Clinical and Research Methods for Analysis and Study of Platelet Populations.

Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are among the most commonly diagnosed forms of cancer in the United States. Due to their widespread prevalence and high mortality rate, it is vital to develop effective therapeutic drugs to combat these deadly diseases. In both CRC and PDAC, the multifunctional factor nuclear factor kappa B (NF-kB), a central coordinator of immune responses, is activated abnormally, leading to tumorigenesis and cancer progression. Therefore, controlling NF-kB activity is critical in the treatment of these cancers. Previously, we discovered a novel mechanism by which NF-kB is activated through methylation by an epigenetic enzyme known as protein arginine methyltransferase 5 (PRMT5). We showed that overexpression of PRMT5 significantly promoted several characteristics associated with cancer, including increased cell proliferation, migration, and anchorage-independent growth in both CRC and PDAC cells, thus, putting forward PRMT5 as a novel therapeutic target in these cancers. In this study, we successfully adapted AlphaLISA technique into a high-throughput screen platform, and further employed this approach to successfully identify PR5-LL-FDA1 as a potent PRMT5 inhibitor from commercially-available screening libraries. Furthermore, we confirmed that treatment of PDAC and CRC cells with PR5-LL-FDA1 led to decreased NF-kB activation, and reduced cancer associated properties in PDAC and CRC cells. Importantly, we show that PR5-LL-FDA1 is more efficacious than the commercial PRMT5 inhibitor, EPZ015666 in both PDAC and CRC. Our work clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC and hold the promise to establish PR5-LL-FDA1 as a promising basis for new drug development in the future. Citation Format: Lakshmi Prabhu, Lan Chen, Ahmad Safa, Murray Korc, Zhong-Yin Zhang, Tao Lu. Protein arginine methyltransferase 5 as a tumor promoter and therapeutic target in gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4862.

The 26S proteasome is a large intracellular adenosine 5'-triphosphate-dependent protease that identifies and degrades proteins tagged for destruction by the ubiquitin system. The orderly degradation of cellular proteins is critical for normal cell cycling and function, and inhibition of the proteasome pathway results in cell-cycle arrest and apoptosis. Dysregulation of this enzymatic system may also play a role in tumor progression, drug resistance, and altered immune surveillance, making the proteasome an appropriate and novel therapeutic target in cancer. Bortezomib (formerly known as PS-341) is the first proteasome inhibitor to enter clinical practice. It is a boronic aid dipeptide that binds directly with and inhibits the enzymatic complex. Bortezomib has recently shown significant preclinical and clinical activity in several cancers, confirming the therapeutic value of proteasome inhibition in human malignancy. It was approved in 2003 for the treatment of advanced multiple myeloma (MM), with approximately one third of patients with relapsed and refractory MM showing significant clinical benefit in a large clinical trial. Its mechanism of action is partly mediated through nuclear factor-kappa B inhibition, resulting in apoptosis, decreased angiogenic cytokine expression, and inhibition of tumor cell adhesion to stroma. Additional mechanisms include c-Jun N-terminal kinase activation and effects on growth factor expression. Several clinical trials are currently ongoing in MM as well as several other malignancies. This article discusses proteasome inhibition as a novel therapeutic target in cancer and focuses on the development, mechanism of action, and current clinical experience with bortezomib.

The chromatin remodeling SWI/SNF complex is mutated in 20% of all cancers and ARID1A is the most frequently mutated subunit. However, the tumor suppressive functions of ARID1A are not fully understood and no feasible therapeutic strategies are available for ARID1A-mutant cancers. Recent studies found that loss of ARID1A is associated with increased phosphorylation of AKT. We found that from a study that analyzed data from Project Achilles, a broad shRNA screening project, PIK3CA is the number 2 gene essential for survival of ARID1A-mutant cell lines compared to ARID1A-wildtype cell lines (P = 7.37 × 10-6, FDR < 0.001). Based on these findings, we hypothesized that the PI3K pathway is a potential therapeutic target in ARID1A-mutant cancers. We analyzed the Cancer Genome Atlas (TCGA) datasets and found that mutations in the PI3K pathway co-occur with ARID1A mutations. In addition, the number of co-existing PI3K pathway mutations in the same sample is higher when ARID1A is mutated. We knocked down PIK3CA in ARID1A-wildtype cells (RMG1 and OVCAR3) and ARID1A-mutant cells (OVAS and HCH-1). We found that proliferation was impaired more profoundly in ARID1A-mutant cells. Interestingly, HCH-1 cells are wildtype in PIK3CA, PTEN, PIK3R1 and KRAS, but are still sensitive to PIK3CA depletion. For an unbiased approach, we analyzed the Genomics of Drug Sensitivity in Cancer datasets, which contain drug responses of a large cancer cell line panel to 138 anti-cancer drugs. We compared the drug responses of 49 cell lines harboring inactivating ARID1A-mutations with 266 ARID1A-wildtype cell lines. We found that the presence of inactivating ARID1A mutations is highly associated with sensitivity to mTOR inhibitor AZD8055 (ranked 2nd, P = 2.00 × 10-3) and AKT inhibitor MK2206 (ranked 4th, P = 7.98 × 10-3). This association is still significant for MK2206 when we removed cell lines with PIK3CA, KRAS, PTEN, PIK3R1 and TSC1 alterations (P = 1.32 × 10-2). Finally, we investigated how ARID1A loss can directly increase PI3K/mTOR activity. Using microarray analysis, we found that knockdown of ARID1A up-regulated MYC and MYC target genes, including SLC7A5, an amino-acid transporter required for mTOR activation. Analysis of TCGA datasets showed that MYC amplification and ARID1A mutations are mutual exclusive events, suggesting that overexpression of MYC and loss of ARID1A may converge on the same pathway. In conclusion, we found that ARID1A-mutant cells are highly sensitive to PI3K/mTOR inhibition. Although ARID1A mutations frequently co-occur with PI3K pathway mutations, it is not the sole explanation of this specific sensitivity. ARID1A loss may increase mTOR signaling through MYC target gene SLC7A5. However, increase in PI3K/mTOR activity maybe a long term effect of ARID1A loss. Together, our data identified PI3K/mTOR signaling is essential for survival of ARID1A-mutant cancers and PI3K/mTOR inhibitors can be used as therapeutic strategies. Citation Format: Suet-Yan Kwan, Daisy I. Izaguirre, Xuanjin Cheng, Suet-Ying Kwan, Yvonne TM Tsang, Hoi-Shan Kwan, Kwong-Kwok Wong. The PI3K/mTOR pathway is a potential therapeutic target in cancers with ARID1A mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4697. doi:10.1158/1538-7445.AM2015-4697

Over 85% of colorectal cancers is driven by aberrations in the Wnt-signaling pathway. Thus, identifying druggable targets in this pathway can be beneficial for optimizing colorectal cancer treatment. Within this context, a member of the RNA helicase gene family, DDX3, has been identified to exhibit oncogenic properties in breast and lung carcinomas as well as medulloblastomas. Notably, recent studies have identified DDX3 as a multilevel activator of Wnt-signaling in both normal and transformed cells without activating mutations in the Wnt signaling pathway. In this study, we evaluated whether DDX3 also plays a role in the constitutionally activated Wnt-signaling that drives colorectal cancer and therefore could be a potential therapeutic target in this cancer type. To determine if DDX3 is expressed in colorectal cancers, we immunohistochemically stained a cohort of 303 Dutch and German colorectal cancer patients. We found 40.4% of these tumors to overexpress DDX3 in comparison to the surrounding normal tissue. DDX3 expression was found predominantly in the cytoplasm and occasionally in the nucleus. High cytoplasmic DDX3 expression correlated with nuclear Beta-catenin expression, a marker of activated Wnt-signaling. The presence of nuclear DDX3 expression correlated with shorter overall survival (HR = 2.38, 95% CI 1.45-3.93, p < 0.001). Functionally, we validated these findings in vitro and found that inhibition of DDX3 with siRNA resulted in reduced proliferation and a G1-arrest in the HCT116 and HT29 colorectal cancer cell lines. This finding further supports the potential oncogenic role of DDX3 in colorectal cancer. With respect to targeting DDX3, we developed a small molecule inhibitor of DDX3, referred to as RK-33. RK-33 is designed to bind to the ATP-binding site of DDX3 and abrogate its functional activity. As proof of principle, we demonstrated that RK-33 binds preferentially to DDX3 and not to DDX5 and DDX17, other members of the RNA helicase family. Moreover, RK-33 inhibited the helicase activity in an in vitro assay. Furthermore, treatment of colorectal cancer cell lines and patient derived 3D- tumor cell cultures indicated that RK-33 inhibits growth and promotes cell death with IC-50 values ranging from 2.5 to 8 uM. To further elucidate the mechanism of RK-33, we studied if inhibition of DDX3 with RK-33 could cause inhibition of Wnt-signaling in colorectal cancer cell lines. Treatment with RK-33 indeed resulted in reduced TCF-reporter activity and lowered the mRNA expression levels of the Wnt-signaling downstream target genes AXIN-2, C-MYC, CCND1 and BIRC5A. Overall, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes potent inhibition of Wnt-signaling and is a promising future treatment strategy in colorectal cancer. Citation Format: Marise R. Heerma van Voss, Farhad Vesuna, Kari Trumpi, Justin Brilliant, Liudmila L. Kodach, Folkert H.M. Morsink, G. Johan A. Offerhaus, Horst Buerger, Elsken van der Wall, Paul J. van Diest, Venu Raman. Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2015-3570

More than 30 published articles have suggested that a protein kinase called MELK is an attractive therapeutic target in human cancer, but three recent reports describe compelling evidence that it is not. These reports highlight the caveats associated with some of the research tools that are commonly used to validate candidate therapeutic targets in cancer research.

Background: Prostate cancer (PCa) is the second leading cause of cancer death for men in the United States. Up to 70% of primary prostate tumors show loss of heterozygosity (LOH) at the PTEN locus, and loss of PTEN is a key initiation event in PCa development. Synthetic and collateral lethality have provided conceptual frameworks to identify cancer-specific vulnerabilities. Here, we explored an approach to identify potential synthetic lethal interactions through screening mutually exclusive deletion patterns in cancer genomes. Methods: We sought to identify ‘synthetic essential' genes, which might be occasionally deleted in some cancers but almost always retained in the context of a specific tumor suppressor deficiency, and posited that such synthetic essential genes would be therapeutic targets in cancers harboring specific tumor suppressor deficiencies. Results: In addition to known synthetic lethal interactions, this approach uncovered the chromatin helicase DNA-binding factor CHD1 as a putative synthetic essential gene in PTEN-deleted cancers. In PTEN-deleted prostate and breast cancers, the functional analysis showed that CHD1 depletion profoundly and specifically suppressed cell proliferation, survival, and tumorigenic potential. Mechanistically, functional PTEN stimulates GSK3β-mediated phosphorylation of CHD1 degron domains, which promotes CHD1 degradation via β-TrCP-mediated ubiquitination-proteasome pathway. Conversely, PTEN deficiency results in CHD1 protein stabilization, which in turn engages the H3K4me3 mark to activate transcription of the pro-tumorigenic TNFα/NF-κB gene network. In addition, we found CHD1 depletion significantly inhibits the progression of Pten-deficient prostate cancer genetic engineered mouse model. Conclusions: Together, this study identifies CHD1 as a novel downstream effector in PTEN pathway, and verifies CHD1 as a novel therapeutic target in PTEN deficient prostate cancer and breast cancer. Additionally, this study provides a framework for the discovery of trackable targets in cancers harboring specific tumor suppressor deficiencies. Citation Format: Di Zhao. Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN-deficient cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4484.

Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.

Complementary Genomic Screens Identify SERCA as a Therapeutic Target in NOTCH1 Mutated Cancer

Long non-coding RNAs (lncRNAs) have emerged as crucial regulators in various cellular processes, including cancer progression and stress response. Recent studies have demonstrated that copper accumulation induces a unique form of cell death known as cuproptosis, with lncRNAs playing a key role in regulating cuproptosis-associated pathways. These lncRNAs may trigger cell-specific responses to copper stress, presenting new opportunities as prognostic markers and therapeutic targets. This paper delves into the role of lncRNAs in cuproptosis-mediated cancer, underscoring their potential as biomarkers and targets for innovative therapeutic strategies. A thorough review of scientific literature was conducted, utilizing databases such as PubMed, Google Scholar, and ScienceDirect, with search terms like 'lncRNAs,' 'cuproptosis,' and 'cancer.' Studies were selected based on their relevance to lncRNA regulation of cuproptosis pathways and their implications for cancer prognosis and treatment. The review highlights the significant contribution of lncRNAs in regulating cuproptosis-related genes and pathways, impacting copper metabolism, mitochondrial stress responses, and apoptotic signaling. Specific lncRNAs are potential prognostic markers in breast, lung, liver, ovarian, pancreatic, and gastric cancers. The objective of this article is to explore the role of lncRNAs as potential prognostic markers and therapeutic targets in cancers mediated by cuproptosis.

<div>Abstract<p>The polo family serine threonine kinase Plk4 has been proposed as a therapeutic target in advanced cancers based on increased expression in primary human cancers, facilitation of tumor growth in murine xenograft models, and centrosomal amplification induced by its overexpression. However, both the causal link between these phenomena and the feasibility of selective Plk4 inhibition remain unclear. Here we characterize Plk4-dependent cancer cell migration and invasion as well as local invasion and metastasis of cancer xenografts. Plk4 depletion suppressed cancer invasion and induced an epithelial phenotype in poorly differentiated breast cancer cells. In an unbiased BioID screen for Plk4 interactors, we identified members of the Arp2/3 complex and confirmed a physical and functional interaction between Plk4 and Arp2 in mediating Plk4-driven cancer cell movement. This interaction is mediated through the Plk4 Polo-box 1-Polo-box 2 domain and results in phosphorylation of Arp2 at the T237/T238 activation site, which is required for Plk4-driven cell movement. Our results validate Plk4 as a therapeutic target in cancer patients and reveal a new role for Plk4 in regulating Arp2/3-mediated actin cytoskeletal rearrangement. <i>Cancer Res; 77(2); 434–47. ©2016 AACR</i>.</p></div>

BackgroundmiR-200b has been reported to be a tumor suppressor and a promising therapeutic target in cancer. miR-200b has been associated with epithelial-mesenchymal transition and chemo-resistance in cancer. The aim of this study is to investigate the expression of miR-200b, its prognostic roles and its potential targets in breast cancer.MethodsqRT-PCR was used to detect miR-200b expression in breast cancer tissues and cell lines. In situ hybridization of miR-200b on tissue microarray including 134 breast cancer samples was used to evaluate its prognostic role. Novel targets of miR-200b in breast cancer were predicted and confirmed by luciferase reporter assay and western bloting. Immunohistochemical staining was used for protein detection. The biological effects of miR-200b in breast cancer cells were further confirmed by ectopic expression of its mimics followed by MTT assay and invasion test.ResultsmiR-200b was downregulated in breast cancer tissues and cell lines and its low-expression correlated with poor outcome in breast cancer patients. Members of RAB family, RAB21, RAB23, RAB18 and RAB3B were predicted to be the targets of miR-200b. The luciferase reporter assay was performed to certificate this prediction. The expressions of RAB21, RAB23, RAB18 and RAB3B were suppressed by transfection of miR-200b in breast cancer cells. Over-expression of miR-200b or knock-down of RAB21, RAB23, RAB18 and RAB3B inhibited breast cancer cell proliferation and invasion in vitro.ConclusionsOur study provides evidence that miR-200b is a prognostic factor in breast cancer targeting multiple members of RAB family. MiR-200b could be a potential therapeutic target in breast cancer.

The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer.

One-carbon metabolism, which integrates the folate and methionine cycles, is crucial for cancer growth. Methionine synthase (MTR), a key enzyme in this pathway, converts 5-methyltetrahydrofolate (5-CH3-THF) to active tetrahydrofolate (THF), a cofactor required for nucleotide synthesis. Concurrently, MTR catalyzes the conversion of homocysteine to methionine, effectively linking the folate and methionine cycles. Given its central role, MTR has gained attention as a potential therapeutic target in cancer research, though its function in KRAS-driven lung cancer remains poorly understood. Recently, we performed an in vitro CRISPR screening using a metabolism-focused sgRNA library in mouse KrasG12D/+;p53-/- (KP) lung tumor derived cell line to identify potential metabolic therapeutic targets, where MTR emerged as one of top hits. Functional validation showed that MTR knockout (MtrKO) impaired cell proliferation, reduced colony formation, and inhibited allograft tumor growth. To further evaluate MTR's role in vivo, we used genetically engineered mouse models (GEMMs) of KRAS-driven non-small cell lung cancer (NSCLC). Lentiviral delivery of sgRNAs targeting Mtr, along with Cre recombinase, into KrasLSL_G12D/+;Lkb1flox/flox;Rosa26-LSL-Cas9-EGFP (KL_Cas9) and KrasLSL_G12D/+;p53flox/flox;Lkb1flox/flox;Rosa26-LSL-Cas9-EGFP (KPL_Cas9) mice resulted in conditional MTR knockout in lung tumors, significantly extending survival in mice with KL or KPL lung tumors compared to mice bearing wild-type (MtrWT) KL or KPL lung tumors. Furthermore, MtrKO cells showed increased sensitivity to folate and methionine deprivation compared to MtrWT cells in vitro. Folic acid supplementation did not affect the growth of MtrWT;KP allografts but rescued the growth of MtrKO tumors, confirming that MTR is essential for maintaining THF levels in vivo to support metabolic processes critical for KP tumor growth. Further in vivo isotope tracing revealed that purine salvage pathway was upregulated in MtrKO tumors. Targeting purine salvage pathway, but not the pyrimidine salvage pathway, synergistically suppressed MtrKO KP allograft tumor growth. Moreover, MTR knockout induced S-phase arrest, elevated γH2AX levels (indicative of DNA damage), and enhanced KP cell sensitivity to cisplatin. These findings highlight MTR as a critical metabolic vulnerability and promising therapeutic target in KRAS-driven lung cancers. Citation Format: Wenping Wang, Samuel Wang, Ioana Dobrescu, Hyungsin Kim, Kyle Nunn, Joel Lee, Joshua Martorelli, Fuqian Shi, Eduardo Lopes, Victoria Silva, Hua Zhong, Zhixian Hu, Daniel Herranz, Chang Chan, Eileen White, Jessie Yanxiang Guo. CRISPR screening identifies methionine synthase as a potential therapeutic target in KRAS-driven NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3803.

<div>Abstract<p>The polo family serine threonine kinase Plk4 has been proposed as a therapeutic target in advanced cancers based on increased expression in primary human cancers, facilitation of tumor growth in murine xenograft models, and centrosomal amplification induced by its overexpression. However, both the causal link between these phenomena and the feasibility of selective Plk4 inhibition remain unclear. Here we characterize Plk4-dependent cancer cell migration and invasion as well as local invasion and metastasis of cancer xenografts. Plk4 depletion suppressed cancer invasion and induced an epithelial phenotype in poorly differentiated breast cancer cells. In an unbiased BioID screen for Plk4 interactors, we identified members of the Arp2/3 complex and confirmed a physical and functional interaction between Plk4 and Arp2 in mediating Plk4-driven cancer cell movement. This interaction is mediated through the Plk4 Polo-box 1-Polo-box 2 domain and results in phosphorylation of Arp2 at the T237/T238 activation site, which is required for Plk4-driven cell movement. Our results validate Plk4 as a therapeutic target in cancer patients and reveal a new role for Plk4 in regulating Arp2/3-mediated actin cytoskeletal rearrangement. <i>Cancer Res; 77(2); 434–47. ©2016 AACR</i>.</p></div>

Long non-coding RNA PVT1, as an important carcinogenic lncRNA, is highly expressed in many malignant tumors and suggests a poorer prognosis. It can promote the occurrence and development of cancers by affecting cell proliferation, migration, invasion and apoptosis. This article reviews the progress of lncRNA PVT1 on cancer therapy, in order to facilitate the in-depth study of lncRNA PVT1 acting as a promising target for therapy in cancers. We extracted all relevant studies of lncRNA PVT1 on the treatment of cancers by searching electronic databases Pubmed, Embase, Web of Science from inception to November 30, 2017. Accumulating vigorous evidence has shown that lncRNA PVT1 performs a significant carcinogenic activity in various cancers, for instance, negatively modulating miRNA as a ceRNA or a molecular sponge to exert tumor-promoting effect. Based on the critical role of lncRNA PVT1 in the pathogenesis of cancers, numerous studies have already demonstrated that lncRNA PVT1 might serve as a potential therapeutic target for various cancers, such as non-small cell lung cancer, hepatocellular carcinoma, gastric cancer, breast cancer, glioma, etc. Numerous studies have indicated that lncRNA PVT1 will most likely become a novel target for cancer therapy with the deepening systematic research.