High-Throughput DNA-Encoded Libraries Affinity Selection Platform for Binder Identification with Solid Support Protein Immobilization.

DNA-encoded libraries (DELs) have demonstrated to be one of the most powerful technologies within the ligand identification toolbox, widely used either in academia or biotech and pharma companies. DEL methodology utilizes affinity selection (AS) as the approach to interrogate the protein of interest for the identification of binders. Here we present a high-throughput, fully automated AS platform developed to fulfill industrial standards and compatible with different assay formats to improve the reproducibility of the AS process for DEL binders identification. This platform is flexible enough to virtually set aside all kinds of DELs and AS methods and conditions using immobilized proteins. It bears the two main immobilization methods to support of the proteins of interest: magnetic beads or resin tip columns. A combination of a broad variety of protocol options with a wide range of different experimental conditions can be set up with a throughput of 96 samples at the same time. In addition, small modifications of the protocols provide the platform with the versatility to run not only the routine DEL screens, but also test covalent libraries, the successful immobilization of the proteins of interest, and many other experiments that may be required. This versatile AS platform for DEL can be a powerful instrument for direct application of the technology in academic and industry settings.

TPX2 overexpression promotes sensitivity to dasatinib in breast cancer by activating YAP transcriptional signaling.

Chromosomal instability (CIN) is a hallmark of cancer aggressiveness, providing genetic plasticity and tumor heterogeneity that allows the tumor to evolve and adapt to stress conditions. CIN is considered a cancer therapeutic biomarker because healthy cells do not exhibit CIN. Despite recent efforts to identify therapeutic strategies related to CIN, the results obtained have been very limited. CIN is characterized by a genetic signature where a collection of genes, mostly mitotic regulators, are overexpressed in CIN-positive tumors, providing aggressiveness and poor prognosis. We attempted to identify new therapeutic strategies related to CIN genes by performing a drug screen, using cells that individually express CIN-associated genes in an inducible manner. We find that the overexpression of targeting protein for Xklp2 (TPX2) enhances sensitivity to the proto-oncogene c-Src (SRC) inhibitor dasatinib due to activation of the Yes-associated protein 1 (YAP) pathway. Furthermore, using breast cancer data from The Cancer Genome Atlas (TCGA) and a cohort of cancer-derived patient samples, we find that both TPX2 overexpression and YAP activation are present in a significant percentage of cancer tumor samples and are associated with poor prognosis; therefore, they are putative biomarkers for selection for dasatinib therapy.

Design, synthesis and validation of a new Crimped Head-Piece for DNA-Encoded libraries generation.

Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.

Crystal structure of active CDK4-cyclin D and mechanistic basis for abemaciclib efficacy.

Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.

Continuous treatment with abemaciclib leads to sustained and efficient inhibition of breast cancer cell proliferation.

Abemaciclib is an oral, selective cyclin-dependent kinase 4 & 6 inhibitor (CDK4 & 6i), approved for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC) as monotherapy for endocrine refractory disease, and with endocrine therapy (ET) for initial treatment and after progression on ET. Abemaciclib has also shown clinical activity in combination with ET in patients with high risk early BC (EBC). Here, we examined the preclinical attributes of abemaciclib and other CDK4 & 6i using biochemical and cell-based assays. In vitro, abemaciclib preferentially inhibited CDK4 kinase activity versus CDK6, resulting in inhibition of cell proliferation in a panel of BC cell lines with higher average potency than palbociclib or ribociclib. Abemaciclib showed activity regardless of HER2 amplification and phosphatidylinositol 3-kinase (PI3KCA) gene mutation status. In human bone marrow progenitor cells, abemaciclib showed lower impact on myeloid maturation than other CDK4 & 6i when tested at unbound concentrations similar to those observed in clinical trials. Continuous abemaciclib treatment provided profound inhibition of cell proliferation, and triggered senescence and apoptosis. These preclinical results support the unique efficacy and safety profile of abemaciclib observed in clinical trials.

Combined inhibition of PIM and CDK4/6 suppresses both mTOR signaling and Rb phosphorylation and potentiates PI3K inhibition in cancer cells.

Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation. TSC2 or PIK3CA mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways (Akt, PIM, and CDK4) to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of PIK3CA-mutant ER+ breast cancer.

Aurora A-Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy.

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

Cyclin dependent kinase (CDK) inhibitors as anticancer drugs: Recent advances (2015-2019).

Sustained proliferative capacity and gene dysregulation are hallmarks of cancer. In mammalian cells, cyclin-dependent kinases (CDKs) control critical cell cycle checkpoints and key transcriptional events in response to extracellular and intracellular signals leading to proliferation. Significant clinical activity for the treatment of hormone receptor positive metastatic breast cancer has been demonstrated by palbociclib, ribociclib and abemaciclib, dual CDK4/6 inhibitors recently FDA-approved. SY-1365, a CDK7 inhibitor has shown initial encouraging data in phase I for solid tumors treatment. These results have rejuvenated the CDKs research field. This review provides an overview of relevant advances on CDK inhibitor research since 2015 to 2019, with special emphasis on transcriptional CDK inhibitors, new emerging strategies such as target protein degradation and compounds under clinical evaluation.

Aurora A Kinase Inhibition Is Synthetic Lethal with Loss of the RB1 Tumor Suppressor Gene.

Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and leads to durable regression of RB1 mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.

Genomic Aberrations that Activate D-type Cyclins Are Associated with Enhanced Sensitivity to the CDK4 and CDK6 Inhibitor Abemaciclib.

Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3'UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.

Preclinical characterization of abemaciclib in hormone receptor positive breast cancer.

Abemaciclib is an ATP-competitive, reversible kinase inhibitor selective for CDK4 and CDK6 that has shown antitumor activity as a single agent in hormone receptor positive (HR+) metastatic breast cancer in clinical trials. Here, we examined the mechanistic effects of abemaciclib treatment using in vitro and in vivo breast cancer models. Treatment of estrogen receptor positive (ER+) breast cancer cells with abemaciclib alone led to a decrease in phosphorylation of Rb, arrest at G1, and a decrease in cell proliferation. Moreover, abemaciclib exposure led to durable inhibition of pRb, TopoIIα expression and DNA synthesis, which were maintained after drug removal. Treatment of ER+ breast cancer cells also led to a senescence response as indicated by accumulation of ß-galactosidase, formation of senescence-associated heterochromatin foci, and a decrease in FOXM1 positive cells. Continuous exposure to abemaciclib altered breast cancer cell metabolism and induced apoptosis. In a xenograft model of ER+ breast cancer, abemaciclib monotherapy caused regression of tumor growth. Overall these data indicate that abemaciclib is a CDK4 and CDK6 inhibitor that, as a single agent, blocks breast cancer cell progression, and upon longer treatment can lead to sustained antitumor effects through the induction of senescence, apoptosis, and alteration of cellular metabolism.

Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures.

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

Cyclin dependent kinase (CDK) inhibitors as anticancer drugs.

Sustained proliferative capacity is a hallmark of cancer. In mammalian cells proliferation is controlled by the cell cycle, where cyclin-dependent kinases (CDKs) regulate critical checkpoints. CDK4 and CDK6 are considered highly validated anticancer drug targets due to their essential role regulating cell cycle progression at the G1 restriction point. This review provides an overview of recent advances on cyclin dependent kinase inhibitors in general with special emphasis on CDK4 and CDK6 inhibitors and compounds under clinical evaluation. Chemical structures, structure activity relationships, and relevant preclinical properties will be described.

Phenotypic Screening Approaches to Develop Aurora Kinase Inhibitors: Drug Discovery Perspectives.

Targeting mitotic regulators as a strategy to fight cancer implies the development of drugs against key proteins, such as Aurora-A and -B. Current drugs, which target mitosis through a general mechanism of action (stabilization/destabilization of microtubules), have several side effects (neutropenia, alopecia, and emesis). Pharmaceutical companies aim at avoiding these unwanted effects by generating improved and selective drugs that increase the quality of life of the patients. However, the development of these drugs is an ambitious task that involves testing thousands of compounds through biochemical and cell-based assays. In addition, molecules usually target complex biological processes, involving several proteins and different molecular pathways, further emphasizing the need for high-throughput screening techniques and multiplexing technologies in order to identify drugs with the desired phenotype. We will briefly describe two multiplexing technologies [high-content imaging (HCI) and flow cytometry] and two key processes for drug discovery research (assay development and validation) following our own published industry quality standards. We will further focus on HCI as a useful tool for phenotypic screening and will provide a concrete example of HCI assay to detect Aurora-A or -B selective inhibitors discriminating the off-target effects related to the inhibition of other cell cycle or non-cell cycle key regulators. Finally, we will describe other assays that can help to characterize the in vitro pharmacology of the inhibitors.

Preclinical characterization of the CDK4/6 inhibitor LY2835219: in-vivo cell cycle-dependent/independent anti-tumor activities alone/in combination with gemcitabine.

The G1 restriction point is critical for regulating the cell cycle and is controlled by the Rb pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of retinoblastoma protein (Rb) by CDK4/6, which are highly validated cancer drug targets. We present the identification and characterization of a potent CDK4/6 inhibitor, LY2835219. LY2835219 inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show LY2835219 is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of LY2835219 inhibits tumor growth in human tumor xenografts representing different histologies in tumor-bearing mice. LY2835219 is effective and well tolerated when administered up to 56 days in immunodeficient mice without significant loss of body weight or tumor outgrowth. In calu-6 xenografts, LY2835219 in combination with gemcitabine enhanced in vivo antitumor activity without a G1 cell cycle arrest, but was associated with a reduction of ribonucleotide reductase expression. These results suggest LY2835219 may be used alone or in combination with standard-of-care cytotoxic therapy. In summary, we have identified a potent, orally active small-molecule inhibitor of CDK4/6 that is active in xenograft tumors. LY2835219 is currently in clinical development.

Multiparametric cell-based assay for the evaluation of transcription inhibition by high-content imaging.

Loss of normal cell cycle regulation is a hallmark of human cancer. Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and have been actively pursued as promising therapeutic targets. Likewise, members of the CDK family are functionally related to transcriptional modulation, a molecular pathway suitable for therapeutic intervention. We used a set of 2500 compounds in the U2OS cell line to evaluate its effect in the cell division process. Interestingly, out of this analysis, we identified a subpopulation of compounds that are able to inhibit RNA polymerase activity, thus interfering with gene transcription processes. After this finding, we developed, validated, and fully automated a multiparameter high-content imaging (HCI) assay to measure phosphorylation of the RNA polymerase II carboxyl terminal domain (pCTD). Simultaneously, we measured both the DNA content and cell proliferation index in the treated cells. The linear regression analysis comparing the IC50 for pCTD and the 4N EC50 for DNA content or IC50 for cell proliferation showed an excellent agreement (r (2) = 0.84 and r (2) = 0.94, respectively). Our results confirm that this method allows discriminating between cell cycle and transcription inhibition and confirms HCI as a powerful technology for the identification of compounds with an effective and selective pathway phenotype.

Splicing regulation at the second catalytic step by Sex-lethal involves 3' splice site recognition by SPF45.

The Drosophila protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. An unusual sequence arrangement of two AG dinucleotides and an intervening polypyrimidine (Py)-tract at the 3' end of intron 2 is important for Sxl autoregulation. Here we show that U2AF interacts with the Py-tract and downstream AG, whereas the spliceosomal protein SPF45 interacts with the upstream AG and activates it for the second catalytic step of the splicing reaction. SPF45 represents a new class of second step factors, and its interaction with SXL blocks splicing at the second step. These results are in contrast with other known mechanisms of splicing regulation, which target early events of spliceosome assembly. A similar role for SPF45 is demonstrated in the activation of a cryptic 3' ss generated by a mutation that causes human beta-thalassemia.