Cancer selective cell death induction by a bivalent CK2 inhibitor targeting the ATP site and the allosteric αD pocket.

Although the involvement of protein kinase CK2 in cancer is well-documented, there is a need for selective CK2 inhibitors suitable for investigating CK2 specific roles in cancer-related biological pathways and further exploring its therapeutic potential. Here, we report the discovery of AB668, an outstanding selective inhibitor that binds CK2 through a bivalent mode, interacting both at the ATP site and an allosteric αD pocket unique to CK2. Using caspase activation assay, live-cell imaging, and transcriptomic analysis, we have compared the effects of this bivalent inhibitor to representative ATP-competitive inhibitors, CX-4945, and SGC-CK2-1. Our results show that in contrast to CX-4945 or SGC-CK2-1, AB668, by targeting the CK2 αD pocket, has a distinct mechanism of action regarding its anti-cancer activity, inducing apoptotic cell death in several cancer cell lines and stimulating distinct biological pathways in renal cell carcinoma.

A new scaffold-free tumoroid model provides a robust preclinical tool to investigate invasion and drug response in Renal Cell Carcinoma.

Clear cell Renal Cell Carcinoma (ccRCC) is one of the most prevalent kidney cancers, which is often asymptomatic and thus discovered at a metastatic state (mRCC). mRCC are highly heterogeneous tumors composed of subclonal populations that lead to poor treatment response rate. Several recent works explored the potential of ccRCC tumoroids culture derived from patients. However, these models were produced following a scaffold-based method using collagen I or Matrigel that exhibit lot variability and whose complexity could induce treatment response modifications and phenotypic alterations. Following the observation that ccRCC tumoroids can create their own niche by secreting extracellular matrix components, we developed the first scaffold-free tumoroid model of ccRCC tumors. Tumoroids from mice as well as from human tumors were generated with high success rate (≥90%) using a magnetic suspension method and standard culture media. Immunofluorescence analysis revealed their self-organization capacities to maintain multiple tumor-resident cell types, including endothelial progenitor cells. Transcriptomic analysis showed the reproducibility of the method highlighting that the majority of gene expression patterns was conserved in tumoroids compared to their matching tumor tissue. Moreover, this model enables to evaluate drug effects and invasiveness of renal cancer cells in a 3D context, providing a robust preclinical tool for drug screening and biomarker assessment in line with alternative ex vivo methods like tumor tissue slice culture or in vivo xenograft models.

Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction.

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL- cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug-gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

A Nucleolar Isoform of the Drosophila Ubiquitin Specific Protease dUSP36 Regulates MYC-Dependent Cell Growth.

The c-Myc oncogene is a transcription factor that regulates the expression of a very large set of genes mainly involved in cell growth and proliferation. It is overexpressed in more than 70% of human cancers, illustrating the importance of keeping its levels and activity under control. The ubiquitin proteasome system is a major regulator of MYC levels in humans as well as in model organisms such as Drosophila melanogaster. Although the E3 ligases that promote MYC ubiquitination have been largely investigated, the identity and the role of the deubiquitinating enzymes, which counteract their action is only beginning to be unraveled. Using isoform-specific CRISPR-Cas9 mutagenesis, we show that the Drosophila homolog of the Ubiquitin Specific Protease USP36 has different isoforms with specific sub-cellular localizations and that the nucleolar dUSP36-D isoform is specifically required for cell and organismal growth. We also demonstrate that this isoform interacts with dMYC and the E3 ligase AGO and regulates their stability and ubiquitination levels. Furthermore, we show that dUSP36 is ubiquitinated by AGO and is able to self-deubiquitinate. Finally, we provide in vivo evidence supporting the functional relevance of these regulatory relationships. Together these results reveal that dMYC, AGO and dUSP36 form a tripartite, evolutionary conserved complex that acts as a regulatory node to control dMYC protein levels.

Ex-Vivo Treatment of Tumor Tissue Slices as a Predictive Preclinical Method to Evaluate Targeted Therapies for Patients with Renal Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the third type of urologic cancer. At time of diagnosis, 30% of cases are metastatic with no effect of chemotherapy or radiotherapy. Current targeted therapies lead to a high rate of relapse and resistance after a short-term response. Thus, a major hurdle in the development and use of new treatments for ccRCC is the lack of good pre-clinical models that can accurately predict the efficacy of new drugs and allow the stratification of patients into the correct treatment regime. Here, we describe different 3D cultures models of ccRCC, emphasizing the feasibility and the advantage of ex-vivo treatment of fresh, surgically resected human tumor slice cultures of ccRCC as a robust preclinical model for identifying patient response to specific therapeutics. Moreover, this model based on precision-cut tissue slices enables histopathology measurements as tumor architecture is retained, including the spatial relationship between the tumor and tumor-infiltrating lymphocytes and the stromal components. Our data suggest that acute treatment of tumor tissue slices could represent a benchmark of further exploration as a companion diagnostic tool in ccRCC treatment and a model to develop new therapeutic drugs.

Combined inhibition of PI3K and Src kinases demonstrates synergistic therapeutic efficacy in clear-cell renal carcinoma.

Potent inhibitors of PI3K (GDC-0941) and Src (Saracatinib) exhibit as individual agents, excellent oral anticancer activity in preclinical models and have entered phase II clinical trials in various cancers. We found that PI3K and Src kinases are dysregulated in clear cell renal carcinomas (ccRCCs), an aggressive disease without effective targeted therapies. In this study we addressed this challenge by testing GDC-0941 and Saracatinib as either single agents or in combination in ccRCC cell lines, as well as in mouse and PDX models. Our findings demonstrate that combined inhibition of PI3K and Src impedes cell growth and invasion and induces cell death of renal carcinoma cells providing preclinical evidence for a pairwise combination of these anticancer drugs as a rational strategy to improve renal cancer treatment.

Novel synthetic pharmacophores inducing a stabilization of cellular microtubules.

Microtubule drugs have been widely used in cancer chemotherapies. Although microtubules are subject to regulation by signal transduction mechanisms, their pharmacological modulation has so far relied on compounds that bind to the tubulin subunit. Using a cell-based assay designed to probe the microtubule polymerization status, we identified two pharmacophores, CM09 and CM10, as cell-permeable microtubule stabilizing agents. These synthetic compounds do not affect the assembly state of purified microtubules in vitro but they profoundly suppress microtubule dynamics in vivo. Moreover, they exert cytotoxic effects on several cancer cell lines including multidrug resistant cell lines. Therefore, these classes of compounds represent novel attractive leads for cancer chemotherapy.

Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities.

BACKGROUND AND PURPOSE: Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell-permeable microtubule-targeting agents. EXPERIMENTAL APPROACH: Using a cell-based assay designed to probe for microtubule polymerization status, we screened a chemical library and identified two azaindole derivatives, CM01 and CM02, as cell-permeable microtubule-depolymerizing agents. The mechanism of the anti-tumour effects of these two compounds was further investigated both in vivo and in vitro. KEY RESULTS: CM01 and CM02 induced G2/M cell cycle arrest and exerted potent cytostatic effects on several cancer cell lines including multidrug-resistant (MDR) cell lines. In vitro experiments revealed that the azaindole derivatives inhibited tubulin polymerization and competed with colchicines for this effect, strongly indicating that tubulin is the cellular target of these azaindole derivatives. In vivo experiments, using a chicken chorioallantoic xenograft tumour assay, established that these compounds exert a potent anti-tumour effect. Furthermore, an assay probing the growth of vessels out of endothelial cell spheroids showed that CM01 and CM02 exert anti-angiogenic activities. CONCLUSIONS AND IMPLICATIONS: CM01 and CM02 are reversible microtubule-depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including MDR cells. They were also shown to inhibit angiogenesis and tumour growth in chorioallantoic breast cancer xenografts. Hence, these azaindole derivatives are attractive candidates for further preclinical investigations.

Pharmacological inhibition of LIM kinase stabilizes microtubules and inhibits neoplastic growth.

The emergence of tumor resistance to conventional microtubule-targeting drugs restricts their clinical use. Using a cell-based assay that recognizes microtubule polymerization status to screen for chemicals that interact with regulators of microtubule dynamics, we identified Pyr1, a cell permeable inhibitor of LIM kinase, which is the enzyme that phosphorylates and inactivates the actin-depolymerizing factor cofilin. Pyr1 reversibly stabilized microtubules, blocked actin microfilament dynamics, inhibited cell motility in vitro and showed anticancer properties in vivo, in the absence of major side effects. Pyr1 inhibition of LIM kinase caused a microtubule-stabilizing effect, which was independent of any direct effects on the actin cytoskeleton. In addition, Pyr1 retained its activity in multidrug-resistant cancer cells that were resistant to conventional microtubule-targeting agents. Our findings suggest that LIM kinase functions as a signaling node that controls both actin and microtubule dynamics. LIM kinase may therefore represent a targetable enzyme for cancer treatment.