A Vulnerability of a Subset of Colon Cancers with Potential Clinical Utility.

BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.

Locus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly.

Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.

Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair.

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

EGFR activation limits the response of liver cancer to lenvatinib.

Hepatocellular carcinoma (HCC)-the most common form of liver cancer-is an aggressive malignancy with few effective treatment options1. Lenvatinib is a small-molecule inhibitor of multiple receptor tyrosine kinases that is used for the treatment of patients with advanced HCC, but this drug has only limited clinical benefit2. Here, using a kinome-centred CRISPR-Cas9 genetic screen, we show that inhibition of epidermal growth factor receptor (EGFR) is synthetic lethal with lenvatinib in liver cancer. The combination of the EGFR inhibitor gefitinib and lenvatinib displays potent anti-proliferative effects in vitro in liver cancer cell lines that express EGFR and in vivo in xenografted liver cancer cell lines, immunocompetent mouse models and patient-derived HCC tumours in mice. Mechanistically, inhibition of fibroblast growth factor receptor (FGFR)  by lenvatinib treatment leads to feedback activation of the EGFR-PAK2-ERK5 signalling axis, which is blocked by EGFR inhibition. Treatment of 12 patients with advanced HCC who were unresponsive to lenvatinib treatment with the combination of lenvatinib plus gefitinib (trial identifier NCT04642547) resulted in meaningful clinical responses. The combination therapy identified here may represent a promising strategy for the approximately 50% of patients with advanced HCC who have high levels of EGFR.

Mammalian life depends on two distinct pathways of DNA damage tolerance.

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.

Inducing and exploiting vulnerabilities for the treatment of liver cancer.

Liver cancer remains difficult to treat, owing to a paucity of drugs that target critical dependencies1,2; broad-spectrum kinase inhibitors such as sorafenib provide only a modest benefit to patients with hepatocellular carcinoma3. The induction of senescence may represent a strategy for the treatment of cancer, especially when combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Here, using a kinome-focused genetic screen, we show that pharmacological inhibition of the DNA-replication kinase CDC7 induces senescence selectively in liver cancer cells with mutations in TP53. A follow-up chemical screen identified the antidepressant sertraline as an agent that kills hepatocellular carcinoma cells that have been rendered senescent by inhibition of CDC7. Sertraline suppressed mTOR signalling, and selective drugs that target this pathway were highly effective in causing the apoptotic cell death of hepatocellular carcinoma cells treated with a CDC7 inhibitor. The feedback reactivation of mTOR signalling after its inhibition6 is blocked in cells that have been treated with a CDC7 inhibitor, which leads to the sustained inhibition of mTOR and cell death. Using multiple in vivo mouse models of liver cancer, we show that treatment with combined inhibition of of CDC7 and mTOR results in a marked reduction of tumour growth. Our data indicate that exploiting an induced vulnerability could be an effective treatment for liver cancer.

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.

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine.

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.

Systematic screening identifies ABCG2 as critical factor underlying synergy of kinase inhibitors with transcriptional CDK inhibitors.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited treatment options and poor clinical prognosis. Inhibitors of transcriptional CDKs are currently under thorough investigation for application in the treatment of multiple cancer types, including breast cancer. These studies have raised interest in combining these inhibitors, including CDK12/13 inhibitor THZ531, with a variety of other anti-cancer agents. However, the full scope of these potential synergistic interactions of transcriptional CDK inhibitors with kinase inhibitors has not been systematically investigated. Moreover, the mechanisms behind these previously described synergistic interactions remain largely elusive. METHODS: Kinase inhibitor combination screenings were performed to identify kinase inhibitors that synergize with CDK7 inhibitor THZ1 and CDK12/13 inhibitor THZ531 in TNBC cell lines. CRISPR-Cas9 knockout screening and transcriptomic evaluation of resistant versus sensitive cell lines were performed to identify genes critical for THZ531 resistance. RNA sequencing analysis after treatment with individual and combined synergistic treatments was performed to gain further insights into the mechanism of this synergy. Kinase inhibitor screening in combination with visualization of ABCG2-substrate pheophorbide A was used to identify kinase inhibitors that inhibit ABCG2. Multiple transcriptional CDK inhibitors were evaluated to extend the significance of the found mechanism to other transcriptional CDK inhibitors. RESULTS: We show that a very high number of tyrosine kinase inhibitors synergize with the CDK12/13 inhibitor THZ531. Yet, we identified the multidrug transporter ABCG2 as key determinant of THZ531 resistance in TNBC cells. Mechanistically, we demonstrate that most synergistic kinase inhibitors block ABCG2 function, thereby sensitizing cells to transcriptional CDK inhibitors, including THZ531. Accordingly, these kinase inhibitors potentiate the effects of THZ531, disrupting gene expression and increasing intronic polyadenylation. CONCLUSION: Overall, this study demonstrates the critical role of ABCG2 in limiting the efficacy of transcriptional CDK inhibitors and identifies multiple kinase inhibitors that disrupt ABCG2 transporter function and thereby synergize with these CDK inhibitors. These findings therefore further facilitate the development of new (combination) therapies targeting transcriptional CDKs and highlight the importance of evaluating the role of ABC transporters in synergistic drug-drug interactions in general.

High-throughput compound screen reveals mTOR inhibitors as potential therapeutics to reduce (auto)antibody production by human plasma cells.

Antibody production by the B cell compartment is a crucial part of the adaptive immune response. Dysregulated antibody production in the form of autoantibodies can cause autoimmune disease. To date, B-cell depletion with anti-CD20 antibodies is commonly applied in autoimmunity, but pre-existing plasma cells are not eliminated in this way. Alternative ways of more selective inhibition of antibody production would add to the treatment of these autoimmune diseases. To explore novel therapeutic targets in signaling pathways essential for plasmablast formation and/or immunoglobulin production, we performed a compound screen of almost 200 protein kinase inhibitors in a robust B-cell differentiation culture system. This study yielded 35 small cell-permeable compounds with a reproducible inhibitory effect on B-cell activation and plasmablast formation, among which was the clinically applied mammalian target of rapamycin (mTOR) inhibitor rapamycin. Two additional compounds targeting the phosphoinositide 3-kinase-AKT-mTOR pathway (BKM120 and WYE-354) did not affect proliferation and plasmablast formation, but specifically reduced the immunoglobulin production. With this compound screen we successfully applied a method to investigate therapeutic targets for B-cell differentiation and identified compounds in the phosphoinositide 3-kinase-AKT-mTOR pathway that could specifically inhibit immunoglobulin production only. These drugs may well be explored to be of value in current B-cell-depleting treatment regimens in autoimmune disorders.

CDK12 inhibition mediates DNA damage and is synergistic with sorafenib treatment in hepatocellular carcinoma.

OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most frequent malignancies and a major leading cause of cancer-related deaths worldwide. Several therapeutic options like sorafenib and regorafenib provide only modest survival benefit to patients with HCC. This study aims to identify novel druggable candidate genes for patients with HCC. DESIGN: A non-biased CRISPR (clustered regularly interspaced short palindromic repeats) loss-of-function genetic screen targeting all known human kinases was performed to identify vulnerabilities of HCC cells. Whole-transcriptome sequencing (RNA-Seq) and bioinformatics analyses were performed to explore the mechanisms of the action of a cyclin-dependent kinase 12 (CDK12) inhibitor in HCC cells. Multiple in vitro and in vivo assays were used to study the synergistic effects of the combination of CDK12 inhibition and sorafenib. RESULTS: We identify CDK12 as critically required for most HCC cell lines. Suppression of CDK12 using short hairpin RNAs (shRNAs) or its inhibition by the covalent small molecule inhibitor THZ531 leads to robust proliferation inhibition. THZ531 preferentially suppresses the expression of DNA repair-related genes and induces strong DNA damage response in HCC cell lines. The combination of THZ531 and sorafenib shows striking synergy by inducing apoptosis or senescence in HCC cells. The synergy between THZ531 and sorafenib may derive from the notion that THZ531 impairs the adaptive responses of HCC cells induced by sorafenib treatment. CONCLUSION: Our data highlight the potential of CDK12 as a drug target for patients with HCC. The striking synergy of THZ531 and sorafenib suggests a potential combination therapy for this difficult to treat cancer.

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma.

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-ß signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-ß (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-ß results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-ß becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-ß signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a 'drug holiday' and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation.

Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.

Phospho-ERK is a biomarker of response to a synthetic lethal drug combination of sorafenib and MEK inhibition in liver cancer.

BACKGROUND & AIMS: Treatment of liver cancer remains challenging because of a paucity of drugs that target critical dependencies. Sorafenib is a multikinase inhibitor that is approved as the standard therapy for patients with advanced hepatocellular carcinoma, but it only provides limited survival benefit. In this study we aimed to identify potential combination therapies to improve the clinical response to sorafenib. METHODS: To investigate the cause of the limited therapeutic effect of sorafenib, we performed a CRISPR-Cas9 based synthetic lethality screen to search for kinases whose knockout synergizes with sorafenib. Synergistic effects of sorafenib and selumetinib on cell apoptosis and phospho-ERK (p-ERK) were analyzed by caspase-3/7 apoptosis assay and western blot, respectively. p-ERK was measured by immunochemical analysis using a tissue microarray containing 78 liver cancer specimens. The in vivo effects of the combination were also measured in two xenograft models. RESULT: We found that suppression of ERK2 (MAPK1) sensitizes several liver cancer cell lines to sorafenib. Drugs inhibiting the MEK (MEK1/2 [MAP2K1/2]) or ERK (ERK1/2 [MAPK1/3]) kinases reverse unresponsiveness to sorafenib in vitro and in vivo in a subset of liver cancer cell lines characterized by high levels of active p-ERK, through synergistic inhibition of ERK kinase activity. CONCLUSION: Our data provide a combination strategy for treating liver cancer and suggest that tumors with high basal p-ERK levels, which are seen in approximately 30% of liver cancers, are most likely to benefit from such combinatorial treatment. LAY SUMMARY: Sorafenib is approved as the standard therapy for patients with advanced hepatocellular carcinoma, but only provides limited survival benefit. Herein, we found that inhibition of the kinase ERK2 increases the response to sorafenib in liver cancer. Our data indicate that a combination of sorafenib and a MEK inhibitor is most likely to be effective in tumors with high basal phospho-ERK levels.

TRIM28 is an Epigenetic Barrier to Induced Pluripotent Stem Cell Reprogramming.

Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large-scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2017;35:147-157.

Targeting of vulnerabilities of drug-tolerant persisters identified through functional genetics delays tumor relapse.

Drug-tolerant persisters (DTPs) are a rare subpopulation of cells within a tumor that can survive therapy through nongenetic adaptive mechanisms to develop relapse and repopulate the tumor following drug withdrawal. Using a cancer cell line with an engineered suicide switch to kill proliferating cells, we perform both genetic screens and compound screens to identify the inhibition of bromodomain and extraterminal domain (BET) proteins as a selective vulnerability of DTPs. BET inhibitors are especially detrimental to DTPs that have reentered the cell cycle (DTEPs) in a broad spectrum of cancer types. Mechanistically, BET inhibition induces lethal levels of ROS through the suppression of redox-regulating genes highly expressed in DTPs, including GPX2, ALDH3A1, and MGST1. In vivo BET inhibitor treatment delays tumor relapse in both melanoma and lung cancer. Our study suggests that combining standard of care therapy with BET inhibitors to eliminate residual persister cells is a promising therapeutic strategy.

A proof-of-concept study of sequential treatment with the HDAC inhibitor vorinostat following BRAF and MEK inhibitors in BRAFV600mutated melanoma.

PURPOSE: Development of resistance limits the clinical benefit of BRAF and MEK inhibitors (BRAFi/MEKi) in BRAFV600 mutated melanoma. It has been shown that short-term treatment (14 days) with vorinostat was able to initiate apoptosis of the resistant tumor cells. We aimed to assess the anti-tumor activity of sequential treatment with vorinostat following BRAFi/MEKi in patients with BRAFV600 melanoma who progressed after initial response to BRAFi/MEKi. PATIENTS AND METHODS: Patients with BRAFi/MEKi resistant BRAFV600 melanoma were treated with vorinostat 360 mg QD for 14 days followed by BRAFi/MEKi. The primary endpoint was an objective response rate of progressive lesions of at least 30% according to RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), safety, pharmacokinetics of vorinostat and translational molecular analyses using ctDNA and tumor biopsies. RESULTS: Twenty-six patients with progressive BRAFi/MEKi resistant BRAFV600 mutated melanoma received treatment with vorinostat. Twenty-two patients were evaluable for response. The ORR was 9% (one complete response for 31.2 months and one partial response for 14.9 months. Median PFS and OS were 1.4 and 5.4 months, respectively. Common adverse events were fatigue (23%) and nausea (19%). ctDNA analysis showed emerging secondary mutations in NRAS and MEK in eight patients at time of BRAFi/MEKi resistance. Elimination of these mutations by vorinostat treatment was observed in three patients. CONCLUSIONS: Intermittent treatment with vorinostat in patients with resistant BRAFV600mutated melanoma is well tolerated. Although the primary endpoint of this study was not met, durable anti-tumor responses were observed in a minority of patients (9%).

Paradoxical Activation of Oncogenic Signaling as a Cancer Treatment Strategy.

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance. Significance: A therapy consisting of deliberate hyperactivation of oncogenic signaling combined with perturbation of the stress responses that result from this is very effective in animal models of colon cancer. Resistance to this therapy is associated with loss of oncogenic signaling and reduced oncogenic capacity, indicative of tumor-suppressive drug resistance.

Sustained activation of EGFR-ERK1/2 signaling limits the response to tigecycline-induced mitochondrial respiratory deficiency in liver cancer.

BACKGROUND: Identification of tumor dependencies is important for developing therapeutic strategies for liver cancer. METHODS: A genome-wide CRISPR screen was performed for finding critical vulnerabilities in liver cancer cells. Compounds screen, RNA sequencing, and human phospho-receptor tyrosine kinase arrays were applied to explore mechanisms and search for synergistic drugs. FINDINGS: We identified mitochondrial translation-related genes associated with proliferation for liver cancer cells. Tigecycline induced deficiency of respiratory chain by disturbing mitochondrial translation process and showed therapeutic potential in liver cancer. For liver cancer cells extremely insensitive to tigecycline, a compounds screen was applied to identify MEK inhibitors as synergistic drugs to tigecycline-insensitive liver cancer cells. Mechanistically, sustained activation of EGFR-ERK1/2-MYC cascade conferred the insensitivity to tigecycline, which was mediated by enhanced secretion of EREG and AREG. Moreover, glycolytic enzymes, such as HK2 and PKM2 were upregulated to stimulate glycolysisin a MYC-dependent manner. Tigecycline induced respiratory chain deficiency in combination with cutting off EGFR-ERK1/2-MYC cascade by MEK inhibitors or EGFR inhibitors, resulting in decrease of both oxidative phosphorylation and glycolysis in liver cancer cells. INTERPRETATION: Our study proved that blocking EGFR-ERK1/2-MYC cascade combined with tigecycline could be a potential therapeutic strategy for liver cancer. FUNDING: This work was funded by grants from the National Natural Science Foundation of China (82073039,82222047, 81920108025), Program of Shanghai Academic/Technology Research Leader (22XD1423100), Shanghai Municipal Science and Technology Project (20JC1411100), 111 Project (B21024), Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZDCX20212700, SHSMU-ZDCX20210802) and Shanghai Jiao Tong University School of Medicine (YG2019GD01).