Novel non-covalent LSD1 inhibitors endowed with anticancer effects in leukemia and solid tumor cellular models

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.

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

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

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.

Objective To investigate the effects of matrine on proliferation and apoptosis of human rhabdom-yosarcoma(RMS) RD cells line in vitro, to study the regulatory mechanism of Wnt/β-catenin pathway-influenced apoptosis of RD cells line by detecting the expressions of β-catenin protein, Bcl-2 protein and caspase-3 protein, and to explore Wnt/β-catenin mechanism during the process of RMS. Methods The human RMS RD cells line was treated with matrine of different concentrations (0.5, 1.0, 1.5, 2.0 g/L)for 48 hours respectively, and the proliferation inhibition rates of different concentrations of matrine on RD cells were detected by methyl thiazolyl tetrazolium assay, while the apoptosis rates by flow cytometry(FCM) and the expressions of β-catenin, Bcl-2 and caspase-3 by Wes-tern blot. Results The proliferation inhibition rates between control group and different concentrations of matrine groups were(13.70±0.25)%, (33.16±0.11)%, (42.96±0.90)%, (56.26±0.79)% and (67.89±0.63)%, respectively.The apoptosis rates were (5.49±0.96)%, (17.23±5.03)%, (25.84±4.17)%, (36.08±3.68)% and (47.79±4.82) %, respectively.The highest expression of β-catenin and Bcl-2 proteins and the minimum amount of caspase-3 protein were found in the control group.After intervention of matrine, the expressions of β-catenin and Bcl-2 reduced while the amount of caspase-3 rose significantly, which was concentration-dependent obviously.Differences were found between every concentration of matrine group with control group according to statistics (all P<0.05). Conclusions Matrine can inhibit proliferation and induce apoptosis of RD cells.Matrine can down-regulate the expression of β-catenin and Bcl-2 proteins in RD cells, while the amount of caspase-3 protein rises.Wnt/β-catenin signal pathway plays an important role in the apoptosis of RD cell induced by matrine, and its downstream proteins Bcl-2 and caspase-3 are also involved in the regulation of this process. Key words: Matrine; Rhabdomyosarcoma; Wnt Proteins; Cell apoptosis

The combined effects of matrine (Mat) and cisplatin on the survival and apoptosis of rhabdomyosarcoma (RMS) RD cells, as well as the possible mechanism of the synergistic effect of Mat and cisplatin were investigated in the present study. RMS RD cells were divided and treated as follows: control group, 5 mg/l cisplatin group, Mat groups (0.5, 1.0 and 1.5 g/l), and Mat (0.5, 1.0 and 1.5 g/l) combined with 5 mg/l cisplatin groups. An MTT assay and flow cytometry were applied to detect the survival and apoptotic rates, respectively, while RT-PCR was applied to detect the expression levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA in the RD cells of each group. The survival rates of RD cells in each experimental group were lower than in the control group, and the apoptotic rates were higher than those in the control group (P<0.05). An increase in drug concentrations led to the cell proliferation inhibitory and apoptotic rates of the single Mat groups increasing as a function of dose (pairwise comparison among the groups, P<0.05), while the proliferation inhibitory and apoptotic rates of Mat combined with the cisplatin groups under different concentration were significantly higher than those of the single Mat and single cisplatin groups under the same concentration (P<0.01). The expression levels of XIAP mRNA in the RD cells of each experimental group were lower than those in the control group (P<0.05). Additionally, the expression levels of XIAP mRNA in the group treated with Mat and cisplatin were significantly lower than those of the single cisplatin and single Mat groups (P<0.01). In conclusion, Mat and cisplatin are capable of inhibiting the proliferation of RD cells and inducing apoptosis by suppressing the XIAP mRNA expression levels.

Androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) and remains critical for more aggressive castration-resistant PCa (CRPC) through transactivation of multiple genes. Although CRPC is generally responsive to further CYP17A1 inhibition (abiraterone) or more potent AR antagonist (enzalutamide) treatments, patients treated with these agents still relapse within 1-2 years, and high levels of AR and AR regulated genes in many of these relapsed tumors indicate that AR activity has again been restored. Therefore, there remains a need for further novel AR targeted therapies. Initially identified as a component in the REST complex that functions as a transcriptional suppressor, Lysine Specific Demethylase 1 (LSD1) removes the methyl group from histone 3 lysine 4 (H3K4me1 and 2), and turns off the marks of active transcription. Our recent genome-wide integrated analysis of LSD1 and AR chromatin bindings and gene expression profiles indicates that LSD1 is broadly associated with AR regulated enhancers, and LSD1 functions as a coactivator on AR-stimulated genes through demethylating novel histone or nonhistone substrates in addition to H3K4me. LSD1 is also associated with FOXA1 at AR regulated enhancer sites, and an LSD1-FOXA1 interaction enhances binding of both proteins at these sites. These results clearly demonstrated LSD1 as a therapeutic target in prostate cancer. We then accessed if LSD1 enzymatic activity is required for its global AR coactivator function. AR transcription activity is significantly impaired in cells overexpressing a catalytic-deficient LSD1 mutant. In addition, this mutant also decreased FOXA1 binding on AR-regulated enhancers. We then selected of a panel of LSD1 inhibitors (a few of them have been used in clinical trials for lung cancer and leukemia) to assess the effects on AR activity and PCa cell growth. Inhibition of LSD1 consistently impaired AR activity through abrogating acetylated H3K27 at AR-regulated enhancers. LSD1 inhibitor also decreased FOXA1 binding prior to androgen stimulation. While most of inhibitors showed relatively low efficacy in PCa cells (&amp;gt;50μM), LSD1-C12 is highly potent and specific at μM range to dramatically decrease AR activity and PCa cell growth. Overall, our study suggests that the AR coactivator function of LSD1 depends on its enzymatic activity and targeting LSD1 with more specific and potent inhibitors is a promising therapeutic approach to treat PCa. Furthermore, understanding the mechanism of LSD1 coactivator function will potentially advance PCa therapy by targeting LSD1. Citation Format: Shuai Gao, Yanfei Gao, Hansen He, Myles Brown, Steve Balk, Changmeng Cai. LSD1 functions as a global androgen receptor coactivator and is a therapeutic target in prostate 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 966. doi:10.1158/1538-7445.AM2015-966

The zinc-finger (ZnF) transcriptional repressor Growth Factor Independence (GFI) 1 is a master regulator of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. Transcriptional repression by GFI1 requires a SNAG domain at its N-terminus and a concatemer of six ZnFs at its C-terminus. The SNAG domain recruits Lysine Specific Demethylase (LSD) 1, a dominant effector of transcriptional repression by GFI1, while ZnFs 3-5 bind a consensus response element in the promoters of GFI1 target genes. A linker region separates the SNAG and ZnF domains in GFI1, and likely provides sites for protein—protein interactions and post-translational modifications that modulate GFI1 functions in cell fate determination. In hematopoiesis, GFI1 regulates self-renewal and maintenance within the hematopoietic stem cell (HSC) compartment, supports early events in lymphopoiesis and is required for maturation of granulocytic precursors. Mutations in GFI1 cause severe congenital neutropenia (SCN) type II, a pre-leukemic condition, and recent evidence shows that GFI1 maintains the malignant phenotype in lymphoid leukemias and lymphomas. These findings intimate that regulators and effectors of GFI1 may be attractive candidates for therapeutic development in hematologic malignancy and maturational failure syndromes. How GFI1 is regulated to control cell fates during hematopoiesis is unknown. We show that GFI1 contains a consensus SUMOylation motif, centered on lysine (K) 239 within its linker region that is required for GFI1 conjugation by SUMO/Ubiquitin-like (UBL) proteins. K239 is found within the binding site for PIAS3, an E3 SUMO ligase and known GFI1 binding protein. PIAS3 antagonizes GFI1-mediated transcriptional repression. Arginine substitution at K239 (GFI1-K239R) preserves transcriptional repression by GFI1 yet resists the inhibitory effects of PIAS3. Moreover, the GFI1-K239R derivative displays a prolonged half-life and preferentially segregates with chromatin in fractionated cells. These findings suggest that GFI1-mediated transcriptional repression is limited by SUMO/UBL protein conjugation and that its roles in cell fate determination may be altered by factors governing these modifications. Among these factors is the Notch1 intracellular domain (N1-ICD), which we show interacts with the same region in GFI1 that binds PIAS3 and competes with PIAS3 for GFI1 binding. Furthermore, N1-ICD abolishes GFI1 conjugation by SUMO/UBL proteins, prolongs GFI1 half-life and reverses the inhibitory effect of PIAS3 toward GFI1-mediated transcriptional repression. In so doing, N1-ICD supports the function of GFI1 as a transcriptional repressor. We also show that Gfi1 depletion causes profound apoptosis in T-ALL cells driven by N1-ICD, suggesting GFI1 inhibitors and/or small molecule inhibitors of its effectors may prove efficacious in T-ALL treatment. To address this hypothesis, we have developed reversible small molecule inhibitors of LSD1. These inhibitors display sub-micromolar IC50 toward T-ALL cell lines and primary patient isolates in the ex vivo setting, including those resistant to γ-secretase inhibitors. These findings suggest that the GFI1—LSD1 axis has significant influence over T-ALL viability and that LSD1 may be a novel therapeutic target in cancers driven by Notch activating mutations. Citation Format: Jason Singer, Daniel Andrade, Diana Bareyan, David McClellan, Helena Lucente, Matthew Velinder, Luke Maese, Mahesh Chandrasekharan, Emily Theisen, Fang Liu, Sunil Sharma, Michael Engel. Lysine specific demethylase-1 inhibition as a therapeutic strategy that leverages the requirement for growth factor independence-1 in Notch-driven T-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B19.

Objective To investigate the effect of Matrine on the expression of X-linked inhibitor of apoptosis protein (XIAP) in human rhabdomyosarcoma RD cells in vitro. Methods Cultured human rhabdomyosarcoma RD cells were divided into Matrine intervention groups (0.5 g/L, 1.0 g/L and 1.5 g/L) and a control group.The proliferation-inhibition rates in RD cells treated with different concentrations of matrine were detected by methylthiazolyl blue colorimetric assay.Flow cytometry analysis was performed for the apoptosis rates of RD cells.Reverse transcription-polymerase chain reaction analysis was used to measure the XIAP mRNA expression. Results There was a significant difference in the proliferation-inhibition rates [0.5 g/L Matrine group: (15.84±2.58)%, 1.0 g/L Matrine group: (23.13±4.19)%, 1.5 g/L Matrine group: (30.32±3.02)%, and the control group: (8.92±1.23)%]; apoptotic rates [0.5 g/L Matrine group: (12.33±1.15)%, 1.0 g/L Matrine group: (16.67±0.99)%, 1.5 g/L Matrine group: (25.33±1.91)%, and the control group: (9.47±0.96)%]; XIAP mRNA expression(0.5 g/L Matrine group: 0.633±0.046, 1.0 g/L Matrine group: 0.441±0.055, 1.5 g/L Matrine group: 0.326±0.065, control group: 0.794±0.029)in RD cells among 0.5 g/L, 1.0 g/L, 1.5 g/L Matrine groups and the control group (F=14.15, 83.37, 50.57, all P<0.05). The proliferation-inhibition and apoptotic rates in RD cells were gradually increased with the increasing Matrine concentration.The expression of XIAP mRNA was significantly decreased in different Matrine groups compared with the control group, exhibiting a dose-dependent manner. Conclusions Matrine can inhibit the proliferation of RD cells and induce the apoptosis in a dose-dependent manner, which may be related to the down-regulated XIAP mRNA. Key words: Human rhabdomyosarcoma RD cell; Matrine; X-linked inhibitor of apoptosis protein

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.

Purpose: It is difficult to prioritize potential therapeutic targets from thousands of differentially expressed genes identified by genome-wide gene expression profiling in cancer. The vast array of in silico resources currently available in life sciences research offer the possibility of aiding drug discovery process. Here we propose to take advantage of these resources to develop a genetic network-based model to comprehensively and effectively identify potential therapeutic targets in several cancer types. Method: A whole-genome genetic network, which can reveal the tendency for genes to operate in the same or similar pathways, is first constructed from heterogeneous data using a developed machine learning approach, RVM-based ensemble. A tumor-specific network can then be generated by mapping the differentially expressed genes in a tumor to the whole-genome network. Finally, potential therapeutic targets can be identified as hub genes that are functionally associated to multiple existing cancer pathways in the tumor-specific network. Result: Here, the approach is applied to Breast, Colon, and Lung Cancer separately. In each case, differentially expressed genes are all ranked based on the extent of their functional association with multiple known cancer pathways in the tumor-specific network. The result in each case shows that higher ranked genes are cited by more literature respectively related to the three cancers (Spearman's Rank Correlations, R&amp;gt;0.2 with p&amp;lt;1×10−10); that is, they likely play more important roles in these cancers, compared to lower ranked genes. While mapping the results to gene annotation, we find that many kinase, receptor, and transcription factor related genes, which are often proposed as possible molecular targets, are ranked highly in all cases. We also find that the effective targets detected by siRNA screens tend to be ranked highly in each case (the area under the ROC curve, AUC&amp;gt;0.75). Additionally, we also identified drugs and compounds that can target the highly ranked genes based on known drug-target information. Targets of many drugs, already in clinical trials and used for treatment of the three cancers, are all highly ranked in each case. Other drugs and compounds identified but not in clinical trials have also shown anti-cancer effect and could be considered as potential novel drug for these cancers. Moreover, we also find several novel targets in each case, which are not yet identified as cancer genes, are highly ranked and also increase cancer cell death in siRNA screens. One example is CSNK1G2 (casein kinase 1, gamma 2) in Colon cancer. Conclusion: Our approach has demonstrated the ability to identify potential therapeutic targets in cancer systematically and comprehensively using integrated functional genomic and proteomic data. It also implies that the proposed approach could be utilized to generate personal therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 109.

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

&lt;div&gt;Abstract&lt;p&gt;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. &lt;i&gt;Cancer Res; 77(2); 434–47. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;