Search

Supplementary Figure 3

Supplementary Table 3

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors.Significance:In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.

Supplementary Table 1

Supplementary Figure 2

Supplementary Table 2

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. Significance: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.

Superior knowledge of cancer biology has enabled unprecedented innovations in therapies targeting mutated driver genes. Despite the attempt of targeting cancer-inducing genes such as KRAS, the life expectancy of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) has poorly improved. We performed an unbiased genome wide in-vivo loss-of-function CRISPR screen to discover novel oncogenes and identified heat-shock protein HSPE1, whose function is still unknown in PDAC. By decreasing HSPE1 expression with shRNA and CRISPR technology, we detected a slowdown in cell growth of PDAC cancer cells. HSPE1 knock-out cells injected in mice displayed a drastic reduction in tumor volume. We exploited this vulnerability by disrupting HSPE1 function by using KHS101, a validated HSPD1-HSPE1 complex inhibitor. Several PDAC cell lines cultured in vitro were sensitive upon KHS101 treatment and in vivo administration of KHS101 reduced tumorigenesis. Compiling the negative fitness scores from DepMap database revealed a co-dependency between HSPE1 and PLK1, a protein involved in regulating G2/M checkpoint. Indeed, we detected a protein-level decrease of PLK1 in our experimental model of PDAC cells having either a drug-induced HSPE1 deficiency by KHS101 exposure or a HSPE1 gene-expression reduction by shRNA. We found that the cell cycle was disrupted through G2/M arrest in PDAC cells treated with KHS101. Our findings highlight a new role underlying PDAC tumorigenesis for HSPE1 and could unlock a new area of research towards precision medicine. Citation Format: Julien Boudreault, Ni Wang, Gang Yan, Meiou Dai, Sophie Poulet, Girija Daliah, Jean-Jacques Lebrun. In vivo genome-wide CRISPR screen in pancreatic ductal adenocarcinoma defines HSPE1 as a potential oncogene by acting through G2/M cell cycle arrest. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3938.

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Dedifferentiation increased cellular plasticity and stemness are established derivers of tumor heterogeneity, metastasis and therapeutic failure resulting in incurable cancers. Therefore, it is essential to decipher pro/forward-differentiation mechanisms in cancer that may serve as therapeutic targets. We found that interfering with expression of the receptor for the lactogenic hormone prolactin (PRLR) in breast cancer cells representative of the luminal and epithelial breast cancer subtypes (hormone receptor positive (HR+) and HER2-enriched (HER2-E) resulted in loss of their differentiation state, enriched for stem-like cell subpopulations, and increased their tumorigenic capacity in a subtype-specific manner. Loss of PRLR expression in HR+ breast cancer cells caused their dedifferentiation generating a mesenchymal-basal-like phenotype enriched in CD44+ breast cancer stem-like cells (BCSCs) showing high tumorigenic and metastatic capacities and resistance to anti-hormonal therapy. Whereas loss of PRLR expression in HER2-E breast cancer cells resulted in loss of their luminal differentiation yet enriched for epithelial ALDH+ BCSC population showing elevated HER2-driven tumorigenic, multi-organ metastatic spread, and resistance to anti-HER2 therapy. Collectively, this study defines PRLR as a driver of precise luminal and epithelial differentiation limiting cellular plasticity, stemness, and tumorigenesis and emphasizing the function of pro/forward-differentiation pathways as a foundation for the discovery of anti-cancer therapeutic targets.

Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer., Triple negative breast cancer (TNBC) lack effective therapies. Here, through an in vivo genome-wide CRISPR screen in TNBCs, the authors identify tumorigenic functions for components of the mTORC1/2 complex and of the YAP/Hippo pathway, and demonstrate that pharmacological inhibition of mTOR and YAP reduces tumour growth in vivo.

Breast cancer consists of a range of tumor subtypes with different clinical characteristics, disease prognosis, and treatment-response. Luminal breast cancer has the best prognosis while basal-like breast cancer (BLBC) represents the worst subtype. Transforming growth factor-beta (TGFβ) plays a prominent role in stimulating the migration and invasion of malignant breast cancer cells contributing to tumor progression. In this study, we identified the Ephrin type-A receptor 4 (EPHA4) as a novel target of TGFβ in breast cancer. Moreover, we show that TGFβ induction of EPHA4 gene expression is specific to basal-like tumors and is required for TGFβ-mediated cell migration. We further addressed the mechanism and found EPHA4 to be required for TGFβ-mediated cell migration in breast cancer through TGFβ-induced short term and long term activation of RhoGTPases. Finally, our data revealed a strong association between high EPHA4 expression and advanced tumor stage, aggressive BLBC molecular subtype and poor prognosis. Importantly, we found significant co-expression of EPHA4 and the TGFβ receptor type-2 (TGFβR2) in breast cancer subtypes associated with increased tumor relapse and drug resistance. Together, this study highlight the important role of the TGFβ/EPHA4 signaling axis in mediating tumor aggressiveness and poor patient survival in human breast cancer.

Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of riboswitches, it is important to monitor in real time the riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study riboswitch conformational changes as it allows to both monitor steady-state populations of riboswitch conformers and associated interconversion dynamics. Since FRET fluorophores can be attached to virtually any nucleotide position, FRET assays can be adapted to monitor specific conformational changes, thus enabling to deduce complex riboswitch folding pathways. Herein, we show how to employ sm-FRET to study the folding pathway of the S-adenosylmethionine (SAM) and how this can be used to understand very specific conformational changes that are at the heart of riboswitch regulation mechanism.

Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of riboswitches, it is important to monitor in real time the riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study riboswitch conformational changes as it allows to both monitor steady-state populations of riboswitch conformers and associated interconversion dynamics. Since FRET fluorophores can be attached to virtually any nucleotide position, FRET assays can be adapted to monitor specific conformational changes, thus enabling to deduce complex riboswitch folding pathways. Herein, we show how to employ sm-FRET to study the folding pathway of the S-adenosylmethionine (SAM) and how this can be used to understand very specific conformational changes that are at the heart of riboswitch regulation mechanism.