ADARp150 counteracts whole genome duplication.

Impaired control of the G1/S checkpoint allows initiation of DNA replication under non-permissive conditions. Unscheduled S-phase entry is associated with DNA replication stress, demanding for other checkpoints or cellular pathways to maintain proliferation. Here, we uncovered a requirement for ADARp150 to sustain proliferation of G1/S-checkpoint-defective cells under growth-restricting conditions. Besides its well-established mRNA editing function in inversely oriented short interspersed nuclear elements (SINEs), we found ADARp150 to exert a critical function in mitosis. ADARp150 depletion resulted in tetraploidization, impeding cell proliferation in mitogen-deprived conditions. Mechanistically we show that ADAR1 depletion induced aberrant expression of Cyclin B3, which was causative for mitotic failure and whole-genome duplication. Finally, we find that also in vivo ADAR1-depletion-provoked tetraploidization hampers tumor outgrowth.

Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing.

The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.

Widespread chromatin context-dependencies of DNA double-strand break repair proteins.

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. Examples of the former are BRCA2 and POLL, and of the latter the FANC complex and ATM. Moreover, in a diversity of human cancer types, loss of several of these proteins alters the distribution of pathway-specific mutations between heterochromatin and euchromatin. Together, these results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner.

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.

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.

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.

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.

A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator.

The crosstalk between prostate cancer (PCa) cells and the tumor microenvironment plays a pivotal role in disease progression and metastasis and could provide novel opportunities for patient treatment. Macrophages are the most abundant immune cells in the prostate tumor microenvironment (TME) and are capable of killing tumor cells. To identify genes in the tumor cells that are critical for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified AR, PRKCD, and multiple components of the NF-κB pathway as hits, whose expression in the tumor cell are essential for being targeted and killed by macrophages. These data position AR signaling as an immunomodulator, and confirmed by androgen-deprivation experiments, that rendered hormone-deprived tumor cells resistant to macrophage-mediated killing. Proteomic analyses showed a downregulation of oxidative phosphorylation in the PRKCD- and IKBKG-KO cells compared to the control, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Furthermore, phosphoproteomic analyses revealed that all hits impaired ferroptosis signaling, which was validated transcriptionally using samples from a neoadjuvant clinical trial with the AR-inhibitor enzalutamide. Collectively, our data demonstrate that AR functions together with the PRKCD and the NF-κB pathway to evade macrophage-mediated killing. As hormonal intervention represents the mainstay therapy for treatment of prostate cancer patients, our findings may have direct implications and provide a plausible explanation for the clinically observed persistence of tumor cells despite androgen deprivation therapy.

Multi-omics analysis reveals distinct non-reversion mechanisms of PARPi resistance in BRCA1- versus BRCA2-deficient mammary tumors.

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanisms that do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance in vivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response.

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.

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.

Subtype-specific kinase dependency regulates growth and metastasis of poor-prognosis mesenchymal colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al., Cell Death Differ 25:616-33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020). METHODS: To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns. RESULTS: PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162-72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2. CONCLUSION: Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.

Enhancer profiling identifies epigenetic markers of endocrine resistance and reveals therapeutic options for metastatic castration-resistant prostate cancer patients.

Androgen Receptor (AR) signaling inhibitors, including enzalutamide, are treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC), but resistance inevitably develops. Using metastatic samples from a prospective phase II clinical trial, we epigenetically profiled enhancer/promoter activities with H3K27ac chromatin immunoprecipitation followed by sequencing, before and after AR-targeted therapy. We identified a distinct subset of H3K27ac-differentially marked regions that associated with treatment responsiveness. These data were successfully validated in mCRPC patient-derived xenograft models (PDX). In silico analyses revealed HDAC3 as a critical factor that can drive resistance to hormonal interventions, which we validated in vitro . Using cell lines and mCRPC PDX tumors in vitro , we identified drug-drug synergy between enzalutamide and the pan-HDAC inhibitor vorinostat, providing therapeutic proof-of-concept. These findings demonstrate rationale for new therapeutic strategies using a combination of AR and HDAC inhibitors to improve patient outcome in advanced stages of mCRPC.

Inhibitor of Apoptosis Proteins Antagonist Induces T-cell Proliferation after Cross-Presentation by Dendritic Cells.

Cross-presentation of tumor antigens by dendritic cells (DC) is crucial to prime, stimulate and restimulate CD8+ T cells. This process is important in initiating and maintaining an antitumor response. Here, we show that the presence of conventional type 1 DCs (cDC1), a DC subtype that excels in cross-presentation, in the tumor correlated with response to neoadjuvant immune checkpoint blockade (ICB) in melanoma. This led us to hypothesize that patients failing to respond to ICB could benefit from enhanced cross-presentation of tumor antigens. We therefore established a cross-presentation assay to screen over 5,500 compounds for enhancers of DC cross-presentation using induced T-cell proliferation as the readout. We identified 145 enhancers, including AZD5582, an antagonist of inhibitor of apoptosis proteins (IAP) cIAP1, cIAP2, and XIAP. AZD5582 treatment led to DC activation of the noncanonical NF-kB pathway, enhanced antigen import from endolysosomes into the cytosol, and increased expression of genes involved in cross-presentation. Furthermore, it upregulated expression of CD80, CD86, MHC class II, CD70 and secretion of TNF by DCs. This enhanced DC activation and maturation program was observed also in tumor-bearing mice upon AZD5582 treatment, culminating in an increased frequency of systemic tumor antigen-specific CD8+ T cells. Our results merit further exploration of AZD5582 to increase antigen cross-presentation for improving the clinical benefit of ICB in patients who are unlikely to respond to ICB.

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.

Rational combination of SHP2 and mTOR inhibition for the treatment of hepatocellular carcinoma.

Liver cancer is the fourth most common cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the main primary malignancy affecting the liver. Unfortunately, there are still limited therapeutic options for HCC, and even the latest advances have only increased the overall survival modestly. Thus, new treatment strategies and rational drug combinations are urgently needed. Reactivation of receptor tyrosine kinases (RTK) has been described as a mechanism of intrinsic resistance to targeted therapies in a variety of cancers, including inhibitors of mTOR. The design of rational combination therapies to overcome this type of resistance is complicated by the notion that multiple RTK can be upregulated during the acquisition of resistance. SHP2, encoded by the gene PTPN11, acts downstream of virtually all RTK, and has proven to be a good target for small molecule inhibitors. Here, we report activation of multiple RTK upon mTOR inhibition in HCC which, through SHP2, leads to reactivation of the mTOR pathway. We show that co-inhibition of both mTOR and SHP2 is highly synergistic in vitro by triggering apoptosis. More importantly, the combination is well-tolerated and outperforms the monotherapies in impairing tumor growth in multiple HCC mouse models. Our findings suggest a novel rational combination therapy for the treatment of HCC.

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).

Inhibition of casein kinase 2 sensitizes mantle cell lymphoma to venetoclax through MCL-1 downregulation.

BCL-2 family proteins are frequently aberrantly expressed in mantle cell lymphoma (MCL). Recently, the BCL-2-specific inhibitor venetoclax has been approved by the US Food and Drug Administration for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). In MCL, venetoclax has shown promising efficacy in early clinical trials; however, a significant subset of patients is resistant. By conducting a kinome-centered CRISPR-Cas9 knockout sensitizer screen, we identified casein kinase 2 (CK2) as a major regulator of venetoclax resistance in MCL. Interestingly, CK2 is over-expressed in MCL and high CK2 expression is associated with poor patient survival. Targeting of CK2, either by inducible short hairpin RNA (shRNA)-mediated knockdown of CK2 or by the CK2-inhibitor silmitasertib, did not affect cell viability by itself, but strongly synergized with venetoclax in both MCL cell lines and primary samples, also if combined with ibrutinib. Furthermore, targeting of CK2 reduced MCL-1 levels, which involved impaired MCL-1 translation by inhibition of eIF4F complex assembly, without affecting BCL-2 and BCL-XL expression. Combined, this results in enhanced BCL-2 dependence and, consequently, venetoclax sensitization. In cocultures, targeting of CK2 overcame stroma-mediated venetoclax resistance of MCL cells. Taken together, our findings indicate that targeting of CK2 sensitizes MCL cells to venetoclax through downregulation of MCL-1. These novel insights provide a strong rationale for combining venetoclax with CK2 inhibition as therapeutic strategy for MCL patients.

An Unbiased CRISPR-Cas9 Screening Method for the Identification of Positive and Negative Regulatory Proteins of Cell Adhesion.

Mature B-cell lymphomas are highly dependent upon the protective lymphoid organ microenvironment for their growth and survival. Targeting integrin-mediated homing and retention of the malignant B cells in the lymphoid organs, using the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, is a highly efficacious FDA-approved therapy for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM). Unfortunately, a significant subset of patients is intrinsically resistant to ibrutinib or will develop resistance upon prolonged treatment. Here, we describe an unbiased functional genomic CRISPR-Cas9 screening method to identify novel proteins involved in B-cell receptor-controlled integrin-mediated adhesion, which provides novel therapeutic targets to overcome ibrutinib resistance. This screening method is highly flexible and can be easily adapted to identify cell adhesion-regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types. Graphical abstract.

cFLIP suppression and DR5 activation sensitize senescent cancer cells to senolysis.

Senolytics, drugs that kill senescent cells, have been proposed to improve the response to pro-senescence cancer therapies; however, this remains challenging due to a lack of broadly acting senolytic drugs. Using CRISPR/Cas9-based genetic screens in different senescent cancer cell models, we identify loss of the death receptor inhibitor cFLIP as a common vulnerability of senescent cancer cells. Senescent cells are primed for apoptotic death by NF-κB-mediated upregulation of death receptor 5 (DR5) and its ligand TRAIL, but are protected from death by increased cFLIP expression. Activation of DR5 signaling by agonistic antibody, which can be enhanced further by suppression of cFLIP by BRD2 inhibition, leads to efficient killing of a variety of senescent cancer cells. Moreover, senescent cells sensitize adjacent non-senescent cells to killing by DR5 agonist through a bystander effect mediated by secretion of cytokines. We validate this 'one-two punch' cancer therapy by combining pro-senescence therapy with DR5 activation in different animal models.