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Cyclin-dependent kinase 2 (CDK2) is a potential therapeutic target for the treatment of cancer. Development of CDK2 inhibitors has been extremely challenging as its ATP-binding site shares high similarity with CDK1, a related kinase whose inhibition causes toxic effects. Here, we report the development of TMX-2172, a heterobifunctional CDK2 degrader with degradation selectivity for CDK2 and CDK5 over not only CDK1, but transcriptional CDKs (CDK7 and CDK9) and cell cycle CDKs (CDK4 and CDK6) as well. In addition, we demonstrate that antiproliferative activity in ovarian cancer cells (OVCAR8) depends on CDK2 degradation and correlates with high expression of cyclin E1 (CCNE1), which functions as a regulatory subunit of CDK2. Collectively, our work provides evidence that TMX-2172 represents a lead for further development and that CDK2 degradation is a potentially valuable therapeutic strategy in ovarian and other cancers that overexpress CCNE1.

Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).

Proliferation is a fundamental trait of cancer cells, but its properties and spatial organization in tumours are poorly characterized. Here we use highly multiplexed tissue imaging to perform single-cell quantification of cell cycle regulators and then develop robust, multivariate, proliferation metrics. Across diverse cancers, proliferative architecture is organized at two spatial scales: large domains, and smaller niches enriched for specific immune lineages. Some tumour cells express cell cycle regulators in the (canonical) patterns expected of freely growing cells, a phenomenon we refer to as 'cell cycle coherence'. By contrast, the cell cycles of other tumour cell populations are skewed towards specific phases or exhibit non-canonical (incoherent) marker combinations. Coherence varies across space, with changes in oncogene activity and therapeutic intervention, and is associated with aggressive tumour behaviour. Thus, multivariate measures from high-plex tissue images capture clinically significant features of cancer proliferation, a fundamental step in enabling more precise use of anti-cancer therapies.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

Homologous recombination (HR)-deficient cancers are sensitive to inhibitors of Poly-ADP Ribose Polymerase (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive, TP53−/− and BRCA1−/− epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison to a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained pre-existing drug tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical BRCA1-mutant HGSC patient with acquired PARPi resistance were observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance.

Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis1. To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC. We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations. We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells2. Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program. Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models. Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models. Our work contributes to resolving the HSGOC landscape3-5 and provides a resource for the development of novel therapeutic approaches.

The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo–expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages. Significance: HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor–resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.

Doublecortin-like kinase 1 (DCLK1) is critical for neurogenesis, but overexpression is also observed in multiple cancers and is associated with poor prognosis. Nevertheless, the function of DCLK1 in cancer, especially the context-dependent functions, are poorly understood. We present a "toolkit" that includes the DCLK1 inhibitor DCLK1-IN-1, a complementary DCLK1-IN-1-resistant mutation G532A, and kinase dead mutants D511N and D533N, which can be used to investigate signaling pathways regulated by DCLK1. Using a cancer cell line engineered to be DCLK1 dependent for growth and cell migration, we show that this toolkit can be used to discover associations between DCLK1 kinase activity and biological processes. In particular, we show an association between DCLK1 and RNA processing, including the identification of CDK11 as a potential substrate of DCLK1 using phosphoproteomics.

Summary 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.

SUMMARY Evidence that some high-impact biomedical results cannot be repeated has stimulated interest in practices that generate findable, accessible, interoperable, and reusable (FAIR) data. Multiple papers have identified specific examples of irreproducibility, but practical ways to make data more reproducible have not been widely studied. Here, five research centers in the NIH LINCS Program Consortium investigate the reproducibility of a prototypical perturbational assay: quantifying the responsiveness of cultured cells to anti-cancer drugs. Such assays are important for drug development, studying cellular networks, and patient stratification. While many experimental and computational factors impact intra- and inter-center reproducibility, the factors most difficult to identify and control are those with a strong dependency on biological context. These factors often vary in magnitude with the drug being analyzed and with growth conditions. We provide ways to identify such context-sensitive factors, thereby improving both the theory and practice of reproducible cell-based assays., Graphical Abstract, In Brief Factors that impact the reproducibility of experimental data are poorly understood. Five NIH-LINCS centers performed the same set of drug-response measurements and compared results. Technical and biological variables that impact precision and reproducibility and are also sensitive to biological context were the most problematic.

Mammography is used to screen a large fraction of the population for breast cancer, and mammography quality X rays are speculated to be more damaging than the higher energy X rays used for other diagnostic procedures. The radiation dose delivered to breast cells as a result of these screening exposures may be a concern. The purpose of this current study was to determine the relative biological effectiveness (RBE) of low-energy mammography X rays for radiation-induced DNA double-strand breaks evaluated using a highly sensitive automated 53BP1 assay. Automation of the 53BP1 assay enabled the quantification and analysis of meaningful image-based features, including foci counting, within the cell nuclei. Nontumorigenic, human breast epithelial MCF-10A cells were irradiated in the low-dose range with approximately 3-30 mGy of 29 kVp mammography X rays or (137)Cs (662 keV) gamma rays. The induction and resolution of the 53BP1 foci did not differ significantly between exposures to (137)Cs gamma rays and 29 kVp X rays. The RBE was calculated to be 1.1 with a standard deviation of 0.2 for the initial number of radiation-induced double-strand breaks. The radiation dose from a single mammogram did not yield a significant change in the number of detectable foci. However, analysis of additional features revealed subtle differences in the distribution of 53BP1 throughout the nuclei after exposure to the different radiation qualities. A single mammogram was sufficient to alter the distribution of 53BP1 within the nuclear area, but not into discrete foci, while a dose-matched gamma exposure was not sufficient to alter the distribution of 53BP1. Our results indicate that exposure to clinically relevant doses of low-energy mammography quality X rays does not induce more DNA double-strand breaks than exposure to higher energy photons.

SUMMARYThe target profiles of many drugs are established early in their development and are not systematically revisited at the time of FDA approval. Thus, it is often unclear whether therapeutics with the same nominal targets but different chemical structures are functionally equivalent. In this paper we use five different phenotypic and biochemical assays to compare approved inhibitors of cyclin-dependent kinases 4/6 – collectively regarded as breakthroughs in the treatment of hormone receptor-positive breast cancer. We find that transcriptional, proteomic and phenotypic changes induced by palbociclib, ribociclib, and abemaciclib differ significantly; abemaciclib in particular has advantageous activities partially overlapping those of alvocidib, an older polyselective CDK inhibitor. In cells and mice, abemaciclib inhibits kinases other than CDK4/6 including CDK2/Cyclin A/E – implicated in resistance to CDK4/6 inhibition – and CDK1/Cyclin B. The multi-faceted experimental and computational approaches described here therefore uncover under-appreciated differences in CDK4/6 inhibitor activities with potential importance in treating human patients.

Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

A 4 x 500 mm2 "cloverleaf" low energy germanium detector array has been assembled for the purpose of in vivo bone lead measurement through x-ray fluorescence. Using 109Cd as an exciting source, results are reported from a leg phantom simulating measurement of lead in a human tibia. For high activity (4.0-4.4 GBq) and low activity (0.18-0.19 GBq) sources, measurement results are reported for both the cloverleaf system and a conventional single detector system of equivalent surface area (2000 mm2). The mean uncertainty and reproducibility of measurement were both significantly improved for the cloverleaf system with a high activity 109Cd source. When using a source activity of 4.4 GBq, measurement of the phantom resulted in an average bone lead uncertainty of 0.79 microg/g and a reproducibility of 0.84 microg/g. These results represent the highest precision yet reported from a bone lead x-ray fluorescence system.