CCNE1 amplification is synthetic-lethal with PKMYT1 kinase inhibition

Amplification of the gene encoding cyclin E (CCNE1) is an oncogenic driver in several malignancies and is associated with chemoresistance and poor prognosis. To uncover therapeutic targets for CCNE1-amplified tumors, we undertook genome-scale CRISPR/Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here, we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumor regressions when combined with gemcitabine in models of CCNE1-amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1 overexpressing-cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the FOXM1/MYBL2/MuvB-dependent mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

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KRASG12C inhibition produces a driver-limited state revealing collateral dependencies

Science Signaling ◽

10.1126/scisignal.aaw9450 ◽

2019 ◽

Vol 12 (583) ◽

pp. eaaw9450 ◽

Cited By ~ 28

Author(s):

Kevin Lou ◽

Veronica Steri ◽

Alex Y. Ge ◽

Y. Christina Hwang ◽

Christopher H. Yogodzinski ◽

...

Keyword(s):

Cysteine Residue ◽

Mutant Form ◽

Cellular Models ◽

Guanosine Diphosphate ◽

Survival Pathways ◽

A Genome ◽

Oncogenic Driver ◽

Guanosine Triphosphatase ◽

Genome Scale

Inhibitors targeting KRASG12C, a mutant form of the guanosine triphosphatase (GTPase) KRAS, are a promising new class of oncogene-specific therapeutics for the treatment of tumors driven by the mutant protein. These inhibitors react with the mutant cysteine residue by binding covalently to the switch-II pocket (S-IIP) that is present only in the inactive guanosine diphosphate (GDP)–bound form of KRASG12C, sparing the wild-type protein. We used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with a KRASG12C inhibitor in cellular models of KRASG12C mutant lung and pancreatic cancer. Our data revealed genes that were selectively essential in this oncogenic driver–limited cell state, meaning that their loss enhanced cellular susceptibility to direct KRASG12C inhibition. We termed such genes “collateral dependencies” (CDs) and identified two classes of combination therapies targeting these CDs that increased KRASG12C target engagement or blocked residual survival pathways in cells and in vivo. From our findings, we propose a framework for assessing genetic dependencies induced by oncogene inhibition.

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The Yeast Ser/Thr Phosphatases Sit4 and Ppz1 Play Opposite Roles in Regulation of the Cell Cycle

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2408 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2408-2415 ◽

Cited By ~ 56

Author(s):

Josep Clotet ◽

Eloi Garí ◽

Martí Aldea ◽

Joaquín Ariño

Keyword(s):

Cell Cycle ◽

Protein Phosphatase ◽

Slow Growth ◽

Negative Regulator ◽

Yeast Cells ◽

Growth Defect ◽

Gene Encoding ◽

Synthetic Lethal ◽

Bud Emergence ◽

Normal Transition

ABSTRACT Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from α-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 onsit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression ofPPZ1 is intensified in strains with low G1cyclin levels (such as bck2Δ or cln3Δ mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.

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Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth.

Genetics ◽

10.1093/genetics/135.3.693 ◽

1993 ◽

Vol 135 (3) ◽

pp. 693-709 ◽

Cited By ~ 1

Author(s):

T S Karpova ◽

M M Lepetit ◽

J A Cooper

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Synthetic Lethality ◽

Actin Binding ◽

Cortical Actin ◽

Gene Encoding ◽

Synthetic Lethal ◽

Capping Protein ◽

Colony Color ◽

Actin Cables

Abstract Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the beta subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.

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DDRE-06. CELLULAR STRESS RESPONSE IN DIPG THERAPY

Neuro-Oncology ◽

10.1093/neuonc/noaa215.251 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii62-ii62

Author(s):

Sreepradha Sridharan ◽

Arif Harmanci ◽

Robert Siddaway ◽

Tara Dobson ◽

Jyothishmathi Swaminathan ◽

...

Keyword(s):

Stress Response ◽

Single Cell ◽

Synthetic Lethality ◽

Clonal Evolution ◽

Single Agent ◽

Objective Response ◽

Hdac Inhibitors ◽

Pediatric Brain Tumor ◽

Dominant Negative ◽

Cellular Stress Response

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor of the pons and brainstem. Therefore, there is a desperate need for new therapeutics. Genomic profiling of tumors identified a highly prevalent dominant negative somatic mutation at lysine (K)-27 in histone genes HIST1H3B and H3F3A. Clonal evolution modeling suggests these mutations are truncal, and studies have demonstrated their contribution to tumorigenesis. ONC201, a first-in-class DRD2 antagonist and ClpP agonist is an anticancer drug developed by Oncoceutics, which targets the unfolded protein response (UPR) and integrated stress response (ISR) signaling and is actively being investigated in patients with recurrent H3 K27M-mutant gliomas. In adults with recurrent glioma, single agent studies showed benign-safety, no dose-limiting toxicities and a durable objective response when administered orally. In addition, intra-tumoral drug levels exceeded therapeutic thresholds, and induced tumor cell apoptosis. Based on this and response seen in a pediatric patient with DIPG for whom compassionate use of ONC201 was approved, a multi-arm, non-randomized multi-institutional Phase I clinical trial (NCT03416530) is actively accruing patients. However, the strength of UPR and ISR in DIPGs and their effect on DIPG response to ONC201 is not known. Our group employed bulk/single cell transcriptomic and single cell proteomic approaches to demonstrate substantial heterogeneity in UPR and ISR signaling in human DIPG samples. Consistent with this, DIPG cell lines exhibited considerable variability in sensitivity to ONC201. Single cell profiling identified tumor sub-populations with significant proliferative capacity even after ONC201 exposure. Incomplete response promotes recurrence. To target these cells, we performed a synthetic lethality screen with a library of 360 FDA-approved CNS penetrant compounds, which identified HDAC inhibitors and DNA damage-inducing chemotherapy as having synergy with ONC201. Thus, we suggest that tumor heterogeneity impacts sensitivity to ONC201 and that this can be reduced by combination treatments.

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Controlled Transcription of Regulator Gene carS by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in Fusarium fujikuroi

Microorganisms ◽

10.3390/microorganisms9010071 ◽

2020 ◽

Vol 9 (1) ◽

pp. 71

Author(s):

Julia Marente ◽

Javier Avalos ◽

M. Carmen Limón

Keyword(s):

Wild Strain ◽

Ring Finger ◽

Carotenoid Biosynthesis ◽

Negative Regulator ◽

Fusarium Fujikuroi ◽

Structural Genes ◽

Gene Encoding ◽

In The Wild ◽

Set Up

Carotenoid biosynthesis is a frequent trait in fungi. In the ascomycete Fusarium fujikuroi, the synthesis of the carboxylic xanthophyll neurosporaxanthin (NX) is stimulated by light. However, the mutants of the carS gene, encoding a protein of the RING finger family, accumulate large NX amounts regardless of illumination, indicating the role of CarS as a negative regulator. To confirm CarS function, we used the Tet-on system to control carS expression in this fungus. The system was first set up with a reporter mluc gene, which showed a positive correlation between the inducer doxycycline and luminescence. Once the system was improved, the carS gene was expressed using Tet-on in the wild strain and in a carS mutant. In both cases, increased carS transcription provoked a downregulation of the structural genes of the pathway and albino phenotypes even under light. Similarly, when the carS gene was constitutively overexpressed under the control of a gpdA promoter, total downregulation of the NX pathway was observed. The results confirmed the role of CarS as a repressor of carotenogenesis in F. fujikuroi and revealed that its expression must be regulated in the wild strain to allow appropriate NX biosynthesis in response to illumination.

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MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903150116 ◽

2019 ◽

Vol 116 (35) ◽

pp. 17438-17443 ◽

Cited By ~ 6

Author(s):

Gayathri Srinivasan ◽

Elizabeth A. Williamson ◽

Kimi Kong ◽

Aruna S. Jaiswal ◽

Guangcun Huang ◽

...

Keyword(s):

Dna Repair ◽

Cancer Cells ◽

Synthetic Lethality ◽

Negative Regulator ◽

Nonhomologous End Joining ◽

Chromosomal Translocations ◽

Replication Forks ◽

Repair Pathway ◽

End Joining ◽

Parp1 Inhibitors

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223–3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

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Using Yeast Synthetic Lethality To Inform Drug Combination for Malaria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01533-17 ◽

2018 ◽

Vol 62 (4) ◽

Author(s):

Suvitha Subramaniam ◽

Christoph D. Schmid ◽

Xue Li Guan ◽

Pascal Mäser

Keyword(s):

Plasmodium Falciparum ◽

Drug Combination ◽

Synthetic Lethality ◽

Drug Combinations ◽

Synthetic Lethal Interaction ◽

Content Type ◽

Synthetic Lethal ◽

Gene Pairs ◽

Phosphatidylinositol 4

ABSTRACT Combinatorial chemotherapy is necessary for the treatment of malaria. However, finding a suitable partner drug for a new candidate is challenging. Here we develop an algorithm that identifies all of the gene pairs of Plasmodium falciparum that possess orthologues in yeast that have a synthetic lethal interaction but are absent in humans. This suggests new options for drug combinations, particularly for inhibitors of targets such as P. falciparum calcineurin, cation ATPase 4, or phosphatidylinositol 4-kinase.

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Abstract 2139: Enabling cancer drug target discovery through genome-scale identification of synthetic lethal paralog pairs

10.1158/1538-7445.am2021-2139 ◽

2021 ◽

Author(s):

Phoebe C. Parrish ◽

James D. Thomas ◽

Shriya Kamlapurkar ◽

Robert K. Bradley ◽

Alice H. Berger

Keyword(s):

Drug Target ◽

Cancer Drug ◽

Drug Target Discovery ◽

Target Discovery ◽

Synthetic Lethal ◽

Genome Scale

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A Screen for Dynein Synthetic Lethals in Aspergillus nidulans Identifies Spindle Assembly Checkpoint Genes and Other Genes Involved in Mitosis

Genetics ◽

10.1093/genetics/149.1.101 ◽

1998 ◽

Vol 149 (1) ◽

pp. 101-116

Author(s):

Vladimir P Efimov ◽

N Ronald Morris

Keyword(s):

Aspergillus Nidulans ◽

Spindle Assembly Checkpoint ◽

Genetic Interaction ◽

Synthetic Lethality ◽

Spindle Assembly ◽

Nuclear Migration ◽

Cytoplasmic Dynein ◽

Vesicle Transport ◽

Synthetic Lethal ◽

Checkpoint Genes

Abstract Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.

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Cohesin mutations are synthetic lethal with stimulation of WNT signaling

eLife ◽

10.7554/elife.61405 ◽

2020 ◽

Vol 9 ◽

Author(s):

Chue Vin Chin ◽

Jisha Antony ◽

Sarada Ketharnathan ◽

Anastasia Labudina ◽

Gregory Gimenez ◽

...

Keyword(s):

Wnt Signaling ◽

Therapeutic Strategy ◽

Synthetic Lethality ◽

Mcf10a Cell ◽

Synthetic Lethal ◽

Deletion Mutations ◽

Damage Repair ◽

Genes Encoding ◽

Mutant Cells ◽

Gsk3 Inhibitor

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers.

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