The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1

Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry. This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression. We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase

Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling.

The nuclear factor κB (NF-κB) signalling pathway controls important cellular events such as cell proliferation, differentiation, apoptosis and immune responses. Pathway activation occurs rapidly upon TNFα stimulation and is highly dependent on ubiquitination events. Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling. We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling. F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity

Cloning, expression and chromosomal localization of a new putative receptor-like protein tyrosine phosphatase

AbstractWe have isolated a mouse cDNA of 5.7 kb, encoding a new member of the family of receptor-like protein tyrosine phosphatases, termed mRPTPμ. The cDNA predicts a protein of 1432 amino acids (not including signal peptide) with a calculated Mr of 161 636. In addition, we have cloned the human homologue, hRPTPμ, which shows 98.7% amino acid identity to mRPTPμ. The predicted mRPTPμ protein consists of a 722 amino acid extracellular region, containing 13 potential N-glycosylation sites, a single transmembrane domain and a 688 amino acid intracellular part containing 2 tandem repeats homologous to the catalytic domains of other tyrosine phosphatases. The N-terminal extracellular part contains a region of about 170 amino acids with no sequence similarities to known proteins, followed by one Ig-like domain and 4 fibronectin type III-like domains. The intracellular part is unique in that the region between the transmembrane domain and the first catalytic domain is about twice as large as in other receptor-like protein tyrosine phosphatases. RNA blot analysis reveals a single transcript, that is most abundant in lung and present in much lower amounts in brain and heart. Transfection of the mRPTPμ cDNA into COS cells results in the synthesis of a protein with an apparent Mr of 195 000, as detected in immunoblots using an antipeptide antibody. The human RPTPμ gene is localized on chromosome 18pter-q11, a region with frequent abnormalities implicated in human cancer

The RECQL helicase prevents replication fork collapse during replication stress

Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy

Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization.

ABSTRACT The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization

Parallel In Vivo and In Vitro Melanoma RNAi Dropout Screens Reveal Synthetic Lethality between Hypoxia and DNA Damage Response Inhibition

SummaryTo identify factors preferentially necessary for driving tumor expansion, we performed parallel in vitro and in vivo negative-selection short hairpin RNA (shRNA) screens. Melanoma cells harboring shRNAs targeting several DNA damage response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for the identification of pharmacologically tractable targets

PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

How steroid hormone receptors (SHRs) regulate transcriptional activity remains partly understood. Upon activation, SHRs bind the genome together with a co-regulator repertoire, crucial to induce gene expression. However, it remains unknown which components of the SHR-recruited co-regulator complex are essential to drive transcription following hormonal stimuli. Through a FACS-based genome-wide CRISPR screen, we functionally dissected the Glucocorticoid Receptor (GR) complex. We describe a functional cross-talk between PAXIP1 and the cohesin subunit STAG2, critical for regulation of gene expression by GR. Without altering the GR cistrome, PAXIP1 and STAG2 depletion alter the GR transcriptome, by impairing the recruitment of 3D-genome organization proteins to the GR complex. Importantly, we demonstrate that PAXIP1 is required for stability of cohesin on chromatin, its localization to GR-occupied sites, and maintenance of enhancer-promoter interactions. In lung cancer, where GR acts as tumor suppressor, PAXIP1/STAG2 loss enhances GR-mediated tumor suppressor activity by modifying local chromatin interactions. All together, we introduce PAXIP1 and STAG2 as novel co-regulators of GR, required to maintain 3D-genome architecture and drive the GR transcriptional programme following hormonal stimuli.</p

Intrinsic resistance to PIM kinase inhibition in AML through p38α-mediated feedback activation of mTOR signaling

Although conventional therapies for acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) are effective in inducing remission, many patients relapse upon treatment. Hence, there is an urgent need for novel therapies. PIM kinases are often overexpressed in AML and DLBCL and are therefore an attractive therapeutic target. However, in vitro experiments have demonstrated that intrinsic resistance to PIM inhibition is common. It is therefore likely that only a minority of patients will benefit from single agent PIM inhibitor treatment. In this study, we performed an shRNA-based genetic screen to identify kinases whose suppression is synergistic with PIM inhibition. Here, we report that suppression of p38α (MAPK14) is synthetic lethal with the PIM kinase inhibitor AZD1208. PIM inhibition elevates reactive oxygen species (ROS) levels, which subsequently activates p38α and downstream AKT/mTOR signaling. We found that p38α inhibitors sensitize hematological tumor cell lines to AZD1208 treatment in vitro and in vivo. These results were validated in ex vivo patient-derived AML cells. Our findings provide mechanistic and translational evidence supporting the rationale to test a combination of p38α and PIM inhibitors in clinical trials for AML and DLBCL

Intrinsic Resistance to MEK Inhibition in KRAS Mutant Lung and Colon Cancer through Transcriptional Induction of ERBB3

Summary There are no effective therapies for the ∼30% of human malignancies with mutant RAS oncogenes. Using a kinome-centered synthetic lethality screen, we find that suppression of the ERBB3 receptor tyrosine kinase sensitizes KRAS mutant lung and colon cancer cells to MEK inhibitors. We show that MEK inhibition results in MYC-dependent transcriptional upregulation of ERBB3, which is responsible for intrinsic drug resistance. Drugs targeting both EGFR and ERBB2, each capable of forming heterodimers with ERBB3, can reverse unresponsiveness to MEK inhibition by decreasing inhibitory phosphorylation of the proapoptotic proteins BAD and BIM. Moreover, ERBB3 protein level is a biomarker of response to combinatorial treatment. These data suggest a combination strategy for treating KRAS mutant colon and lung cancers and a way to identify the tumors that are most likely to benefit from such combinatorial treatment