Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules.

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

Discovery of MFH290: A Potent and Highly Selective Covalent Inhibitor for Cyclin-Dependent Kinase 12/13.

Genetic depletion of cyclin-dependent kinase 12 (CDK12) or selective inhibition of an analog-sensitive CDK12 reduces DNA damage repair gene expression, but selective inhibition of endogenous CDK12 is difficult. Here, we report the development of MFH290, a novel cysteine (Cys)-directed covalent inhibitor of CDK12/13. MFH290 forms a covalent bond with Cys-1039 of CDK12, exhibits excellent kinome selectivity, inhibits the phosphorylation of serine-2 in the C-terminal domain (CTD) of RNA-polymerase II (Pol II), and reduces the expression of key DNA damage repair genes. Importantly, these effects were demonstrated to be CDK12-dependent as mutation of Cys-1039 rendered the kinase refractory to MFH290 and restored Pol II CTD phosphorylation and DNA damage repair gene expression. Consistent with its effect on DNA damage repair gene expression, MFH290 augments the antiproliferative effect of the PARP inhibitor olaparib.

Discovery of a selective inhibitor of doublecortin like kinase 1.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

CDK7 Inhibition Potentiates Genome Instability Triggering Anti-tumor Immunity in Small Cell Lung Cancer.

Cyclin-dependent kinase 7 (CDK7) is a central regulator of the cell cycle and gene transcription. However, little is known about its impact on genomic instability and cancer immunity. Using a selective CDK7 inhibitor, YKL-5-124, we demonstrated that CDK7 inhibition predominately disrupts cell-cycle progression and induces DNA replication stress and genome instability in small cell lung cancer (SCLC) while simultaneously triggering immune-response signaling. These tumor-intrinsic events provoke a robust immune surveillance program elicited by T cells, which is further enhanced by the addition of immune-checkpoint blockade. Combining YKL-5-124 with anti-PD-1 offers significant survival benefit in multiple highly aggressive murine models of SCLC, providing a rationale for new combination regimens consisting of CDK7 inhibitors and immunotherapies.

Development of a Selective CDK7 Covalent Inhibitor Reveals Predominant Cell-Cycle Phenotype.

Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.

Targeting MYC dependency in ovarian cancer through inhibition of CDK7 and CDK12/13.

High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.

The dTAG system for immediate and target-specific protein degradation.

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic Isoform.

The SRPK family of kinases regulates pre-mRNA splicing by phosphorylating serine/arginine (SR)-rich splicing factors, signals splicing control in response to extracellular stimuli, and contributes to tumorigenesis, suggesting that these splicing kinases are potential therapeutic targets. Here, we report the development of the first irreversible SRPK inhibitor, SRPKIN-1, which is also the first kinase inhibitor that forms a covalent bond with a tyrosine phenol group in the ATP-binding pocket. Kinome-wide profiling demonstrates its selectivity for SRPK1/2, and SRPKIN-1 attenuates SR protein phosphorylation at submicromolar concentrations. Vascular endothelial growth factor (VEGF) is a known target for SRPK-regulated splicing and, relative to the first-generation SRPK inhibitor SRPIN340 or small interfering RNA-mediated SRPK knockdown, SRPKIN-1 is more potent in converting the pro-angiogenic VEGF-A165a to the anti-angiogenic VEGF-A165b isoform and in blocking laser-induced neovascularization in a murine retinal model. These findings encourage further development of SRPK inhibitors for treatment of age-related macular degeneration.

Extracellular signal regulated kinase 5 and inflammasome in progression of mesothelioma.

Malignant mesothelioma is an aggressive cancer in desperate need of treatment. We have previously shown that extracellular signaling regulated kinase 5 (ERK5) plays an important role in mesothelioma pathogenesis using ERK5 silenced human mesothelioma cells exhibiting significantly reduced tumor growth in immunocompromised mice. Here, we used a specific ERK 5 inhibitor, XMD8-92 in various in vitro and in vivo models to demonstrate that inhibition of ERK5 can slow down mesothelioma tumorigenesis. First, we show a dose dependent toxicity of XMD8-92 to 2 human mesothelioma cell lines growing as a monolayer. We also demonstrate the inhibition of ERK5 phosphorylation in various human mesothelioma cell lines by XMD8-92. We further confirmed the toxicity of XMD8-92 towards mesothelioma cell lines grown as spheroids in a 3-D model as well as in intraperitoneal (immune-competent) and intrapleural (immune-deficient) mouse models with and without chemotherapeutic drugs. To ascertain the mechanism, we explored the role of the nod-like receptor family member containing a pyrin domain 3 (NLRP3) inflammasome in the process. We found XMD8-92 attenuated naïve and chemotherapeutic-induced inflammasome priming and activation in mesothelioma cells. It can thus be concluded that ERK5 inhibition attenuates mesothelioma tumor growth and this phenomenon in part is regulated by the inflammasome.

EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma.

Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity.

Suppression of Adaptive Responses to Targeted Cancer Therapy by Transcriptional Repression.

Acquired drug resistance is a major factor limiting the effectiveness of targeted cancer therapies. Targeting tumors with kinase inhibitors induces complex adaptive programs that promote the persistence of a fraction of the original cell population, facilitating the eventual outgrowth of inhibitor-resistant tumor clones. We show that the addition of a newly identified CDK7/12 inhibitor, THZ1, to targeted therapy enhances cell killing and impedes the emergence of drug-resistant cell populations in diverse cellular and in vivo cancer models. We propose that targeted therapy induces a state of transcriptional dependency in a subpopulation of cells poised to become drug tolerant, which THZ1 can exploit by blocking dynamic transcriptional responses, promoting remodeling of enhancers and key signaling outputs required for tumor cell survival in the setting of targeted therapy. These findings suggest that the addition of THZ1 to targeted therapies is a promising broad-based strategy to hinder the emergence of drug-resistant cancer cell populations.Significance: CDK7/12 inhibition prevents active enhancer formation at genes, promoting resistance emergence in response to targeted therapy, and impedes the engagement of transcriptional programs required for tumor cell survival. CDK7/12 inhibition in combination with targeted cancer therapies may serve as a therapeutic paradigm for enhancing the effectiveness of targeted therapies. Cancer Discov; 8(1); 59-73. ©2017 AACR.See related commentary by Carugo and Draetta, p. 17This article is highlighted in the In This Issue feature, p. 1.

Inflammasome Modulation by Chemotherapeutics in Malignant Mesothelioma.

Malignant mesothelioma (MM) is a fatal disease in dire need of therapy. The role of inflammasomes in cancer is not very well studied, however, literature supports both pro-and anti-tumorigenic effects of inflammasomes on cancer depending upon the type of cancer. Asbestos is a causative agent for MM and we have shown before that it causes inflammasome priming and activation in mesothelial cells. MM tumor cells/tissues showed decreased levels of inflammasome components like NLRP3 and caspase-1 as compared to human mesothelial cells or normal tissue counterpart of tumor. Based on our preliminary findings we hypothesized that treatment of MMs with chemotherapeutic drugs may elevate the levels of NLRP3 and caspase-1 resulting in increased cell death by pyroptosis while increasing the levels of IL-1ß and other pro-inflammatory molecules. Therefore, a combined strategy of chemotherapeutic drug and IL-1R antagonist may play a beneficial role in MM therapy. To test our hypothesis we used two human MM tumor cell lines (Hmeso, H2373) and two chemotherapeutic drugs (doxorubicin, cisplatin). Through a series of experiments we showed that both chemotherapeutic drugs caused increases in NLRP3 levels, caspase-1 activation, pyroptosis and pro-inflammatory molecules released from MM cells. In vivo studies using SCID mice and Hmeso cells showed that tumors were smaller in combined treatment group of cisplatin and IL-1R antagonist (Anakinra) as compared to cisplatin alone or untreated control groups. Taken together our study suggests that chemotherapeutic drugs in combination with IL-1R antagonist may have a beneficial role in MM treatment.