Proteomic Landscape of Tissue-Specific Cyclin E Functions in Vivo

E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their ‘core’ cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners in vivo. We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer

The cyclin D1-CDK4 oncogenic interactome enables identification of potential novel oncogenes and clinical prognosis

<div><p>Overexpression of cyclin D1 and its catalytic partner, CDK4, is frequently seen in human cancers. We constructed cyclin D1 and CDK4 protein interaction network in a human breast cancer cell line MCF7, and identified novel CDK4 protein partners. Among CDK4 interactors we observed several proteins functioning in protein folding and in complex assembly. One of the novel partners of CDK4 is FKBP5, which we found to be required to maintain CDK4 levels in cancer cells. An integrative analysis of the extended cyclin D1 cancer interactome and somatic copy number alterations in human cancers identified BAIAPL21 as a potential novel human oncogene. We observed that in several human tumor types BAIAPL21 is expressed at higher levels as compared to normal tissue. Forced overexpression of BAIAPL21 augmented anchorage independent growth, increased colony formation by cancer cells and strongly enhanced the ability of cells to form tumors in vivo. Lastly, we derived an Aggregate Expression Score (AES), which quantifies the expression of all cyclin D1 interactors in a given tumor. We observed that AES has a prognostic value among patients with ER-positive breast cancers. These studies illustrate the utility of analyzing the interactomes of proteins involved in cancer to uncover potential oncogenes, or to allow better cancer prognosis.</p></div

Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition

Breast cancer is the most common type of cancer in women and the second leading cause of cancer death among women. While significant progress has been made in the treatment of hormone receptor (HR) positive, HER2 negative and HER2-amplified breast cancer in the last decades, Triple negative breast cancer (TNBC) is lacking behind. Still today, chemotherapy is the only treatment option, but response rates are low and only a small fraction of patients can successfully be treated. Here we show, that CDK4/6 inhibitors which are FDA-approved for the treatment of HR+/HER2negative breast cancer and significantly improved treatment outcome in this type of breast cancer, also represent a promising therapy option for TNBC in contrast to the current view. We show, that a subset of TNBCs despite being resistant to chemical inhibition of CDK4/6 strictly depend on CDK4/6 kinases for proliferation. We identified lysosomal sequestration of the drug and therefore limited bioavailability at its target site responsible for the discrepancy. We show that increased number of lysosomes correlates with resistance and inhibition of the lysosome renders these cells fully sensitive to CDK4/6 inhibitors. We provide strategies how to overcome resistance by means of FDA-approved lysosomotropic agents that raise lysosomal pH, co-inhibition of CDK2 or the use of structurally altered CDK4/6 inhibitors with decreased basic characteristics. We also provide a biomarker to stratify patients for successful CDK4/6 inhibitor therapy. Moreover, we show that this mechanism of resistance also underlies cases of acquired resistance in HR+/HER2negative breast cancer and importantly we provide evidence that increased lysosomal sequestration operates in patients presenting resistant to treatment with the CDK4/6 inhibitor palbociclib. Our study therefore suggests that CDK4/6 inhibitor therapy could be a treatment option for a subset of TNBCs where new targeted therapeutic intervention is so urgently needed and furthermore, offers strategies how to overcome resistance in HR+/HER2-negative breast cancer where CDK4/6 inhibition is already successfully applied. In summary, our study provides ways on how to improve therapeutic use of this promising new class of anti-cancer agents

Cyclin A2 promotes DNA repair in the brain during both development and aging

Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation in CCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice

Generation of tagged cyclin E1 knock-In mice and analyses of cyclin E1-containing protein complexes.

<p>(A and B) Targeting strategy to knock-in Flag and HA tags into <i>the cyclin E1</i> locus to generate N-terminally tagged <i>cyclin E1</i>^<i>Ntag</i> (A) and C-terminally tagged <i>cyclin E1</i>^<i>Ctag</i> alleles (B). The exons are shown as green boxes, Flag tag as a blue box, and HA tag as a red box. Start and stop codons are marked with orange and yellow arrowheads, respectively. The hygromycin resistance cassette (Hyg) with flanking loxP sequences (filled arrows) is also indicated. Restriction enzyme recognition sites: E, EcoRI; A, AflII; Sc, ScaI; N, NotI; X, XhoI; K, KpnI; S, SpeI; P, PmeI; Hp, HpaI. Note that panel (A) was shown in ref [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (C) Western blot analysis of wild-type control (Ctrl), heterozygous cyclin <i>E1</i>^{<i>+/Ntag</i>}, <i>cyclin E1</i>^{<i>+/Ctag</i>}, and <i>cyclin E1</i>^{<i>Ntag/Ctag</i>} embryonic stem cells probed with anti-cyclin E1 and -HA antibodies. Actin served as a loading control. Forth panel: cyclin E1 was immunoprecipitated with anti-Flag antibody and the immunoblots were probed with anti-Cdk2 antibody. Fifth panel: anti-Flag immunoprecipitates were used for <i>in vitro</i> kinase reactions using histone H1 as a substrate. (D) Same analyses as in (C) using spleens of homozygous knock-in mice. Lanes 1–2 in panels (C and D) were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (E) Cyclin E levels detected by western blotting in the indicated organs of 1-month-old mice and in embryonic brain (day E14.5). Actin served as a loading control. The last two lanes (Brain) were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (F) Quantification of cyclin E levels in different organs, normalized against actin (from E). (G) Protein lysates from brains and testes of adult tagged cyclin E1 knock-in mice were separated by gel-filtration chromatography. Fractions containing protein complexes of the indicated molecular weights were analyzed by western blotting for cyclin E using an anti-HA antibody. (H) Cyclin E1-associated proteins were purified from the indicated organs of tagged cyclin E1 knock-in (KI) mice, or from control mice (Ctrl, ‘mock’ purifications) by sequential immunoaffinity purifications with anti-Flag and -HA antibodies, and 10% of the final eluate was resolved on PAGE gels and silver-stained. Arrows indicate bands corresponding to cyclin E1. Panels representing embryonic and adult brains were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>].</p

Targeting the cyclin-dependent kinase 5 in metastatic melanoma

© 2020 National Academy of Sciences. All rights reserved. The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells

Cyclin E1-interactomes.

<p>(A) Diagrams depicting cyclin E1-interacting proteins in the indicated mouse organs. Cyclin E1 is shown as a red node. Green nodes denote highest-confidence ‘core’ interactors (Category 1, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Yellow and blue nodes represent, respectively, lower confidence Categories 2 and 3 interactors that were included to the interactome based on their reported interaction with core interactors in the STRING database (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Solid lines depict STRING-verified interactions. Dashed lines depict an interaction derived from our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other core interactors. (B) A combined diagram depicting cyclin E1-interacting proteins from all five organs analyzed. Cyclin E1 is shown as a red node. Green nodes denote highest-confidence core (Category 1) interactors. Yellow and blue nodes denote, respectively, Categories 2 and 3 interactors, which were included into the interactome based on their ability to interact with core interactors as revealed by STRING (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Solid blue lines depict STRING-verified interactions between pairs of proteins that were identified by us as cyclin E1-interacting proteins within the same organ. Gray dotted lines depict STRING-verified interactions between pairs of proteins identified as cyclin E1-interators in different organs. Blue dashed lines depict interactions detected in our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other core proteins within the same organ interactome.</p

Analyses of cyclin E1-interactomes.

<p>(A) Venn diagram depicting the numbers of unique and shared cyclin E1 interactors in the indicated organs. (B) Fraction of unique interactors in the indicated organs. (C) Pairwise comparisons of the fraction of cyclin E1-interactors shared between the indicated organs. (D) The fraction of cyclin E1 interactors in the indicated organs that were assigned to a given Gene Ontology category (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s008" target="_blank">S2 Table</a>). Categories assigned at least 10% of the interactors in a given organ are marked in red. CC, cell cycle; TX, transcription; Neuro, neuronal function; Cyto, microtubules/cytoskeleton; Ubiq, ubiquitination; Metab, metabolism. (E) Heatmap displaying functional enrichment of cyclin E1 interactors in Gene Ontology classes of biological processes. The five columns correspond to the five organs analyzed, and each horizontal row denotes a distinct biological process. Colors depict fold-enrichment for the cyclin E1 interactors from the particular organ in a given biological process, between green (fold-enrichment one or lower) to red (fold-enrichment five or higher). Only categories in which at least one organ had an EASE score of 0.2 or lower are shown (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.s012" target="_blank">S1 Appendix</a>). Left panel shows a complete heatmap, right panels show selected common and organ-specific biological processes: cell cycle (red box, enriched in all organs), neurogenesis and synaptic plasticity (blue box, shared between embryonic and adult brains) and regulation of microtubule-based processes and microtubule cytoskeleton (green box, specific to adult brain).</p

A Sequentially Priming Phosphorylation Cascade Activates the Gliomagenic Transcription Factor Olig2

During development of the vertebrate CNS, the basic helix-loop-helix (bHLH) transcription factor Olig2 sustains replication competence of progenitor cells that give rise to neurons and oligodendrocytes. A pathological counterpart of this developmental function is seen in human glioma, wherein Olig2 is required for maintenance of stem-like cells that drive tumor growth. The mitogenic/gliomagenic functions of Olig2 are regulated by phosphorylation of a triple serine motif (S10, S13, and S14) in the amino terminus. Here, we identify a set of three serine/threonine protein kinases (glycogen synthase kinase 3α/β [GSK3α/β], casein kinase 2 [CK2], and cyclin-dependent kinases 1/2 [CDK1/2]) that are, collectively, both necessary and sufficient to phosphorylate the triple serine motif. We show that phosphorylation of the motif itself serves as a template to prime phosphorylation of additional serines and creates a highly charged acid blob in the amino terminus of Olig2. Finally, we show that small molecule inhibitors of this forward-feeding phosphorylation cascade have potential as glioma therapeutics