overexpressed in 143B and U2OS cells, suppressing the expression of Smad3 protein in osteosarcoma cells by Smad3 target inhibitor (E)-SIS3 or lentivirus can inhibit the proliferation, migration, invasion, promote cell apoptosis, arrest cell G1 cycle in osteosarcoma cells in vitro, and suppress tumor growth in vivo. Furthermore, the bioinformatics analysis demonstrated that high expression of Smad3 is closely associated with low immune status in TARGET-Osteosarcoma and GSE19276 databases. Our study suggested that Smad3 could contribute positively to osteosarcoma progression via the regulation of tumor immune microenvironment, and Smad3 may represent as an valuable potential therapeutic target in osteosarcoma therapy.

VprBP/DCAF1 Triggers Melanomagenic Gene Silencing through Histone H2A Phosphorylation.

Vpr binding protein (VprBP), also known as DDB1- and CUL4-associated factor1 (DCAF1), is a recently identified atypical kinase and plays an important role in downregulating the transcription of tumor suppressor genes as well as increasing the risk for colon and prostate cancers. Melanoma is the most aggressive form of skin cancer arising from pigment-producing melanocytes and is often associated with the dysregulation of epigenetic factors targeting histones. Here, we demonstrate that VprBP is highly expressed and phosphorylates threonine 120 (T120) on histone H2A to drive the transcriptional inactivation of growth-regulatory genes in melanoma cells. As is the case for its epigenetic function in other types of cancers, VprBP acts to induce a gene silencing program dependent on H2AT120 phosphorylation (H2AT120p). The significance of VprBP-mediated H2AT120p is further underscored by the fact that VprBP knockdown- or VprBP inhibitor-induced lockage of H2AT120p mitigates melanoma tumor growth in xenograft models. Collectively, our results establish VprBP-mediated H2AT120p as a key epigenetic signal for melanomagenesis and suggest the therapeutic potential of targeting VprBP kinase activity for effective melanoma treatment.

Abstract B039: Influence of tumor-associated fibroblasts and their exosomes in the development and progression of prostate cancer

Abstract The unique tumor microenvironment (TME) of the prostate and in particular fibroblasts are known to play an essential role in the growth and progression of prostate cancer (PCa). Earlier studies from our group have shown that co-injection of patient-derived prostate cancer-associated fibroblasts (CAFs) and non-cancer associated fibroblasts (NCAFs) with tumor cells stimulated tumor growth in xenograft models compared to the injection of tumor cells alone. Our present study aims to investigate the effect of patient-derived prostate CAFs and NCAFs on the functional aspects of tumor cells in-vitro and their role in epigenetic regulation. Three prostate CAFs and NCAFs pairs were isolated from tissues of patients who had undergone radical prostatectomy and cultivated in-vitro. We then examined the influence of these fibroblast pairs on the viability, proliferation and migration of prostate cancer cell lines LNCaP and LNCaP C4-2. Cells were co-cultured in a transwell system with tumor cells in the lower chamber and fibroblasts in the upper chamber for 2 and 4 days and the viability of the tumor cells was measured using WST-1 assay and proliferation using BrdU colorimetric assay. For migration assay, the tumor cells were pretreated with serum-deficient medium and were co-cultured in a transwell system as mentioned above for 4 days. The tumor cells were collected after co-culture and the migration was measured using the transwell migration system as well as Xcelligence real-time assays in parallel. Further, extracellular vesicles (EV) were enriched from fibroblasts by ultracentrifugation. The expression of miRNAs isolated from cells and EVs was studied by microarray analysis and RT-qPCR. Patient-derived fibroblasts significantly increased the viability and proliferation of the tumor cells compared to the tumor cells alone. The influence of NCAFs on both viability and proliferation was more pronounced compared to CAFs on the LNCaP cells. However, the influence of the fibroblasts was vice versa on the LNCaP-C4-2 cells. The LNCaP cells exhibited no migration whereas the LNCaP C4-2 cells exhibited slight migration. The effect of different fibroblast pairs on the migration of LNCaP C4-2 cells varied among the pairs. 103 miRNAs and 6 miRNAs were found to be differentially expressed between CAFs and NCAFs and between their EVs, respectively. miR-10b-5p and miR-210-3p were confirmed by RT-qPCR to be upregulated in the CAFs. Our in-vitro data confirmed the in-vivo results. Fibroblasts modulate viability, proliferation and migration of PCa. However, the effects are heterogeneous due to patient and cell line-specific characteristics. Further studies will focus on the possible effects of fibroblasts on apoptosis in correlation to viability and invasion. CAFs and their EVs are characterized by specific miRNA expression supporting an epigenetic regulation of fibroblast characteristics and functions. Therefore, we will further investigate the mechanism of tumor progression induced by fibroblasts and their EVs via miRNA regulation. Citation Format: Aishwarya Tagat, Hiresh Ayoubian, Angela Zaccagnino, Michael Stöckle, Johannes Linxweiler, Kerstin Junker. Influence of tumor-associated fibroblasts and their exosomes in the development and progression of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B039.

Abstract B024: Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and RAS pathway mutations are the known driver mutations in the majority of fusion-negative (FN) RMS. Studies have demonstrated that HRAS mutations are enriched in infants with FN-RMS and can be associated with inferior outcomes. Using HRAS-mutant RMS models, we have demonstrated that tipifarnib (farnesyl transferase inhibitor, FTI) decreases ERK signaling and decreases in vitro and in vivo tumor growth. The effects of tipifarnib can be incomplete, and limitations may be due to adaptive or acquired resistance, or the presence of concurrent mutations that limit the response to single agents. These features suggest that HRAS-mutated FN-RMS may be sensitive to inhibition with combination therapy that prevents or delays the emergence of resistance. We examined the transcriptional effects of tipifarnib in FN-RMS cell lines using bulk RNA-sequencing to identify therapeutic vulnerabilities that may be exploited by RAS-directed therapies. Analysis of RNA sequencing data revealed downregulation of ERK transcriptional output genes upon treatment with tipifarnib, confirming the role of the MEK-ERK pathway in mediating the response to FTI. Trametinib (MEKi) inhibits tumor growth in xenograft models of FN-RMS but has only modest activity as a single agent. The efficacy of inhibition with FTI and MEKi has not been previously explored in RAS-driven FN-RMS. We therefore tested tipifarnib in combination with trametinib in HRAS-mutant FN-RMS cell lines and utilized in vitro assays to evaluate the effects of the combination on cell growth, differentiation and signaling via RAS effector pathways. In HRAS-mutant cells, we observed additive dose-dependent 2D and 3D growth inhibition in response to the combination. Further, tipifarnib and trametinib more potently reduced ERK phosphorylation than either drug individually, indicating effective RAS pathway inhibition. Additionally, we found that the combination of tipifarnib and trametinib induced myosin heavy chain expression, suggesting both inhibition of proliferation and promotion of myogenic differentiation. In an in vivo model, the combination of tipifarnib and trametinib led to tumor regression which was not seen with either drug alone. Our data suggest that the combination of tipifarnib and trametinib is active in models of HRAS-driven FN-RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with FN-RMS. Citation Format: Patience Odeniyide, Alla Lisok, Elana J. Fertig, Alyza Skaist, Christine A. Pratilas. Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B024.

LINC01002 Targets miR-650/FLNA Pathway to Suppress Prostate Cancer Progression

Introduction: In view of the vital implication of long noncoding RNAs in tumorigenesis, we possess the aim to determine the action effects and mechanisms of LINC01002 in prostate cancer (PCa). Methods: Expression level of LINC01002, miR-650, or filamin A (FLNA) in PCa tissues and cells was assessed using quantitative real-time PCR or Western blotting. Cell proliferative and migratory capacities were investigated by Cell Counting Kit-8 (CCK-8) and wound healing assays. Cell apoptosis was investigated by the levels of Bax and Bcl-2. Xenograft models were constructed to testify the role of LINC01002 in vivo. The anticipated binding of miR-650 to LINC01002 or FLNA was confirmed by dual-luciferase reporter or RNA binding protein immunoprecipitation assays. Results: Relatively poor expression of LINC01002 and FLNA, and high expression of miR-650 were identified in PCa tumor specimens and cells. Ectopic LINC01002 expression restrained PCa cell proliferation/migration and provoked apoptosis in vitro, and blocked solid tumor growth in Xenograft models. MiR-650 was directly targeted by LINC01002, and it also directly bound to FLNA. MiR-650 reintroduction in PCa cells overexpressing LINC01002 or FLNA partly reversed the anticancer effects of LINC01002 or FLNA overexpression, thus recovering PCa cell proliferation/migration and repressing apoptosis. Conclusion: LINC01002 deregulation was linked to PCa development. LINC01002 exerted potential anticancer effects in PCa via targeting the miR-650/FLNA pathway, which, at least in part, provided a basis for the involvement of LINC01002 as a therapeutic target in PCa.

Fusobacterium nucleatum promotes esophageal squamous cell carcinoma progression and chemoresistance by enhancing the secretion of chemotherapy-induced senescence-associated secretory phenotype via activation of DNA damage response pathway

ABSTRACT Senescence frequently occurs in cancer cells in response to chemotherapy (called therapy-induced senescence). Senescent cells can exert paracrine effects through the senescence-associated secretory phenotype (SASP) promoting cancer recurrence and chemoresistance. The altered gut microbiota has been closely associated with cancer progression through the direct interaction with cancer cells. However, little is known about the relationship between the gut microbiota and therapy-induced senescent cells. This study aimed to explore the impact of the gut microbiota on therapy-induced senescent cells and the SASP. We found that esophageal squamous cell carcinoma (ESCC) cells were induced into senescence following platinum-based chemotherapy, accompanied by the secretion of a robust SASP. Furthermore, senescent ESCC cells exerted a tumor-promoting effect through the SASP both in vitro and in vivo. Through 16S rRNA gene sequencing and fluorescence in situ hybridization, we identified that Fusobacterium nucleatum (F. nucleatum) was abundant in human ESCC cancerous tissues and correlated with poor prognosis in ESCC patients. Notably, F. nucleatum further promoted the secretion of the SASP by senescent ESCC cells. Compared with the conditioned medium from senescent ESCC cells, the conditioned medium from F. nucleatum-treated senescent ESCC cells accelerated tumor growth in xenograft models, enhanced migration and invasion abilities, and potentiated chemoresistance both in vitro and in vivo. Mechanistically, F. nucleatum invaded and survived in senescent ESCC cells and induced an increase in DNA damage to further activate the DNA damage response pathway, thus enhancing the SASP. Altogether, these findings reveal for the first time that F. nucleatum promotes the secretion of chemotherapy-induced SASP to drive ESCC progression and chemoresistance, which supports F. nucleatum as a potential target for ESCC therapy.

Abstract 2647: Novel FGFR2 biparatopic antibodies for the treatment of cholangiocarcinoma

Abstract Translocations involving Fibroblast Growth Factor Receptor 2 (FGFR2) are found in up to 45% of Intrahepatic Cholangiocarcinoma (ICC). Treatment of patients bearing FGFR2 fusions with FGFR kinase inhibitors induces tumor regressions, though response rate and durability of response appear limited due to the emergence of resistance or to sub-optimal dosing which is limited by drug adverse effects. Thus, therapeutics that drive activity specifically towards FGFR2 are needed to enhance drug efficacy and reduce adverse effects. We first asked whether the extracellular domain (ECD) of FGFR2-BICC1 fusion is functional and important for receptor dimerization and signaling using NanoBiT system and western blotting. FGFR ECD consists of 3 Ig-fold domains D1, D2, and D3. Using focus transformation and viability assays, we found that deletions in D1, D2, D3, and D2-3 ECD FGFR2-BICC1 decreased growth and transformation of FGFR2 fusion driven cells. Next, we sought to investigate if antibodies against the ECD could perturb the growth of FGFR2 fusion transformed cells. We identified six FGFR2 specific antibodies that bind to epitopes in the ECD and confirmed the ligand blocking activity of these bivalent antibodies. In the absence of ligand, however, most of the bivalent antibodies were relatively inactive. As standard bivalent antibodies engage two receptors at a time, they are at risk for agonism or the maintenance of tonic signaling. Thus, to enhance efficacy and prevent the potential agonist or tonic activity of bivalent antibodies, all possible biparatopic antibodies (15), recognizing two distinct epitopes on the FGFR2 ECD, were engineered from the six parental antibodies. The biparatopic antibodies exhibited enhanced efficacies in FGFR2-fusion driven NIH3T3/BaF3 and ICC13-7 cancer cell lines and significantly improved binding affinities (~10-fold) as analyzed by Octet, MSD-SET, and SPR. Two biparatopic antibodies (BpAb-1, BpAb-2) showed the highest antiproliferative activity and induced apoptosis in FGFR2 fusion driven cells. BpAb-1, BpAb-2 were superior to their parental antibodies in reducing FGFR2-fusion driven growth, transformation and downstream signaling (pFGFR2, pFRS) in vitro and in reducing tumor growth in xenograft models. Importantly, BpAb-1 and BpAb-2 significantly induced FGFR2 internalization and lysosomal-mediated degradation as compared to their parental antibodies. Moreover, BpAb-1 and BpAb-2 had anti-growth activities against ICC patient-derived oncogenic FGFR2 ECD in-frame deletions, and FGFR kinase resistance mutations, including the gatekeeper mutation V565F. BpAb-1 and BpAb-2 were also active in FGFR2 WT amplified cells SNU-16. These results suggest that these FGFR2 biparatopic antibodies are a novel treatment option for ICC patients with tumors harboring FGFR2 fusions. Citation Format: Saireudee Chaturantabut, Sydney Oliver, John Kim, Dennie Frederick, Foxy Robinson, Alessandro Sinopoli, Diego J. Rodriguez, Liang Chang, Nabeel Bardeesy, William Sellers. Novel FGFR2 biparatopic antibodies for the treatment of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2647.

Abstract 1715: SOS1 inhibition is an effective therapeutic strategy in SOS1-mutant cancer

Abstract SOS1 is a principal guanine nucleotide exchange factor for canonical RAS GTPases and activates RAS signaling. Mutations in SOS1 are found in 3-5% of non-small cell lung cancers (NSCLC), colorectal cancers (CRC) and melanomas. The recent development of novel SOS1 inhibitors, including BI-3406 and BI-1701963, that block interaction between SOS1 and RAS, presents a potential therapy for SOS1-driven cancers. Here we show that the majority of SOS1 mutations observed in patients enhance RAS guanine nucleotide exchange in cells and activate RAS-MAPK signaling. Of the 11 available SOS1-mutant cancer cell lines without RAS co-mutation, 10 were sensitive to SOS1 inhibition in 2D and/or 3D cell culture, while cells with SOS1 and RAS co-mutations were resistant to SOS1 inhibition. Treatment with a SHP2 inhibitor, which is presumed to inhibit the activity of both SOS1 and SOS2, revealed a subset of SOS1-mutant cell lines that were more sensitive to SHP2 inhibition than SOS1 inhibition. This implied that, in certain settings, SOS2 can compensate for the loss of SOS1 activity. Accordingly, these cells exhibited a rebound in RAS signaling after treatment with SOS1i that was dependent on SOS2. SOS2 knock-down further sensitized these cells to SOS1 inhibition, resulting in responses equivalent to SHP2 inhibition. In addition, we show that, as with SOS1, SOS2 is a target for ERK1/2-dependent feedback phosphorylation. Thus, relief of ERK1/2-dependent negative regulation of SOS2, following SOS1 inhibition, likely contributes to receptor tyrosine kinase (RTK)-SOS2 activation that may mediate SOS1 inhibitor resistance in some contexts. In vivo, SOS1i inhibited tumor growth in xenograft models of SOS1-mutant AML and NSCLC. Tumor-suppressive effects of SOS1i in these models were comparable to or stronger than the effects of SHP2i or MEKi. Thus, our results suggest that SOS1 inhibition could be an effective therapeutic approach in RAS wild-type cancer patients with SOS1 mutations. Citation Format: Matthew J. Sale, Neal Mukherjee, Regina Ruzicka, Robin Jacob, Christoph Albrecht, Teresa Zanin, Tom Madensky, Kaja Kostyrko, Robyn L. Schenk, Anke Baum, Daniel Gerlach, Marco H. Hofmann, Frank McCormick. SOS1 inhibition is an effective therapeutic strategy in SOS1-mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1715.

Abstract 1718: Development of potent estrogen receptor beta agonists for treating glioblastoma

Abstract Background: Glioblastoma (GBM) is the most prevalent and aggressive type of adult brain cancers with low 5-year overall survival rates. Epidemiologic data suggest estrogen may decrease brain tumor growth. The lack of powerful, selective brain permeable estrogen receptor beta (ERβ) ligands to promote its anticancer action is limiting the therapeutic promise of ERβ. The goal of this project is to create a new class of brain permeable ERβ agonists for the treatment of GBM. Method: We have used four different assays to identify potent leads. (1) ERβ and estrogen receptor alpha (ERα) reporter assays, (2) Polar Screen Estrogen Receptor (ER) Competitive Binding Assays, (3) Cell viability assays using GBM cells, and (4) Activity in WT and ERβ-KO GBM cells. CellTiter-Glo Cell Viability and colony formation assays were used to test the efficacy of ERβ agonists. Invasion was measured by matrigel invasion chamber assays and apoptosis was measured by Caspase-Glo® 3/7 and Annexin V assays. Flow cytometry was used to evaluate the cell cycle. Western blotting, RNA-Seq, RT-qPCR, and ERβ KO cells were used in the mechanistic studies. Both orthotopic patient-derived xenografts and GBM cell line-derived (CDX) xenografts were used to test the ERβ agonist's activity in vivo. Results: Indanone and tetralone-oximes or keto-oximes have been hypothesized, produced, and evaluated as new ER agonists. Due to its high action in ERβ reporter assays, specific binding to ERβ in polar screen assays (EC50 91 nM), and potent growth inhibitory activity (IC50 7.4) in GBM cells, CIDD-97 was discovered as a possible hit by screening a library of ~60 compounds. CIDD-97 is significantly more selective for ERβ than ERα (40-fold). Treatment with CIDD-97 markedly reduced U251 cell viability while having little to no effect on U251-ER-KO cells and normal human astrocytes. Further, CIDD-97 treatment decreased the expression of stemness markers in patient derived GSC lines and promoted apoptosis. Additionally, CIDD-ERβ agonist improved TMZ's ability to reduce GBM cell survival. The activation of ERβ target genes in cells treated with CIDD-97 was verified by using RNA-Seq and RTqPCR. PK studies confirmed that CIDD-0149897 has systemic exposure, was found in the brain, and its maximal detectable levels suggested that it has good BBB permeability. Mice tolerated daily intraperitoneal treatment of CIDD-0149897 (50 mg/kg) with a 7-day repeat dosage with no evidence of toxicity. Additionally, when compared to vehicle CIDD-97 (50 mg/kg/i.p.) treatment significantly decreased tumor growth in xenograft models and extended the survival of tumor-bearing mice in orthotopic GSC models. Conclusion: Collectively, these findings pointed to CIDD-97 as a possible ERβ agonist and is easily adaptable to clinical use alongside current chemo- and radiation-therapy regimens, offering GBM patients an additional means of improving survival. Citation Format: Uday P. Pratap, Michael Tidwell, Henriette U. Balinda, Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Stanton McHardy, Andrew Brenner, Ratna K. Vadlamudi. Development of potent estrogen receptor beta agonists for treating glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1718.