HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.

Native Size-Exclusion Chromatography-Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes.

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.

Evolution of kinase polypharmacology across HSP90 drug discovery.

Most small molecules interact with several target proteins but this polypharmacology is seldom comprehensively investigated or explicitly exploited during drug discovery. Here, we use computational and experimental methods to identify and systematically characterize the kinase cross-pharmacology of representative HSP90 inhibitors. We demonstrate that the resorcinol clinical candidates ganetespib and, to a lesser extent, luminespib, display unique off-target kinase pharmacology as compared with other HSP90 inhibitors. We also demonstrate that polypharmacology evolved during the optimization to discover luminespib and that the hit, leads, and clinical candidate all have different polypharmacological profiles. We therefore recommend the computational and experimental characterization of polypharmacology earlier in drug discovery projects to unlock new multi-target drug design opportunities.

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.

Fadraciclib (CYC065), a novel CDK inhibitor, targets key pro-survival and oncogenic pathways in cancer.

Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546).

A Genome-scale CRISPR Screen Identifies the ERBB and mTOR Signaling Networks as Key Determinants of Response to PI3K Inhibition in Pancreatic Cancer.

KRAS mutation is a key driver of pancreatic cancer and PI3K pathway activity is an additional requirement for Kras-induced tumorigenesis. Clinical trials of PI3K pathway inhibitors in pancreatic cancer have shown limited responses. Understanding the molecular basis for this lack of efficacy may direct future treatment strategies with emerging PI3K inhibitors. We sought new therapeutic approaches that synergize with PI3K inhibitors through pooled CRISPR modifier genetic screening and a drug combination screen. ERBB family receptor tyrosine kinase signaling and mTOR signaling were key modifiers of sensitivity to alpelisib and pictilisib. Inhibition of the ERBB family or mTOR was synergistic with PI3K inhibition in spheroid, stromal cocultures. Near-complete loss of ribosomal S6 phosphorylation was associated with synergy. Genetic alterations in the ERBB-PI3K signaling axis were associated with decreased survival of patients with pancreatic cancer. Suppression of the PI3K/mTOR axis is potentiated by dual PI3K and ERBB family or mTOR inhibition. Surprisingly, despite the presence of oncogenic KRAS, thought to bestow independence from receptor tyrosine kinase signaling, inhibition of the ERBB family blocks downstream pathway activation and synergizes with PI3K inhibitors. Further exploration of these therapeutic combinations is warranted for the treatment of pancreatic cancer.

CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells.

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

Correction: Signalling involving MET and FAK supports cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The kinase polypharmacology landscape of clinical PARP inhibitors.

Polypharmacology plays an important role in defining response and adverse effects of drugs. For some mechanisms, experimentally mapping polypharmacology is commonplace, although this is typically done within the same protein class. Four PARP inhibitors have been approved by the FDA as cancer therapeutics, yet a precise mechanistic rationale to guide clinicians on which to choose for a particular patient is lacking. The four drugs have largely similar PARP family inhibition profiles, but several differences at the molecular and clinical level have been reported that remain poorly understood. Here, we report the first comprehensive characterization of the off-target kinase landscape of four FDA-approved PARP drugs. We demonstrate that all four PARP inhibitors have a unique polypharmacological profile across the kinome. Niraparib and rucaparib inhibit DYRK1s, CDK16 and PIM3 at clinically achievable, submicromolar concentrations. These kinases represent the most potently inhibited off-targets of PARP inhibitors identified to date and should be investigated further to clarify their potential implications for efficacy and safety in the clinic. Moreover, broad kinome profiling is recommended for the development of PARP inhibitors as PARP-kinase polypharmacology could potentially be exploited to modulate efficacy and side-effect profiles.

Signalling involving MET and FAK supports cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases.

Deregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer; yet, inhibitors against these kinases are currently used only in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and the mechanisms that may undermine such dependency are poorly understood. Here, we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2 activation, involving as critical mechanistic events loss of the CDKI p21CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates the proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic.

Dissecting mechanisms of resistance to targeted drug combination therapy in human colorectal cancer.

Genomic alterations in cancer cells result in vulnerabilities that clinicians can exploit using molecularly targeted drugs, guided by knowledge of the tumour genotype. However, the selective activity of these drugs exerts an evolutionary pressure on cancers that can result in the outgrowth of resistant clones. Use of rational drug combinations can overcome resistance to targeted drugs, but resistance may eventually develop to combinatorial therapies. We selected MAPK- and PI3K-pathway inhibition in colorectal cancer as a model system to dissect out mechanisms of resistance. We focused on these signalling pathways because they are frequently activated in colorectal tumours, have well-characterised mutations and are clinically relevant. By treating a panel of 47 human colorectal cancer cell lines with a combination of MEK- and PI3K-inhibitors, we observe a synergistic inhibition of growth in almost all cell lines. Cells with KRAS mutations are less sensitive to PI3K inhibition, but are particularly sensitive to the combined treatment. Colorectal cancer cell lines with inherent or acquired resistance to monotherapy do not show a synergistic response to the combination treatment. Cells that acquire resistance to an MEK-PI3K inhibitor combination treatment still respond to an ERK-PI3K inhibitor regimen, but subsequently also acquire resistance to this combination treatment. Importantly, the mechanisms of resistance to MEK and PI3K inhibitors observed, MEK1/2 mutation or loss of PTEN, are similar to those detected in the clinic. ERK inhibitors may have clinical utility in overcoming resistance to MEK inhibitor regimes; however, we find a recurrent active site mutation of ERK2 that drives resistance to ERK inhibitors in mono- or combined regimens, suggesting that resistance will remain a hurdle. Importantly, we find that the addition of low concentrations of the BCL2-family inhibitor navitoclax to the MEK-PI3K inhibitor regimen improves the synergistic interaction and blocks the acquisition of resistance.

Small-molecule targeting of brachyury transcription factor addiction in chordoma.

Chordoma is a primary bone cancer with no approved therapy1. The identification of therapeutic targets in this disease has been challenging due to the infrequent occurrence of clinically actionable somatic mutations in chordoma tumors2,3. Here we describe the discovery of therapeutically targetable chordoma dependencies via genome-scale CRISPR-Cas9 screening and focused small-molecule sensitivity profiling. These systematic approaches reveal that the developmental transcription factor T (brachyury; TBXT) is the top selectively essential gene in chordoma, and that transcriptional cyclin-dependent kinase (CDK) inhibitors targeting CDK7/12/13 and CDK9 potently suppress chordoma cell proliferation. In other cancer types, transcriptional CDK inhibitors have been observed to downregulate highly expressed, enhancer-associated oncogenic transcription factors4,5. In chordoma, we find that T is associated with a 1.5-Mb region containing 'super-enhancers' and is the most highly expressed super-enhancer-associated transcription factor. Notably, transcriptional CDK inhibition leads to preferential and concentration-dependent downregulation of cellular brachyury protein levels in all models tested. In vivo, CDK7/12/13-inhibitor treatment substantially reduces tumor growth. Together, these data demonstrate small-molecule targeting of brachyury transcription factor addiction in chordoma, identify a mechanism of T gene regulation that underlies this therapeutic strategy, and provide a blueprint for applying systematic genetic and chemical screening approaches to discover vulnerabilities in genomically quiet cancers.

Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.

Patient-derived organoids (PDOs) have recently emerged as robust preclinical models; however, their potential to predict clinical outcomes in patients has remained unclear. We report on a living biobank of PDOs from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients recruited in phase 1/2 clinical trials. Phenotypic and genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. Molecular profiling of tumor organoids was matched to drug-screening results, suggesting that PDOs could complement existing approaches in defining cancer vulnerabilities and improving treatment responses. We compared responses to anticancer agents ex vivo in organoids and PDO-based orthotopic mouse tumor xenograft models with the responses of the patients in clinical trials. Our data suggest that PDOs can recapitulate patient responses in the clinic and could be implemented in personalized medicine programs.

Oncogenic RAC1 and NRAS drive resistance to endoplasmic reticulum stress through MEK/ERK signalling.

Cancer cells are able to survive under conditions that cause endoplasmic reticulum stress (ER-stress), and can adapt to this stress by upregulating cell-survival signalling pathways and down-regulating apoptotic pathways. The cellular response to ER-stress is controlled by the unfolded protein response (UPR). Small Rho family GTPases are linked to many cell responses including cell growth and apoptosis. In this study, we investigate the function of small GTPases in cell survival under ER-stress. Using siRNA screening we identify that RAC1 promotes cell survival under ER-stress in cells with an oncogenic N92I RAC1 mutation. We uncover a novel connection between the UPR and N92I RAC1, whereby RAC1 attenuates phosphorylation of EIF2S1 under ER-stress and drives over-expression of ATF4 in basal conditions. Interestingly, the UPR connection does not drive resistance to ER-stress, as knockdown of ATF4 did not affect this. We further investigate cancer-associated kinase signalling pathways and show that RAC1 knockdown reduces the activity of AKT and ERK, and using a panel of clinically important kinase inhibitors, we uncover a role for MEK/ERK, but not AKT, in cell viability under ER-stress. A known major activator of ERK phosphorylation in cancer is oncogenic NRAS and we show that knockdown of NRAS in cells, which bear a Q61 NRAS mutation, sensitises to ER-stress. These findings highlight a novel mechanism for resistance to ER-stress through oncogenic activation of MEK/ERK signalling by small GTPases.

Molecular profiling and combinatorial activity of CCT068127: a potent CDK2 and CDK9 inhibitor.

Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small-molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer.

In vitro nuclear magnetic resonance spectroscopy metabolic biomarkers for the combination of temozolomide with PI3K inhibition in paediatric glioblastoma cells.

Recent experimental data showed that the PI3K pathway contributes to resistance to temozolomide (TMZ) in paediatric glioblastoma and that this effect is reversed by combination treatment of TMZ with a PI3K inhibitor. Our aim is to assess whether this combination results in metabolic changes that are detectable by nuclear magnetic resonance (NMR) spectroscopy, potentially providing metabolic biomarkers for PI3K inhibition and TMZ combination treatment. Using two genetically distinct paediatric glioblastoma cell lines, SF188 and KNS42, in vitro 1H-NMR analysis following treatment with the dual pan-Class I PI3K/mTOR inhibitor PI-103 resulted in a decrease in lactate and phosphocholine (PC) levels (P<0.02) relative to control. In contrast, treatment with TMZ caused an increase in glycerolphosphocholine (GPC) levels (P≤0.05). Combination of PI-103 with TMZ showed metabolic effects of both agents including a decrease in the levels of lactate and PC (P<0.02) while an increase in GPC (P<0.05). We also report a decrease in the protein expression levels of HK2, LDHA and CHKA providing likely mechanisms for the depletion of lactate and PC, respectively. Our results show that our in vitro NMR-detected changes in lactate and choline metabolites may have potential as non-invasive biomarkers for monitoring response to combination of PI3K/mTOR inhibitors with TMZ during clinical trials in children with glioblastoma, subject to further in vivo validation.

Pediatric and adult glioblastoma radiosensitization induced by PI3K/mTOR inhibition causes early metabolic alterations detected by nuclear magnetic resonance spectroscopy.

Poor outcome for patients with glioblastomas is often associated with radioresistance. PI3K/mTOR pathway deregulation has been correlated with radioresistance; therefore, PI3K/mTOR inhibition could render tumors radiosensitive. In this study, we show that NVP-BEZ235, a dual PI3K/mTOR inhibitor, potentiates the effects of irradiation in both adult and pediatric glioblastoma cell lines, resulting in early metabolic changes detected by nuclear magnetic resonance (NMR) spectroscopy. NVP-BEZ235 radiosensitises cells to X ray exposure, inducing cell death through the inhibition of CDC25A and the activation of p21cip1(CDKN1A). Lactate and phosphocholine levels, increased with radiation, are decreased after NVP-BEZ235 and combination treatment, suggesting that inhibiting the PI3K/mTOR pathway reverses radiation induced metabolic changes. Importantly, NVP-BEZ235 potentiates the effects of irradiation in a xenograft model of adult glioblastoma, where we observed a decrease in lactate and phosphocholine levels after seven days of combination treatment. Although tumor size was not affected due to the short length of the treatment, a significant increase in CASP3 mRNA was observed in the combination group. Taken together, our data suggest that NMR metabolites could be used as biomarkers to detect an early response to combination therapy with PI3K/mTOR inhibitors and radiotherapy in adult and pediatric glioblastoma patients.

Inhibitors of cyclin-dependent kinases as cancer therapeutics.

Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin-dependent kinases (CDKs). This attention initially stemmed from observations that different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non-selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform-selective inhibitors with accompanying biomarkers has re-ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER+/HER2- advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies.

Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases.

Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.