INX-315, a Selective CDK2 Inhibitor, Induces Cell Cycle Arrest and Senescence in Solid Tumors.

Unlabelled: Cyclin-dependent kinase 2 (CDK2) is thought to play an important role in driving proliferation of certain cancers, including those harboring CCNE1 amplification and breast cancers that have acquired resistance to CDK4/6 inhibitors (CDK4/6i). The precise impact of pharmacologic inhibition of CDK2 is not known due to the lack of selective CDK2 inhibitors. Here we describe INX-315, a novel and potent CDK2 inhibitor with high selectivity over other CDK family members.

Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation.

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-β-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (HS) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death.

Ribosomal proteins and human diseases: molecular mechanisms and targeted therapy.

Ribosome biogenesis and protein synthesis are fundamental rate-limiting steps for cell growth and proliferation. The ribosomal proteins (RPs), comprising the structural parts of the ribosome, are essential for ribosome assembly and function. In addition to their canonical ribosomal functions, multiple RPs have extra-ribosomal functions including activation of p53-dependent or p53-independent pathways in response to stress, resulting in cell cycle arrest and apoptosis.

The RNA polymerase I transcription inhibitor CX-5461 cooperates with topoisomerase 1 inhibition by enhancing the DNA damage response in homologous recombination-proficient high-grade serous ovarian cancer.

Background: Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease.

Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis.

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our previous first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single-agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured.

CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo.

Oncogene-induced senescence: From biology to therapy.

Oncogene-induced senescence (OIS) is a powerful intrinsic tumor-suppressive mechanism, arresting cell cycle progression upon oncogene-activating genomic alterations. The discovery and characterization of the senescence-associated secretome unveiled a rich additional complexity to the senescence phenotype, including extrinsic impacts on the microenvironment and engagement of the immune response. Emerging evidence suggests that senescence phenotypes vary depending on the oncogenic stimulus.

Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation.

A functional genetic screen defines the AKT-induced senescence signaling network.

Exquisite regulation of PI3K/AKT/mTORC1 signaling is essential for homeostatic control of cell growth, proliferation, and survival. Aberrant activation of this signaling network is an early driver of many sporadic human cancers. Paradoxically, sustained hyperactivation of the PI3K/AKT/mTORC1 pathway in nontransformed cells results in cellular senescence, which is a tumor-suppressive mechanism that must be overcome to promote malignant transformation.

Cystathionine -Synthase in Physiology and Cancer.

Cystathionine -synthase (CBS) regulates homocysteine metabolism and contributes to hydrogen sulfide (HS) biosynthesis through which it plays multifunctional roles in the regulation of cellular energetics, redox status, DNA methylation, and protein modification. Inactivating mutations in CBS contribute to the pathogenesis of the autosomal recessive disease CBS-deficient homocystinuria. Recent studies demonstrating that CBS promotes colon and ovarian cancer growth in preclinical models highlight a newly identified oncogenic role for CBS.

The Potential of Targeting Ribosome Biogenesis in High-Grade Serous Ovarian Cancer.

Overall survival for patients with ovarian cancer (OC) has shown little improvement for decades meaning new therapeutic options are critical. OC comprises multiple histological subtypes, of which the most common and aggressive subtype is high-grade serous ovarian cancer (HGSOC). HGSOC is characterized by genomic structural variations with relatively few recurrent somatic mutations or dominantly acting oncogenes that can be targeted for the development of novel therapies.

Combining High-Content Imaging and Phenotypic Classification Analysis of Senescence-Associated Beta-Galactosidase Staining to Identify Regulators of Oncogene-Induced Senescence.

Hyperactivation of the PI3K/AKT/mTORC1 signaling pathway is a hallmark of the majority of sporadic human cancers. Paradoxically, chronic activation of this pathway in nontransformed cells promotes senescence, which acts as a significant barrier to malignant progression. Understanding how this oncogene-induced senescence is maintained in nontransformed cells and conversely how it is subverted in cancer cells will provide insight into cancer development and potentially identify novel therapeutic targets.

Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling.

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne).

β2-adrenoceptor signaling regulates invadopodia formation to enhance tumor cell invasion.

Introduction: For efficient metastatic dissemination, tumor cells form invadopodia to degrade and move through three-dimensional extracellular matrix. However, little is known about the conditions that favor invadopodia formation. Here, we investigated the effect of β-adrenoceptor signaling - which allows cells to respond to stress neurotransmitters - on the formation of invadopodia and examined the effect on tumor cell invasion.

IL2 Inducible T-cell Kinase, a Novel Therapeutic Target in Melanoma.

Purpose: IL2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared with nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation.

Experimental Design: An ITK-specific monoclonal antibody was used to probe sections from deidentified, formalin-fixed paraffin-embedded tumor blocks or cell line arrays and ITK was visualized by IHC.

LKB1 loss in melanoma disrupts directional migration toward extracellular matrix cues.

Somatic inactivation of the serine/threonine kinase gene STK11/LKB1/PAR-4 occurs in a variety of cancers, including ∼10% of melanoma. However, how the loss of LKB1 activity facilitates melanoma invasion and metastasis remains poorly understood. In LKB1-null cells derived from an autochthonous murine model of melanoma with activated Kras and Lkb1 loss and matched reconstituted controls, we have investigated the mechanism by which LKB1 loss increases melanoma invasive motility.

Intravital imaging of a spheroid-based orthotopic model of melanoma in the mouse ear skin.

Multiphoton microscopy is a powerful tool that enables the visualization of fluorescently tagged tumor cells and their stromal interactions within tissues in vivo. We have developed an orthotopic model of implanting multicellular melanoma tumor spheroids into the dermis of the mouse ear skin without the requirement for invasive surgery. Here, we demonstrate the utility of this approach to observe the primary tumor, single cell actin dynamics, and tumor-associated vasculature.

LKB1/STK11 inactivation leads to expansion of a prometastatic tumor subpopulation in melanoma.

Germline mutations in LKB1 (STK11) are associated with the Peutz-Jeghers syndrome (PJS), which includes aberrant mucocutaneous pigmentation, and somatic LKB1 mutations occur in 10% of cutaneous melanoma. By somatically inactivating Lkb1 with K-Ras activation (±p53 loss) in murine melanocytes, we observed variably pigmented and highly metastatic melanoma with 100% penetrance. LKB1 deficiency resulted in increased phosphorylation of the SRC family kinase (SFK) YES, increased expression of WNT target genes, and expansion of a CD24(+) cell population, which showed increased metastatic behavior in vitro and in vivo relative to isogenic CD24(-) cells.

Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin.

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg(30)) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo.

Unraveling the enigma: progress towards understanding the coronin family of actin regulators.

Coronins are a conserved family of actin cytoskeleton regulators that promote cell motility and modulate other actin-dependent processes. Although these proteins have been known for 20 years, substantial progress has been made in the past 5 years towards their understanding. In this review, we examine this progress, place it into the context of what was already known, and pose several questions that remain to be addressed.

Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK).

The coordinated and dynamic regulation of adhesions is required for cell migration. We demonstrated previously that limited proteolysis of talin1 by the calcium-dependent protease calpain 2 plays a critical role in adhesion disassembly in fibroblasts (Franco, S. J.

FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion.

Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells.

Calpain 2 and PTP1B function in a novel pathway with Src to regulate invadopodia dynamics and breast cancer cell invasion.

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity.

Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis.

Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility.

Integrins in cell migration.

Integrins are cell-surface adhesion receptors that play a central role in regulating cell migration by mediating interactions between the extracellular matrix and the actin cytoskeleton. Substantial progress has been made in understanding the mechanisms by which the formation and breakdown of adhesions are regulated. Here we describe general methods used to study integrin-mediated cell migration.