Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. ...We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCβ mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCβ is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.
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•Cancer-associated PKC mutations are LOF and can act in a dominant-negative manner•Correcting a heterozygous PKCβ LOF mutation reduces tumor volume•Hemizygous deletion shows PKC is haploinsufficient for tumor suppression•Therapeutic strategies should aim to restore PKC activity instead of inhibiting it
Cancer-associated kinase mutations have generally been characterized as oncogenic, but an analysis of PKC mutations reveals that the majority are loss of function, indicating a tumor-suppressive role for this kinase and a shift in therapeutic strategies targeting PKC.
Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRASG12D) also regulates tumor cell signaling via stromal cells. By ...combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer.
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•KRASG12D establishes a reciprocal signaling axis via heterotypic stromal cells•Reciprocal signaling further regulates tumor cell signaling downstream of KRASG12D•Reciprocal signaling regulates tumor cell behavior via AXL/IGF1R-AKT•Heterocellularity expands tumor cell signaling beyond cell-autonomous pathways
Cell-specific proteome labeling reveals that oncogenic KRAS stimulates stromal cells to initiate reciprocal signaling back to pancreatic tumor cells, thereby enabling signaling capacity beyond the traditionally studied cell-autonomous pathways.
Small-cell lung cancer (SCLC), an aggressive neuroendocrine tumor with early dissemination and dismal prognosis, accounts for 15-20% of lung cancer cases and ∼200,000 deaths each year. Most cases are ...inoperable, and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs), which are prevalent in SCLC, present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice, and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged, and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms.
Using a mouse model of human MLL-AF9 leukemia, we identified the lysine-specific demethylase KDM1A (LSD1 or AOF2) as an essential regulator of leukemia stem cell (LSC) potential. KDM1A acts at ...genomic loci bound by MLL-AF9 to sustain expression of the associated oncogenic program, thus preventing differentiation and apoptosis. In vitro and in vivo pharmacologic targeting of KDM1A using tranylcypromine analogs active in the nanomolar range phenocopied Kdm1a knockdown in both murine and primary human AML cells exhibiting MLL translocations. By contrast, the clonogenic and repopulating potential of normal hematopoietic stem and progenitor cells was spared. Our data establish KDM1A as a key effector of the differentiation block in MLL leukemia, which may be selectively targeted to therapeutic effect.
► KDM1A is a key effector of the differentiation block in MLL leukemia ► KDM1A sustains expression of the MLL-AF9 oncogenic program ► Nanomolar KDM1A inhibitor concentrations induce differentiation of human AML cells ► KDM1A inhibition in vivo targets MLL-AF9 cells but spares normal repopulating cells
Quality Control is a fundamental aspect of successful microarray data analysis. Simpleaffy is a BioConductor package that provides access to a variety of QC metrics for assessing the quality of RNA ...samples and of the intermediate stages of sample preparation and hybridization. Simpleaffy also offers fast implementations of popular algorithms for generating expression summaries and detection calls. Availability: Simpleaffy can be downloaded from http://www.bioconductor.org Contact: cmiller@picr.man.ac.uk Supplementary information: Additional information can be found on the supplementary website located at http://bioinformatics.picr.man.ac.uk
Droplet Digital PCR (ddPCR) is a sensitive platform used to quantify specific nucleic acid molecules amplified by polymerase chain reactions. Its sensitivity makes it particularly useful for the ...detection of rare mutant molecules, such as those present in a sample of circulating free tumour DNA obtained from cancer patients. ddPCR works by partitioning a sample into individual droplets for which the majority contain only zero or one target molecule. Each droplet then becomes a reaction chamber for PCR, which through the use of fluorochrome labelled probes allows the target molecules to be detected by measuring the fluorescence intensity of each droplet. The technology supports two channels, allowing, for example, mutant and wild type molecules to be detected simultaneously in the same sample. As yet, no open source software is available for the automatic gating of two channel ddPCR experiments in the case where the droplets can be grouped into four clusters. Here, we present an open source R package 'twoddpcr', which uses Poisson statistics to estimate the number of molecules in such two channel ddPCR data. Using the Shiny framework, an accompanying graphical user interface (GUI) is also included for the package, allowing users to adjust parameters and see the results in real-time.
twoddpcr is available from Bioconductor (3.5) at https://bioconductor.org/packages/twoddpcr/ . A Shiny-based GUI suitable for non-R users is available as a standalone application from within the package and also as a web application at http://shiny.cruk.manchester.ac.uk/twoddpcr/ .
ged.brady@cruk.manchester.ac.uk or crispin.miller@cruk.manchester.ac.uk.
anthony.chiu@cruk.manchester.ac.uk.
Fibrinolysisis is essential for vascular blood flow maintenance and is triggered by endothelial and platelet release of tissue plasminogen activator (t-PA). In certain critical conditions, e.g. ...sepsis, acute respiratory failure (ARF) and trauma, the fibrinolytic response is reduced and may lead to widespread thrombosis and multi-organ failure. The mechanisms underpinning fibrinolysis resistance include reduced t-PA expression and/or release, reduced t-PA and/or plasmin effect due to elevated inhibitor levels, increased consumption and/or clearance. This study in critically ill patients with fibrinolysis resistance aimed to evaluate the ability of t-PA and plasminogen supplementation to restore fibrinolysis with assessment using point-of-care ClotPro viscoelastic testing (VET).
In prospective, observational studies, whole-blood ClotPro VET evaluation was carried out in 105 critically ill patients. In 32 of 58 patients identified as fibrinolysis-resistant (clot lysis time > 300 s on the TPA-test: tissue factor activated coagulation with t-PA accelerated fibrinolysis), consecutive experimental whole-blood VET was carried out with repeat TPA-tests spiked with additional t-PA and/or plasminogen and the effect on lysis time determined. In an interventional study in a patient with ARF and fibrinolysis resistance, the impact of a 24 h intravenous low-dose alteplase infusion on coagulation and fibrinolysis was prospectively monitored using standard ClotPro VET.
Distinct response groups emerged in the ex vivo experimental VET, with increased fibrinolysis observed following supplementation with (i) t-PA only or (ii) plasminogen and t-PA. A baseline TPA-test lysis time of > 1000 s was associated with the latter group. In the interventional study, a gradual reduction (25%) in serial TPA-test lysis times was observed during the 24 h low-dose alteplase infusion.
ClotPro viscoelastic testing, the associated TPA-test and the novel experimental assays may be utilised to (i) investigate the potential mechanisms of fibrinolysis resistance, (ii) guide corrective treatment and (iii) monitor in real-time the treatment effect. Such a precision medicine and personalised treatment approach to the management of fibrinolysis resistance has the potential to increase treatment benefit, while minimising adverse events in critically ill patients.
VETtiPAT-ARF, a clinical trial evaluating ClotPro-guided t-PA (alteplase) administration in fibrinolysis-resistant patients with ARF, is ongoing (ClinicalTrials.gov NCT05540834 ; retrospectively registered September 15th 2022).
Microarrays measure the binding of nucleotide sequences to a set of sequence specific probes. This information is combined with annotation specifying the relationship between probes and targets and ...used to make inferences about transcript- and, ultimately, gene expression. In some situations, a probe is capable of hybridizing to more than one transcript, in others, multiple probes can target a single sequence. These 'multiply targeted' probes can result in non-independence between measured expression levels.
An analysis of these relationships for Affymetrix arrays considered both the extent and influence of exact matches between probe and transcript sequences. For the popular HGU133A array, approximately half of the probesets were found to interact in this way. Both real and simulated expression datasets were used to examine how these effects influenced the expression signal. It was found not only to lead to increased signal strength for the affected probesets, but the major effect is to significantly increase their correlation, even in situations when only a single probe from a probeset was involved. By building a network of probe-probeset-transcript relationships, it is possible to identify families of interacting probesets. More than 10% of the families contain members annotated to different genes or even different Unigene clusters. Within a family, a mixture of genuine biological and artefactual correlations can occur.
Multiple targeting is not only prevalent, but also significant. The ability of probesets to hybridize to more than one gene product can lead to false positives when analysing gene expression. Comprehensive annotation describing multiple targeting is required when interpreting array data.
Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue ...fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.
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