1,720,987 research outputs found
Investigating WDR12 as a Therapeutic Target in Breast Cancer
Full text linkBreast cancer is the most diagnosed and lethal cancer globally in women. Presently, there is a need for novel targets for the development of improved therapeutics. Data mining through genetic screens suggests that the ribosome biogenesis protein WDR12 is essential for breast cancer cell proliferation. In this study, we characterize the effects of WDR12 knockdown in a panel of breast cancer cells. We show that WDR12 knockdown induces ribosomal RNA (rRNA) processing defects resulting in strong growth inhibition in all breast cancer cells tested, however only a subset of lines displayed short-term cell cycle defects. To further understand the cellular consequences of WDR12 suppression, we investigated the global proteomic landscape and found that WDR12 knockdown results in a perturbation of translational and ribosome biogenesis-related networks. Furthermore, we performed RNA-sequencing to assess the global transcriptional changes upon WDR12 knockdown and found dysregulation of the endoplasmic reticulum (ER) stress response pathway. Finally, we characterized the effect of long-term WDR12 suppression and found that growth inhibition was in part due to cellular senescence. Our results demonstrate that WDR12 is implicated in breast cancer cell growth and senescence, and its suppression may represent a viable therapeutic target for breast cancer.Ph.D
Characterizing effects of small molecule inhibition of WDR5 in colorectal cancer
Full text linkAbstract
WDR5 is a core subunit of the MLL-WRAD methyltransferases, NSL-MOF acetyltransferases and MYC-MAX transcription factors. Dysregulation of these complexes has been implicated in numerous cancers. Despite links between colorectal cancer (CRC) and WDR5, little is known about its role. Using OICR11824, an internally developed novel chemical antagonist of WDR5, we investigated the biological effects of targeting the arginine binding pocket of WDR5 in CRC. Firstly, we evaluated the ability of OICR11824 to bind and disrupt WDR5-MLL interaction in cells. We then utilized OICR11824 and shRNA knockdown to investigate effects of WDR5 inhibition on proliferation in CRC cell lines. In addition, Western blot and RT-PCR were performed to confirm modulation of H3K4 methylation and expression of MYC-transcriptional targets. Finally, flow cytometry was used to evaluate effects of WDR5 inhibition on cell cycle. Our research shows that OICR11824 binds and inhibits the WDR5-MLL interaction. As well, OICR11824 displays a range of anti-proliferation potencies in CRC cell lines with the greatest sensitivity seen in COLO205 demonstrating a G1 accumulation and S-phase depletion. Finally, OICR11824 affects the actions of WDR5-complexes in MYC-WDR5 gene targets and H3K4 trimethylation. Our research provides new insights into the role of WDR5 in CRC and will contribute to not only developing new therapeutics against CRC, but will also help elucidate the role of WDR5 complexes in cancer.M.Sc
Validating WDR12 as a Potential Drug Target in Triple Negative Breast Cancer
Full text linkCompared to other breast cancers (BC), triple negative breast cancers (TNBCs) confer lower survival and higher disease recurrence. TNBCs lack receptors targeted by current therapies, requiring new therapeutic target identification. Recently, the ribosome biogenesis protein WDR12 was identified as a potential target from RNAi databases. WDR12 belongs to the WD40-repeat family of scaffolding proteins, which have become interesting targets due to their druggable structure. I hypothesized that TNBC cell lines would be more sensitive to WDR12 knockdown (KD) due to their increased rate of ribosome biogenesis. WDR12 depletion led to decreased proliferation in all BC cell lines that could not be attributed to apoptosis or cell cycle arrest. Interestingly, WDR12 KD decreased nucleolar size to a greater extent in TNBC, indicating a potential role for WDR12 in ribosome biogenesis. In conclusion, WDR12 may be a promising therapeutic target in TNBC, although additional confirmatory testing will be required.M.Sc
Validating SCAP as a Therapeutic Target for Colorectal Cancer
Full text linkColorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide with an increased prevalence in adults below 50 years old, highlighting the need to develop more therapeutic options. Data mining from genome-wide dropout screens revealed the essentiality of sterol regulatory element binding protein cleavage activating protein (SCAP) on the proliferation of CRC cell lines. In this study, we demonstrate that SCAP knockdown leads to marked antiproliferation, with variability in the phenotypic strength across the CRC cell lines tested. SCAP knockdown reduced mRNA expression of its transcription factor binding partners SREBP1/2, and their transcriptional target genes, primarily impacting lipid homeostasis. SCAP knockdown induced autophagy and multiple cell-specific disruptions in cell cycle progression, which preceded caspase-independent cell death. Global metabolic profiling was performed to further characterize the growth suppressive mechanism and uncovered significant metabolic perturbations in multiple pathways. A profound shift was observed in lipid pathways including cholesterol and monounsaturated fatty acid (FA) synthesis. The decrease in FA synthesis affected downstream derivatives, diverting most of the remaining FAs towards phosphatidylcholine and decreasing acyl-carnitine formation. Furthermore, nicotinamide and polyamine metabolism were greatly affected, resulting in significantly elevated metabolites, notably 1-methylnicotinamide, acetylated spermidine, and spermine.Despite challenges, we successfully expressed and purified the full-length human SCAP and its WDR domain. While cells do not favor overexpressing full-length SCAP, we found that the membrane fraction was the most productive for isolating SCAP with a mild detergent buffer (2% DDM+ 0.4% CHS). For the WDR domain we further optimized our expression constructs to shorten the flexible loops and greatly improve the purification quality and protein solubility. Although crystal screening of the WDR domain has been unsuccessful so far, the protein behavior is promising, and more screening conditions will be explored.
Our findings underscore the significance of SCAP in CRC cell proliferation, suggesting that targeting SCAP could offer a promising therapeutic intervention for CRC. Additionally, this work provides the starting ground to determine the crystal structure of SCAP, by establishing a robust and stable recombinant protein for future crystallography efforts, and small molecule screening, which are critical for a future drug discovery effort.
Ph.D
DCAF1 as a Novel Therapeutic Target for Lung Cancer
Full text linkLung cancer remains a leading cause of mortality worldwide, ranking as the second most common cancer and deadliest form in Canada. Therefore, it is important to address the urgent unmet need for novel and effective lung cancer treatments. By mining the DepMap genome-wide shRNA dropout screen, we identified a strong dependency in non-small cell lung cancer (NSCLC) on the WD40 and E3 ligase DCAF1 protein. In-house experiments confirmed that knockdown of DCAF1 resulted in a significant suppression of growth in various tested NSCLC cell lines. Moreover, DCAF1 knockdown led to cell death in all tested cell lines, with a cell cycle defect preceding cell death specifically observed in p53 wild-type cell lines. In order to understand the underlying mechanisms of DCAF1 in NSCLC, unbiased approaches such as RNA sequencing and shotgun proteomics were utilized. Our findings revealed that DCAF1 knockdown led to downregulation of pathways associated with DNA damage, cell cycle regulation, translation, and rRNA processing. Our preliminary data indicate that defects in rRNA processing are more likely to precede changes in DNA damage and cell cycle progression and might be the primary cause of our observed growth suppression phenotype. Furthermore, as part of our efforts to develop therapeutic agents for NSCLC, a hit (Z1391232269) against the WD40 domain of DCAF1 with a Kd of 490 nM in SPR was discovered using an experimental DNA-encoded library followed by a machine learning approach. Z1391232269 was successfully co-crystalized with the WD40 domain of DCAF1. A series of structure activity relationship studies to optimize Z1391232269 identified OICR-38268 a more potent analogue with a Kd of 35 nM by SPR and an EC50 of 10 μM in cells. In conclusion, our study highlights the importance of DCAF1 for NSCLC growth and that targeting it might represent a novel therapeutic approach for lung cancer treatment. Lastly, the discovery of a DCAF1 ligand represents a significant step towards the development of targeted therapeutic agents for NSCLC.Ph.D
SCAP in Colorectal Cancer: Target Validation and Assay Development for Drug Discovery
Full text linkColorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related death, underscoring the demand of novel therapeutics. Increasing evidence links CRC tumorigenesis to dysregulated SCAP-SREBP-mediated lipogenesis. I hypothesized that inhibiting SCAP-SREBP interaction will stop CRC cell proliferation. The role of sterol regulatory element binding protein (SREBP) cleavage activating protein (SCAP) in CRC cell proliferation was previously validated by Dr. Aisha Al-Naamani. However, SCAP knockdown alone did not suppress the proliferation of DLD1 in vivo. In contrast, the combined treatment of SCAP knockdown and Fluvastatin reduced tumor growth in vivo, suggesting that targeting SCAP under lipid-limited conditions could offer a promising strategy for CRC treatment.Two cell-based assays, SRE reporter assay and NanoBRET assay, were developed to monitor the SCAP-SREBP activity and interaction. With further investigation and optimization, they could serve as reliable and reproducible tools for future high-throughput screening of compounds targeting SCAP.M.Sc
Characterizing effects of small molecule inhibition of WDR5 in colorectal cancer
No full textAbstract
WDR5 is a core subunit of the MLL-WRAD methyltransferases, NSL-MOF acetyltransferases and MYC-MAX transcription factors. Dysregulation of these complexes has been implicated in numerous cancers. Despite links between colorectal cancer (CRC) and WDR5, little is known about its role. Using OICR11824, an internally developed novel chemical antagonist of WDR5, we investigated the biological effects of targeting the arginine binding pocket of WDR5 in CRC. Firstly, we evaluated the ability of OICR11824 to bind and disrupt WDR5-MLL interaction in cells. We then utilized OICR11824 and shRNA knockdown to investigate effects of WDR5 inhibition on proliferation in CRC cell lines. In addition, Western blot and RT-PCR were performed to confirm modulation of H3K4 methylation and expression of MYC-transcriptional targets. Finally, flow cytometry was used to evaluate effects of WDR5 inhibition on cell cycle. Our research shows that OICR11824 binds and inhibits the WDR5-MLL interaction. As well, OICR11824 displays a range of anti-proliferation potencies in CRC cell lines with the greatest sensitivity seen in COLO205 demonstrating a G1 accumulation and S-phase depletion. Finally, OICR11824 affects the actions of WDR5-complexes in MYC-WDR5 gene targets and H3K4 trimethylation. Our research provides new insights into the role of WDR5 in CRC and will contribute to not only developing new therapeutics against CRC, but will also help elucidate the role of WDR5 complexes in cancer.M.Sc
Validating WDR12 as a Potential Drug Target in Triple Negative Breast Cancer
No full textCompared to other breast cancers (BC), triple negative breast cancers (TNBCs) confer lower survival and higher disease recurrence. TNBCs lack receptors targeted by current therapies, requiring new therapeutic target identification. Recently, the ribosome biogenesis protein WDR12 was identified as a potential target from RNAi databases. WDR12 belongs to the WD40-repeat family of scaffolding proteins, which have become interesting targets due to their druggable structure. I hypothesized that TNBC cell lines would be more sensitive to WDR12 knockdown (KD) due to their increased rate of ribosome biogenesis. WDR12 depletion led to decreased proliferation in all BC cell lines that could not be attributed to apoptosis or cell cycle arrest. Interestingly, WDR12 KD decreased nucleolar size to a greater extent in TNBC, indicating a potential role for WDR12 in ribosome biogenesis. In conclusion, WDR12 may be a promising therapeutic target in TNBC, although additional confirmatory testing will be required.M.Sc
Developing Small Molecule Inhibitors of ALK2: A Serine/Threonine Kinase Implicated in Diffuse Intrinsic Pontine Glioma
Full text linkDiffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we developed two separate series of potent type I inhibitors of ALK2 based on the previously reported inhibitor LDN-214117. The first structure-activity relationship (SAR) study focuses on improving the selectivity, permeability and pharmacokinetic profile of M4K2149, a benzamide analogue with reduced off-target affinity for the hERG potassium channel. The second part of this thesis highlights the efforts made to develop a conformationally constrained inhibitor that was rigidified into the biologically active configuration of M4K2009, the lead compound of this project. Future studies will assess the permeability of select compounds in a Caco-2 permeability assay and their PK profiles in vivo.M.Sc
Validating WD Repeat Proteins as Potential Negative Regulators of T Cell Activation
Full text linkCancer immunotherapies provide benefit to patients with different tumour types, but are hampered by low response rates and resistance, demanding novel therapeutics. A potential link was identified between WD40 repeat domain (WDR) proteins, a family of scaffolding proteins with druggable structures, and T cell activation. Using gene-silencing technologies, their roles in T cell activation were studied. I hypothesized that loss of WDR proteins which are negative regulators will increase Jurkat T cell activation. CRISPR knockout (KO) of RBBP4 or RBBP7 enhanced CD69 expression levels. While silencing of the paralogs RBBP4/7 differentially affected IL2 secretion, they also differentially affected proliferation. Greater toxicity was observed with loss of RBBP4 vs. RBBP7, implying different cellular roles. Immune-protective gene pathways were upregulated after RBBP7 KO, like the known negative regulator Cbl-b. These results support a negative regulatory role for RBBP7 in T cell activation, paving the way to broadening therapeutic targets for cancer immunotherapy.M.Sc
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