Signaling In Cancer Cells Research Articles

Abstract 7080: Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells

Abstract Background and Purpose: Caveolin-1 (Cav-1), a scaffolding protein implicated in cellular signaling and membrane dynamics, undergoes intricate regulation at multiple levels. Recent evidence has suggested a potential association between oncogenic mutations and alterations in Cav-1 expression. Our study aimed to comprehensively explore how RAS/RAF mutations influence Cav-1 expression in cancer cells, as well as the underlying molecular mechanisms, using tetracycline (tet)-inducible cell lines and cancer cell lines harboring endogenous BRAF or KRAS mutations. Experimental Design: We assessed the regulation of Cav-1 expression using tet-inducible cell lines with BRAFV600E, KRASG12D, or KRASG12C mutations. We measured the half-life of Cav-1 at protein levels by using cycloheximide. We monitored Cav-1 localization and degradation dynamics by SNAP-based pulse-chase assay. Additionally, we performed protein and gene expression analyses by immunoblotting and qRT-PCR, luciferase reporter assay, and immunofluorescence to dissect the impact of BRAF and KRAS mutations on Cav-1 regulation at various stages of its life cycle. Results: We found a significant downregulation of Cav-1 at both mRNA and protein levels upon induction of BRAF or KRAS mutations. The use of KRASG12C, BRAFV600E, ERK, and/or MEK inhibitors in tet-inducible cell lines rescued the effects observed upon induction. The rescue effects have also been observed in H23, MiaPaCa-2 and 8505-C cancer cell lines bearing either BRAF or KRAS mutations. Notably, BRAFV600E exhibited a more pronounced reduction in Cav-1 expression than other mutations. We observed shortened half-life of Cav-1 at protein levels after induction of RAS/RAF mutations by using cycloheximide, and SNAP-based pulse-chase. These mutations accelerated lysosomal-mediated degradation of Cav-1 as determined by immunoblotting and immunofluorescence, which was reversed by lysosomal inhibitors. Conclusions: Our study establishes a novel regulatory paradigm wherein RAS/RAF mutations exert a multi-faceted influence on Cav-1 dynamics in cancer cells. The transcriptional and translational downregulation, coupled with accelerated lysosomal degradation, collectively contributes to the decreased half-life of Cav-1 following the induction of BRAF or KRAS mutations. These findings provide insights into the intricate molecular mechanisms underpinning the dysregulation of Cav-1 in the context of RAS/RAF mutations, offering potential avenues for therapeutic interventions. Citation Format: Veronica Castro Aceituno, Tiantian Cui, Haihua Feng, Linlin Yang, Sindhu Nair, Terence M. Williams. Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells [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 7080.

Abstract 3875: Glioblastoma mutational profiles drive cancer cell signaling and immune evasion

Abstract Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with a median survival below two years. Characterization of GBM through gene expression profiling and DNA sequencing has identified disease subgroups and commonly altered genes and pathways, including alterations in the genes encoding neurofibromin-1 (NF1) and the epidermal growth factor receptor (EGFR). NF1 and EGFR are involved in receptor tyrosine kinase (RTK), RAS/MEK/ERK, and PI3K signaling, and together, are genetically altered in over 60% of adult GBMs. Interestingly, NF1 and EGFR alterations in GBM are almost always mutually exclusive. Here, we designed a multimodal approach integrating spatial proteomics, transcriptomics, and genomics to measure cell composition, immune cell function, cancer cell signaling, cellular spatial organization, gene expression, transcriptional subtypes, and genome alterations in 80 NF1 and EGFR altered human GBMs with extensively curated treatment and survival data. We quantified the spatial expression of 42 proteins in over 31 million single cells measuring RTK/RAS/PI3K and Rb signaling and features of the TME, including 23 cell types covering tumor cells, immune cells, and brain resident cells. In addition to tumor genotype labels, we classified each GBM tumor by its transcriptional subtype (i.e., mesenchymal, classical, or proneural). We identified genotype-phenotype associations in GBM including hyperactive RAS signaling cellular niches, a macrophage-rich TME, and an epithelial-mesenchymal signature in NF1 altered tumors and hyperactive RTK signaling and an immune desert TME in EGFR amplified tumors. Furthermore, we observed spatial heterogeneity in cancer cell signaling patterns suggestive of the presence of different tumor cell clones within different regions of each GBM tumor. Together, we identify several genotype-associated features of GBMs, reinforcing the value of integrating spatial multi-omic data for generation of genotype-driven therapeutic hypotheses, with implications for GBM genotype-based therapeutic stratification. Citation Format: Maryam Pourmaleki, Brian D. Greenstein, Caitlin J. Jones, Subhiksha Nandakumar, Daniel A. Navarrete, Smrutiben Mehta, Carl Campos, Travis J. Hollmann, Nicholas D. Socci, Tejus Bale, Sohrab P. Shah, Ingo K. Mellinghoff. Glioblastoma mutational profiles drive cancer cell signaling and immune evasion [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 3875.

Abstract 5272: APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells

Abstract Introduction: Treatment approach combining cancer cell targeted therapy and immunotherapy is an effective strategy to trigger robust tumor-specific immune responses. The aim of this study is to determine the impact of APL-1202, a clinical-stage drug candidate (NCT04813107), on cellular stress-driven immunogenic outcomes in bladder cancer cells. Methods: RNA-seq analysis was performed using bladder cancer and endothelial cell lines (A56, J82, T24, HUVEC) treated with APL-1202. Key determinants of immunogenic cell death pathway were measured by cell-surface translocation of calreticulin (flow cytometry), extracellular ATP release assay (bioluminescence) and HMGB1 secretion (ELISA) in the supernatants of cancer cells after APL-1202 treatment. Effects on the innate immune effector function was measured by flow cytometry-based phagocytosis assay consisting of Far-Red dye-stained, pre-treated bladder cancer cell lines (UMUC3, T24) in coculture with THP1-derived macrophages/immature dendritic cells or human monocyte-derived dendritic cells. In vivo, tumor end-point immune cell analysis, tumor weight and mouse survival were compared in chemically induced (BBN: N-butyl-N-(4-hydroxybutyl)-nitrosamine) orthotopic bladder cancer mice treated with APL-1202 and anti-PD1 antibody. Results: Pathway analysis of RNA-seq data from APL-1202 treated cells suggested upregulation of cellular stress-related gene signatures (oxidative stress, autophagy, P53) and pro-inflammatory responses (IL-6, IL-8, HMGB1 signaling). Conversely, downregulation of cell cycle (E2F targets, G2-M checkpoint) and IL-10 signaling suggested increased genomic stress and inflammatory switching in the treated cancer cells. Acute exposure to APL-1202 increased cell-surface translocation of calreticulin as well as extracellular release of ATP and HMGB1 in the treated T24 and UMUC3 cells. Higher percentages of phagocytic activities were detected in both THP-1-derived macrophages/immature dendritic cells and human monocyte-derived dendritic cells after coculture with APL-1202 treated T24 and UMUC3 cells compared to coculture with DMSO treated control cells. Immune phenotyping in tumors derived from BBN bladder cancer mouse model revealed increased infiltration of NK cells and CD8+ T cells. Furthermore, APL-1202 enhanced the anti-tumor effects of a therapeutic anti-PD1 antibody in mice as reflected by significantly decreased tumor weights and increased survival in a combination group with APL-1202 treatment compared to the anti-PD-1 antibody alone treatment group. Conclusion: APL-1202 may improve the efficacy of immune-checkpoint blockade therapies in bladder cancer patients by enhancing cancer cell immunogenicity and subsequent anti-tumor effector immune cell processes, particularly in tumors with deficits in antigen-specific immunity. Citation Format: Rakesh BAM, Jingmin Guan, Neetha Nanoth Vellichirammal, Murli Manohar, David Mulholland, John Sfakianos, Qiaoling Sun, Zuoan Yi, Alice Chen. APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells [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 5272.

Abstract 4513: Exploration degradation of intrinsically disordered protein YAP induced by PROTACs

Abstract Yes-associated protein (YAP), a potent oncogene and a key player in the Hippo tumor suppression pathway, has long been considered challenging to target due to its partially intrinsically disordered nature. However, recent advancements in High-throughput Screening (HTS) have yielded a breakthrough, as several YAP binders have been identified, igniting new possibilities in the quest to combat YAP-driven malignancies. Building upon this progress, a novel approach utilizing Proteolysis-Targeting Chimera (PROTAC) technology was employed to design and synthesize a series of YAP degraders. Here, our degraders were created by linking NSC682769, a previously reported YAP binder, with either VHL ligand 2 or pomalidomide using various linkers of different lengths and types. Among these degraders, YZ-6 emerges as the most promising, inducing rapid and sustained YAP degradation via promoting its ubiquitination-proteasome-dependent proteolysis. This process effectively suppressed YAP/TEAD-led transcription in both YAP-dependent NCI-H226 and Huh7 cancer cell lines. In addition to its degradation capabilities, YZ-6 also exhibited antiproliferative activity in both cell lines. Importantly, YZ-6 efficiently suppressed tumor development in the Huh7 xenograft mouse model, accompanied by a remarkable decrease in YAP levels, without adverse effects on the mice. These findings highlight the potential of PROTAC-mediated degradation as a viable strategy for reducing oncogenic YAP levels and attenuating downstream signaling in cancer cells. Moreover, the development of PROTACs based on NSC672869 holds promise for treating YAP-driven malignancies, opening new avenues for cancer therapy. Citation Format: Chen Zhou, Chunbao Sun, Liya Pi, Chenglong Li. Exploration degradation of intrinsically disordered protein YAP induced by PROTACs [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 4513.

Abstract 1657: Microfluidics-based screening platform identifies a novel therapeutic approach to targeting EML4-ALK driven cancers

Abstract To achieve proliferative dysregulation, a hallmark of cancer, tumor cells utilize a variety of mechanisms to maintain constitutive oncogenic signaling in the absence of extracellular cues. One such mechanism occurs in cells expressing the EML4-ALK fusion protein. This fusion, found in about 4% of non-small cell lung cancer (NSCLC), is a result of an inversion within chromosome 2, placing the 3’ end of the ALK (anaplastic lymphoma kinase) gene downstream of the 5’ end of the EML4 (echinoderm microtubule associated protein-like 4) gene. The resulting fusion protein contains the oligomerization domain and additional disordered sequence elements of EML4 upstream of a functional ALK kinase domain. EML4-ALK is a critical driver for these tumors, promoting aberrant ALK-dependent oncogenic signaling. While tumors harboring the EML4-ALK fusion initially respond to ALK tyrosine kinase inhibitors (TKIs), resistance remains a significant clinical challenge requiring new therapeutic approaches. Recently, it has come to be understood that rather than responding through extracellular proliferative cues, the EML4-ALK fusion protein elicits constitutive ALK signaling through the formation of cytoplasmic biomolecular condensates. These condensates are the result of phase separation and have been shown to be causal for disease phenotypes in cell and animal models driven by EML4-ALK, establishing condensate modulation as a novel means by which to treat TKI-resistant tumors. Here, we have developed a novel proprietary microfluidics-based high throughput screening platform (termed PhaseScanTM) to identify small-molecule modulators of EML4-ALK condensates. Using this technology, we identified compounds that disrupt EML4-ALK condensates in vitro and in cells through a novel mechanism, distinct from that of known ALK TKIs, providing utility in both TKI-resistant as well as TKI-naïve tumors. Further profiling of the mechanism of action (MoA) revealed specific modification within the disordered region of EML4, disruption of EML4 oligomerization, and defective ALK signaling in cancer cells. Thus, our PhaseScanTM methodology enabled the identification of novel, functionally active chemical matter that would have otherwise been challenging to identify utilizing any conventional hit identification methodology. Citation Format: Richard C. Centore, Matthew Watson, Julia Doh, Jerome Cattin, Cinzia Sgambato, Amal Alex, Janhavi Sawant, Prathima Radhakrishnan, Jasmine Cornish, Alex Howarth, Nagakumar Bharatham, William E. Arter, Seema Qamar, Kadi L. Saar, Douglas Williamson, Andrew Seeber, Neils Groenewegen, Magdalena Czekalska, Tadas Kartanas, Niklas Ermann, Ahmed Taher, Tuomas Knowles, Shilpi Arora. Microfluidics-based screening platform identifies a novel therapeutic approach to targeting EML4-ALK driven cancers [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 1657.

Abstract 7628: Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy

Abstract Metastatic disease drastically reduces clinical prognosis, and remains the primary cause of mortality from solid cancers, such as breast and pancreatic cancer. Since targeted therapeutic options are limited, we developed small molecule compounds to block activation (GTP loading) of the Rho GTPases Rac and Cdc42. Rac and Cdc42 are ideal molecular targets due to their regulation of cell migration, proliferation, invasion, and signaling in cancer and immunosuppressive cells in the tumor microenvironment. Our lead compound MBQ-167 is a dual Rac and Cdc42 inhibitor with IC50 of ~100 nM for inhibition of Rac/Cdc42 activation and the phosphorylation of their common downstream effector p21-activated kinase (PAK). MBQ-167 is highly effective in mouse models of breast cancer metastasis and is currently undergoing a Phase I clinical trial in advanced breast cancer patients. The purpose of this study is to investigate potential target molecules that can be used as biomarkers predictive of Rac/Cdc42 inhibitor efficacy. We recently published that in addition to inhibiting the growth and migration of breast and pancreatic cancer cells, MBQ-167 inhibits macrophage cell migration, immunosuppressive myeloid cell activation and release of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cancer. Transcriptomic data of tumors from mice treated with vehicle or MBQ-167 identified a number of immunomodulatory pathways downregulated by MBQ-167, including IL-6 and S100A family signaling, and Chitinase 3-like (CHI3L1/CHI4L1) genes coding for the human YKL-40 protein. We tested for the activation status of PAK (phospho (p)-PAK) as a diagnostic marker for Rac/Cdc42 inhibitor efficacy using breast cancer patient tissue in ex vivo culture treated with vehicle or MBQ-167 and found decreased p-PAK staining following immunohistochemistry of MBQ-167-treated tissue. From flow cytometry, using Alexa-488-tagged anti p-PAK antibody, we found that p-PAK was reduced from peripheral blood mononuclear cells (PBMCs) following MBQ-167. ELISA assays for YKL-40/CHI3L1 proteins after treatment of pancreatic cancer cell lines and macrophage-like cells with Rac/Cdc42 inhibitors show that MBQ-167 and the derivative MBQ-168 reduced CHI3L1 levels from conditioned media. These results demonstrate the potential of p-PAK and YKL-40/CHI3L1 from blood as biomarkers for Rac/Cdc42 inhibitor efficacy. This study impacts pharmacodynamic biomarker design for the planned Phase 2 clinical trials for MBQ-167 in breast and pancreatic cancer. Citation Format: Anamaris Torres-Sanchez, Ailed M. Cruz-Collazo, Michael Rivera-Robles, Olga Katsara, Stephanie Dorta-Estremera, Viviana Negron, Victor P. Carlo, Robert J. Schneider, Suranganie Dharmawardhane. Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy [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 7628.

Abstract 2077: Exploring the multifaceted efficacy of de novo lipogenesis inhibitor in cancer therapy

Abstract Cancer cells frequently exhibit altered lipid metabolism to sustain their rapid growth and proliferation.Sterol Regulatory Element-Binding Proteins (SREBPs) are key transcription factors that orchestratelipid biosynthesis and uptake in cells. Dysregulation of SREBPs is a common feature of many cancertypes, making them attractive targets for therapeutic intervention. In this study, we present acomprehensive exploration of the potential and rationale for targeting lipid metabolism in cancerthrough the use of a small molecule inhibitor that targets activation of SREBP. We illustrate that oursmall molecule inhibitor (SMI) exhibits potent anticancer activity and effectively targets SREBPsignaling in a variety of prostate cancer models as well as in non-prostate cancer models that arelipogenesis driven. Our novel SMI induces apoptosis and reduces PCa cell proliferation, suppressescell invasion and migration. Furthermore, we illustrate that in addition to suppressing the transcriptionfactor activity of SREBPs, these novel SMIs are highly effective in suppressing both the full length andvariant androgen receptor and their signaling in prostate cancer cells. Notably, in cell and organoidmodels, these novel inhibitors exhibit synergistic anticancer activity in combination with the ARsignaling inhibitor, Enzalutamide. The anticancer activity of these inhibitors extend beyond ARdependent prostate cancer and are also effective in organoid models of neuroendocrine PCa, aparticularly drug-resistant and aggressive PC subtype. Lastly, we illustrate excellent pharmacokineticsand potent anti-tumor effects of these novel SREBP activation inhibitors using in vivo castration-resistant prostate cancer CDX tumors. Given the urgent need for novel targets in PCa, our first-in-class inhibitor represents a promising therapeutic strategy with the potential to effectively targetdysregulated lipogenesis in cancer patients. Citation Format: Dallin Lowder, Jin Na Shin, Maria E. Ruiz-Echartea, Jenny Deng, Aleksandra Rusin, Darlene Skapura, Michael Ittmann, Salma Kaochar. Exploring the multifaceted efficacy of de novo lipogenesis inhibitor in cancer therapy [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 2077.

Abstract 5147: Identification of pharmacodynamic biomarkers in PBMCs and cancer cells for SIK2 kinase inhibitor therapy

Abstract Salt-inducible kinase 2 (SIK2) is an AMPK family kinase involved in multiple signaling pathways including the LBK1-SIK-HDAC, LKB1-SIK-CRTC/CREB, PI3K-PKA-mTOR and Hippo pathways. The expression of SIK2 is significantly upregulated in fraction of ovarian and breast cancers, and associated with poor prognosis. Inhibition of SIK2 kinase activity suppresses growth of ovarian and breast cancers as well as progression of acute myeloid leukemia. SIK2 inhibitors synergistically enhance sensitivity of ovarian and breast cancer cells to paclitaxel, carboplatin and PARP inhibitors. Utilization of SIK2 inhibitors to treat cancers has been investigated in preclinical studies and in a Phase I clinical trial (NCT04711161). To enable pharmacodynamic (PD) analysis in clinical studies, we have developed biomarkers that measure the effect of GRN-300, a SIK2/3 inhibitor, on SIK2 signaling in ovarian cancer cells. Class IIa histone deacetylases (HDAC4/5/7) are SIK2 substrates in cancer cells; and are also expressed in peripheral blood mononuclear cells (PBMCs). We found that GRN-300 decreased phosphorylated HDAC4/5/7 levels in ovarian and breast cancer cells (Lu et al. J Clin Invest, 2022) and now report that this SIK2 inhibitor also decreases pHDAC4/5/7 in PBMCs ex vivo in a dose-dependent manner using Western blot analysis. We also identified three mRNA markers in PBMCs by RNAseq analysis - SEPP1, RNASE1 and MS4A6A - which are up-regulated by GRN-300 in PBMCs isolated from healthy donors. However, these mRNA markers were either expressed at very low levels or not dramatically changed in cancer cells. We identified an additional mRNA marker panel using RNAseq technology by screening seven ovarian and six breast cancer cell lines: A2780, Caov3, IGROV1, OC316, OVCAR3, OVCAR8, SKOv3ip, BT549, Cal51, HCC1937, MDA-MB-231, MDA-MB-436 and MDA-MB-468. mRNA expression of three genes - DDIT4, NUPR1 and IL6 - is upregulated by GRN-300 in more than 9 of 13 cell lines tested. Five genes - ID1, ID2, ID3, ID4 and TGFBI - are down-regulated in response to GRN-300 treatment in more than 10 of 13 cell lines. The decrease in HDAC4/5/7 phosphorylation, and alterations in mRNA markers we discovered could serve as PD biomarkers for clinical trials to evaluate the effects of SIK2 inhibitors and provide insights in the mechanisms of drug action. Citation Format: Hailing Yang, Weiqun Mao, Philip Rask, Markus Peter, Gayathri Swaminath, Stephan W. Morris, Zhen Lu, Robert C. Bast. Identification of pharmacodynamic biomarkers in PBMCs and cancer cells for SIK2 kinase inhibitor therapy [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 5147.

Abstract 1678: Docetaxel induces Rac1 and Cdc42 Rho GTPase activation in prostate cancer cells

Abstract Prostate cancer (PCa) is a major health burden that affects the quality of life of men worldwide. In the United States, PCa is the second leading cause of mortality among cancers that affect men. Treatment of advanced PCa aims to suppress androgen signaling and proliferation in cancer cells using anti-androgens and chemotherapy. However, the development of drug resistance is a major limitation to curing advanced prostate cancer and strategies to block mechanisms of therapy resistance would enhance treatment efficacy and potentially improve patient prognosis. Cdc42 and Rac1, members of the family of Rho GTPases, have been associated with the progression of different types of hormone-associated cancer, such as breast, ovary, and prostate, among others. The current study was designed to evaluate the role of Cdc42 and Rac1 in chemoresistant prostate cancer cells, including under castration-resistant and androgen-independent conditions. We did not observe significant differences in active (Rac-GTP) or total Rac1 levels in chemoresistant compared to parental PC3 or DU145 cells; similar findings were observed for Ccd42. Treatment of drug-naïve cells with a sublethal dose of docetaxel (10uM) induced gradual increases in Rac1-GTP up to 30 minutes in 22rv1, 60 minutes in LNCaP and DU145, and 24 hours in PC3 cells. Cdc42-GTP levels also increased with subacute docetaxel treatment up to 24 hours in 22rv1 and DU145. Chemoresistant 22rv1, PC3, and DU145 exhibited similar sensitivity to Rac1 and Ccd42 inhibitors including MB1-167, MBQ-168, EHop-097, with an IC50 of about 1uM for each compound and line. Ongoing studies are evaluating the role of these Rho-GTPases in 3-dimensional growth and survival. Citation Format: Andrea Paola López González, Jessica Colón González, María C. Santa María Fuentes, Surangani Dharmawardhane, Carlos J. Diaz Osterman. Docetaxel induces Rac1 and Cdc42 Rho GTPase activation in prostate cancer cells [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 1678.

Abstract 203: Promotion of metastasis of ERBB2-positive breast cancer by mesenchymal stem cells via exosomes-mediated activation of ERBB2/ERBB3→PI3K/Akt singaling pathway

Abstract Elevated expression of ERBB3 receptor correlates with increased distant metastasis of breast cancers with amplification and/or overexpression of ERBB2 (HER2/neu), which the underlying molecular mechanism remains unclear. In recent years, cumulative studies have demonstrated that mesenchymal stem cells (MSCs), one of the important cellular components in tumor microenvironment, play important roles in multiple hallmarks of cancer including invasion and metastasis. However, its role in metastasis of ERBB2-positive breast cancer as well as the precise mechanisms are largely unrevealed. In our current study, we found that co-culture with MSCs or treatment with exosomes derived from MSCs can significantly promote the epithelial-mesenchymal transition (EMT), migration and invasion of ERBB2-positive breast cancer cells. MSCs exhibited significantly higher expression of NRG-1, a known ligand of ERBB3, as compared with ERBB2-positive breast cancer cells. The presence of NRG-1 in exosomes derived from MSCs was also evidenced. Mechanistically, co-culture with MSCs or treatment with exosomes derived from MSCs induced upregulation of ZEB1 and Slug via activation of downstream PI3K/Akt singaling to promote EMT of ERBB2-positive breast cancer cells. In addition, the expression of MMP2 was also upregulated by treatment with MSCs-derived exosomes, which enhanced the migration and invasion of ERBB2-positive breast cancer cells simultaneously. Interestingly, our study further demonstrated that treatment with MSCs-derived exosomes could significantly downregulate the expression of PTEN, the key negative regulator of PI3K/Akt singaling, via transferring three PTEN-targeting miRNAs (miR-21-5p, miR-23a-3p, and miR-148a-3p). Taken together, we presented here that MSCs may, on the one hand, directly activate the ERBB2/ERBB3→PI3K/Akt signaling pathway in ERBB2-positive breast cancer cells through the expression and secretion of NRG-1 via exosomes. Meanwhile, miR-21-5p, miR-23a-3p, and miR-148a-3p targeted delivery by exosomes specifically downregulated the expression of PETN, thus reliefs the negative regulation of PI3K/Akt signaling in ERBB2-positive breast cancer cells. These effects of MSCs act in concert to promote the metastasis of ERBB2-positive breast cancer. Our study lays a theoretical foundation and preliminary experimental basis for the development of novel combined therapies for patient with ERBB2-positive breast cancer through co-targeting MSC-derived NRG-1 and miR-21-5p, miR23a-3p, miR148a-3p/PTEN axis in tumor microenvironment in the future. Citation Format: Yating Wu, Li Chen, Yue Cao, Qinfeng Huang, Huimin Niu, Xiaofeng Lai, Meng Zhao, Huihui Yan, Hui Lyu, Bolin Liu, Shenghang Zhang, Shuiliang Wang. Promotion of metastasis of ERBB2-positive breast cancer by mesenchymal stem cells via exosomes-mediated activation of ERBB2/ERBB3→PI3K/Akt singaling pathway [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 203.

Abstract 2167: Microbial bile acid, 3-oxo-LCA, inhibits colorectal cancer progression

Abstract Colorectal cancer (CRC) affects a significant number of individuals each year, ranking as a leading cause of cancer-related deaths in the U.S. Bile acids (BAs), natural compounds crucial for digesting dietary fats, not only influence the gut microbiota but are also involved in gut health through their interaction with the Farnesoid X Receptor (FXR). Recent findings have highlighted certain microbiota-generated BAs like 3-oxo-lithocholic acid (LCA) and IsoLCA, modulate host immunity, hinder the expansion of intestinal pathogens, and potentially anti-aging. Despite the advancements in identifying the gut microbes behind these BAs, their precise impact on intestinal cells (IECs) and their role in disease progression are largely unexplored. Our pilot investigation unveiled 3oxoLCA as an FXR agonist, restoring FXR signaling in both cancer cells and APCmin/+ mice (a classic CRC mice model). As a result, 3-oxo-LCA inhibits the growth of both human and mouse CRC cells, such as MC38, CT26 and HCT116. Additionally, 3-oxo-LCA also restrained the intestinal stem cells (ISCs)’ proliferation in organoids from both wild-type and APCmin/+ mice, and patient-derived CRC organoids (PDCOs). Remarkably, treatment with 3-oxo-LCA decreased BAs levels, improved gut barrier function, reduced tumor load, and inhibited tumor progression in APCmin/+ mice. Furthermore, 3-oxo-LCA significantly suppressed tumor growth in cancer cell-derived xenograft model mice. Crucially, the 3-oxo-LCA-FXR interaction transcriptionally regulated key apoptotic genes, encouraging cancer cell death. These discoveries highlight the therapeutic promise of incorporating 3-oxo-LCA into strategies for treating CRC. Citation Format: Fei Sun, Xingchen Dong, Ming Qi, Ting Fu. Microbial bile acid, 3-oxo-LCA, inhibits colorectal cancer progression [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 2167.

Abstract 5975: Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family

Abstract Antibodies targeting HER receptors have led to successful therapeutics for multiple human malignancies. Targeting the family of HER ligands provides an alternative strategy to inhibit signaling and subsequent proliferation of cancer cells. We have engineered ligand traps that bind the growth factors activating the HER family and have characterized a series of molecules (EN2000-Fc) using Surface Plasmon Resonance (SPR). Five distinct molecules were further evaluated for improved in vivo pharmacokinetics in mice that express human neonatal Fc receptor (FcRn). Mutations in the IgG Fc known to extend the in-vivo half-life of monoclonal antibodies were incorporated into the parental EN2000-Fc to generate 3 more distinct pan HER ligand traps. In addition, an EN2000-Fc variant with a full-length hinge was engineered, which is 5-amino-acid longer N-terminally than the parental molecule. These five molecules were synthesized (> 300 mg of each), purified (>98%), and characterized for in vitro and in vivo testing. Two variants were compared by evaluating the ligand binding affinity to 11 HER ligands using SPR. Both molecules showed comparable affinity against all ligands tested, confirming reproducibility of the pan HER ligand trap engineering technology. Among the ligands, HB-EGF had the highest affinity. High binding affinities to EGF, NRG1, NRG2, betacellulin and TGFα were also measured. The serum pharmacokinetics of the different ligand traps were compared in 6- to 8-week-old female C57BL/6 mice engineered to express human FcRn. Ligand traps were administered by single tail vein injection, and serum samples collected over a 28-day interval were analyzed by ELISA. Ligand traps with engineered Fc-mutations had 3- to 4-fold greater systemic exposure (area-under-the-curve) compared to ligand traps with the parental sequence, demonstrating improved in vivo characteristics of selected engineered fusion proteins. These results provide useful information for product candidate selection and for further preclinical development of a ligand trap targeting HER family receptors. Citation Format: Daniel C. Maneval, Cindy Yang, Cynthia Trevisiol, H. Michael Shepard, Pei Jin. Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family [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 5975.

Abstract 455: Identification of hsa-miR-6794-3p as a key regulatory factor in human pancreatic cancer metastasis

Abstract Metastasis is a major factor in treatment failure in patients with pancreatic cancer, but the underlying mechanisms remain unclear, and new, effective treatment strategies are needed. Here, we sought to identify novel microRNAs (miRNAs) with a key role in pancreatic cancer metastasis. Using a microarray analysis to evaluate miRNA expression in metastatic and non-metastatic pancreatic cancer, we found that expression of the miRNA, hsa-miR-6794-3p, was significantly lower in metastatic pancreatic cancer tissues than in non-metastatic pancreatic cancer tissues. Methylation-specific polymerase chain reaction analyses revealed that hsa-miR-6794-3p expression was downregulated in metastatic pancreatic cancer cells by hypermethylation of CpG islands within the hsa-miR-6794-3p promoter. Gain- and loss-of-function approaches using the human pancreatic cancer cell lines, MIA-PaCa-2 and HPAF-II, expressing low and high levels of hsa-miR-6794-3p, respectively, suggested a role for hsa-miR-6794-3p in decreasing cell invasion, migration, and epithelial-mesenchymal transition (EMT) signaling. Notably, we found that hsa-miR-6794-3p exerts its effects by inhibiting expression of the chromatin remodeling factor, RBBP4, through direct binding to the 3’-untranslated region of its mRNA. The resulting suppression of RBBP4 expression increases levels of the transcription factor GRHL2, which is involved in regulating invasion, migration, and EMT signaling in metastatic pancreatic cancer cells. Thus, lower levels of hsa-miR-6794-3p disrupt GRHL2-mediated regulation of these pathways by disinhibiting expression of the GRHL2 negative regulator, RBBP4. Consistent with these findings, an investigation of the relationship between hsa-miR-6794-3p and clinicopathological features in pancreatic cancer patients using publicly available datasets and immunohistochemistry showed that low levels of hsa-miR-6794-3p are correlated with a poorer prognosis in pancreatic cancer patients. Additional preclinical experiments in nude mice clearly demonstrated that hsa-miR-6794-3p is capable of suppressing pancreatic cancer cell metastasis. Taken together, these results suggest that hsa-miR-6794-3p suppresses tumor metastasis and thus represents a promising prognostic biomarker and therapeutic target in pancreatic cancer. Citation Format: Ha Gyeong Kim, Eun-Taex Oh, Yunmi Cho, Heon Joo Park. Identification of hsa-miR-6794-3p as a key regulatory factor in human pancreatic cancer metastasis [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 455.

Abstract 1903: Zenocutuzumab, a HER2xHER3 bispecific antibody, is effective in cancer models with high NRG1 expression

Abstract Neuregulin 1 (NRG1) is a ligand that binds to HER3, promotes HER2/HER3 dimerization and PI3K/AKT/mTOR signaling, and causes malignant transformation. Zenocutuzumab (Zeno, MCLA-128) is a humanized IgG1 bispecific antibody that docks on HER2, preventing heterodimerization with HER3, while also blocking NRG1 interaction with HER3, and potently inhibiting NRG1-induced HER2:HER3 proliferation and survival signaling of cancer cells. High NRG1 expression arising from autocrine signaling or gene amplification is associated with poor prognosis in certain cancers, and resistance to standard therapies. The clinical efficacy and safety of Zeno in patients with NRG1 fusion-driven cancer who were enrolled in the ongoing global multicenter eNRGy study and Early Access Program were recently presented. Treatment with Zeno led to durable responses across multiple different previously treated NRG1 fusion tumor types. Zeno utilizes the Merus Dock & Block® mechanism to inhibit HER2/HER3 dimerization and the NRG1/HER3 tumor-signaling pathway and is effective at high NRG1 concentrations. Here, we examined the efficacy of Zeno in preclinical models representing different tumor types expressing high NRG1 levels. Zeno treatment of the lung cancer cell line HCC95, which harbors an NRG1 gene amplification, effectively inhibited signaling pathways involved in the regulation of proliferation and survival, including the PI3K/AKT/mTOR pathway and inhibited cell proliferation. The anti-tumor efficacy of Zeno in vivo against a panel of 28 patient-derived xenograft (PDX) models representing different tumor types was examined. Zeno significantly inhibited tumor growth in seven high-NRG1-expressing PDX models. These results show that Zeno is effective in tumor cell killing in vitro and in vivo in high NRG1-expressing cancer models representing multiple different tumor types. Citation Format: Marie N. O'Connor, Ron C. Schackmann, Igor Odintsov, Szabolcs Fatrai, Arie B. Brinkman, Sidney Walker, Jeroen J. Lammerts van Bueren, Cecile A. Geuijen. Zenocutuzumab, a HER2xHER3 bispecific antibody, is effective in cancer models with high NRG1 expression [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 1903.

Abstract 4588: Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient pancreatic cancer

Abstract KRAS is mutationally activated in 95% of pancreatic ductal adenocarcinoma (PDAC) patients. Direct KRAS inhibitors are under intense preclinical and clinical development, with two KRASG12C mutant-selective inhibitors (G12Ci) now approved. However, treatment-associated resistance to KRAS inhibitors has been reported in the clinic highlighting an urgent need to identify both additional therapeutic targets and novel combination treatment strategies with KRAS inhibitors. To that end, we performed a CRISPR/Cas9 loss-of-function screen using a library targeting ~2,500 druggable genes in KRASG12C/D-mutant PDAC cell lines. We identified multiple genes that were essential for PDAC cell growth and selected PRMT5, a protein arginine methyltransferase, to assess as a novel therapeutic target for pancreatic cancer. We found that suppression of PRMT5 activity using two distinct first-generation clinical candidate small molecule inhibitors (JNJ-64619178 and GSK3326595) demonstrated single agent activity and reduced PDAC cell growth. We further assessed a mechanistically distinct MTA-cooperative clinical candidate PRMT5 inhibitor, MRTX1719, that is selective for MTAP-deleted tumors, which occur in ~25% of PDAC patients. We determined that MRTX1719 exhibited low nanomolar GI50 activities in MTAP-deficient but not MTAP wild-type KRAS-mutant cell lines. Unexpectedly, PDAC cells treated with MRTX1719 exhibited a paradoxical activation of ERK, suggesting that combined inhibition of PRMT5 and KRAS might be therapeutically advantageous. In support of our hypothesis, we demonstrated that combination treatment with MRTX1719 and mutant selective KRAS inhibitors (G12Ci/MRTX849 and G12Di/MRTX1133) further sensitized KRASG12C/D-mutant PDAC cells to KRAS inhibition in short- and long-term growth assays as well as in vivo xenograft studies. Our ongoing studies are evaluating the consequences of co-targeting PRMT5 and KRAS on gene expression changes using RNA-Seq and cancer cell signaling pathways using RPPA analyses. In summary, our data support concurrent inhibition of PRMT5 and KRAS as a promising therapeutic strategy for MTAP-deficient KRAS-mutant pancreatic cancer. Citation Format: Kristina Drizyte-Miller, Wen-Hsuan Chang, Andrew M. Waters, Clint A. Stalnecker, Junning Wang, Andrew J. Aguirre, Adrienne D. Cox, Channing J. Der. Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient 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 4588.

Retinoblastoma-associated protein is important for TRIM24-mediated activation of the mTOR signaling pathway through DUSP2 action in prostate cancer.

RB transcriptional corepressor 1 (RB) deletion is the most important genomic factor associated with the prognosis of castration-resistant prostate cancer (CRPC) patients receiving androgen receptor (AR) signaling inhibitor therapy. Loss of RB could support prostate cancer cell growth in a hormone-independent manner, but the underlying mechanism by which RB regulates tumor progression extends far beyond the cell cycle pathway. A previous study indicated that RB inactivates AKT signaling but has no effect on mTOR signaling in cancer cells. Here, we found that the S249/T252 site in RB is key to regulating the transcriptional activity of the tumor-promoting factor TRIM24 in CRPC, as identified through FXXXV mapping. The RB/TRIM24 complex functions through DUSP2, which serves as an intermediate bridge, to activate the mTOR pathway and promote prostate cancer progression. Accordingly, we designed RB-linker-proteolysis-targeting chimera (PROTAC) molecules, which decreased TRIM24 protein levels and inactivated the mTOR signaling pathway, thereby inhibiting prostate cancer. Therefore, this study not only elucidates the novel function of RB but also provides a theoretical basis for the development of new drugs for treating prostate cancer.

Identification and Development of Cyclic Peptide Inhibitors of Hypoxia Inducible Factors 1 and 2 That Disrupt Hypoxia-Response Signaling in Cancer Cells.

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-regulated α subunit and a constitutively expressed β subunit that serves as the master regulator of the cellular response to low oxygen concentrations. The HIF transcription factor senses and responds to hypoxia by significantly altering transcription and reprogramming cells to enable adaptation to a hypoxic microenvironment. Given the central role played by HIF in the survival and growth of tumors in hypoxia, inhibition of this transcription factor serves as a potential therapeutic approach for treating a variety of cancers. Here, we report the identification, optimization, and characterization of a series of cyclic peptides that disrupt the function of HIF-1 and HIF-2 transcription factors by inhibiting the interaction of both HIF-1α and HIF-2α with HIF-1β. These compounds are shown to bind to HIF-α and disrupt the protein-protein interaction between the α and β subunits of the transcription factor, resulting in disruption of hypoxia-response signaling by our lead molecule in several cancer cell lines.

Structural Insights on the Role of Halogen Bonding in Protein MEK Kinase-Inhibitor Complexes.

Kinases are enzymes that play a critical role in governing essential biological processes. Due to their pivotal involvement in cancer cell signaling, they have become key targets in the development of anti-cancer drugs. Among these drugs, those containing the 2,4-dihalophenyl moiety demonstrated significant potential. Here we show how this moiety, particularly the 2-fluoro-4-iodophenyl one, is crucial for the structural stability of the formed drug-enzyme complexes. Crystallographic analysis of reported kinase-inhibitor complex structures highlights the role of the halogen bonding that this moiety forms with specific residues of the kinase binding site. This interaction is not limited to FDA-approved MEK inhibitors, but it is also relevant for other kinase inhibitors, indicating its broad relevance in the design of this class of drugs.

Riboflavin protects against pancreatic cancer metastasis by targeting TGF-β receptor 1

The inhibition of transforming growth factor-β1 (TGF-β1) signaling by targeting TGF-β receptor 1 (TβR1) has been considered as an ideal approach for the prevention of pancreatic cancer metastasis. Utilizing a pharmacophore model for TβR1 inhibitors, candidate compounds with the potential TβR1 binding ability were screened from the U.S. Food and Drug Administration (FDA) database, and riboflavin (RF) with a highest fit value was chosen to investigate its binding ability to TβR1 and effect on TGF-β1 signaling in pancreatic cancer cells. Molecular docking and cellular thermal shift assay (CETSA) proved that RF at pharmacological concentrations could directly bind to TβR1. Further studies showed that pharmacological concentrations of RF in vitro could block TGF-β1 signaling, suppress the migration and invasion, and prevent epithelial-mesenchymal transition (EMT) process of pancreatic cancer cells in the absence or presence of TGF-β1 stimulation, indicating that RF presented anti-metastatic effect in pancreatic cancer cells. Knockdown of TβR1 could significantly attenuate the effects of RF on the migration and EMT process in pancreatic cancer cells, further confirming that the anti-metastatic effect of RF was achieved by blocking TGF-β1 signaling after binding to TβR1. Moreover, in a mouse model of pancreatic cancer metastasis, it was certified that RF administration could block lung and liver metastases, TGF-β1 signaling and EMT process of pancreatic cancer in vivo. In summary, our findings showed that RF could block TGF-β1 signaling by directly binding to TβR1, thereby suppressing the metastasis of pancreatic cancer cells by inhibiting EMT process both in vitro and in vivo.

Ubiquitin ligase subunit FBXO9 inhibits V-ATPase assembly and impedes lung cancer metastasis

BackgroundThe evolutionarily conserved protein FBXO9 acts as a substrate receptor for the SKP1-cullin-1-RBX1 ubiquitin ligase and is implicated in cancer, exhibiting either tumor-suppressive or oncogenic effects depending on the specific tumor type. However, their role in lung cancer metastasis remains unclear.MethodsLentiviral vectors carrying miRNA-based shRNA sequences for gene-specific knockdown were generated, and Lenti-CRISPR-Cas9 vectors containing gene-specific sgRNA sequences were designed. Gene overexpression was achieved using doxycycline-inducible lentiviral constructs, while gene knockdown or knockout cells were generated using shRNA and CRISPR-Cas9, respectively. Functional assays included migration, clonogenic survival assays, tumor sphere assays, and protein interaction studies using mass spectrometry, immunoprecipitation, and immunoblot analysis.ResultsThis study identified FBXO9 as a crucial regulator that suppresses lung cancer cell migration, tumor sphere growth and restricts metastasis. We showed that FBXO9 facilitates the ubiquitination of the catalytic subunit A (ATP6V1A) of the Vacuolar-type H+-ATPase (V-ATPase), resulting in its interaction with the cytoplasmic chaperone HSPA8 and subsequent sequestration within the cytoplasm. This process hinders the assembly of functional V-ATPase, resulting in reduced vesicular acidification. In contrast, depletion of FBXO9 reduced ATP6V1A ubiquitination, resulting in increased V-ATPase assembly and vesicular acidification, thus promoting pro-metastatic Wnt signaling and metastasis of lung cancer cells. Furthermore, we demonstrated the effectiveness of inhibitors targeting V-ATPase in inhibiting lung cancer metastasis in a mouse model. Finally, we established a correlation between lower FBXO9 levels and poorer survival outcomes in patients with lung cancer.ConclusionThese findings collectively elucidate the critical role of FBXO9 in regulating V-ATPase assembly and provide a molecular basis for FBXO9’s function in inhibiting lung cancer metastasis. This highlights the potential therapeutic opportunities of FBXO9 supplementation.