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.

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.

Drug-repurposing screen on patient-derived organoids identifies therapy-induced vulnerability in KRAS-mutant colon cancer.

Patient-derived organoids (PDOs) are widely heralded as a drug-screening platform to develop new anti-cancer therapies. Here, we use a drug-repurposing library to screen PDOs of colorectal cancer (CRC) to identify hidden vulnerabilities within therapy-induced phenotypes. Using a microscopy-based screen that accurately scores drug-induced cell killing, we have tested 414 putative anti-cancer drugs for their ability to switch the EGFRi/MEKi-induced cytostatic phenotype toward cytotoxicity. A majority of validated hits (9/37) are microtubule-targeting agents that are commonly used in clinical oncology, such as taxanes and vinca-alkaloids. One of these drugs, vinorelbine, is consistently effective across a panel of >25 different CRC PDOs, independent of RAS mutational status. Unlike vinorelbine alone, its combination with EGFR/MEK inhibition induces apoptosis at all stages of the cell cycle and shows tolerability and effective anti-tumor activity in vivo, setting the basis for a clinical trial to treat patients with metastatic RAS-mutant CRC.

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer.

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity in vitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.

Truncated FGFR2 is a clinically actionable oncogene in multiple cancers.

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1-9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.

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.

Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer.

BACKGROUND: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype. METHODS: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors. RESULTS: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors. CONCLUSION: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

QPCTL regulates macrophage and monocyte abundance and inflammatory signatures in the tumor microenvironment.

The enzyme glutaminyl-peptide cyclotransferase-like protein (QPCTL) catalyzes the formation of pyroglutamate residues at the NH2-terminus of proteins, thereby influencing their biological properties. A number of studies have implicated QPCTL in the regulation of chemokine stability. Furthermore, QPCTL activity has recently been shown to be critical for the formation of the high-affinity SIRPα binding site of the CD47 "don't-eat-me" protein. Based on the latter data, interference with QPCTL activity -and hence CD47 maturation-may be proposed as a means to promote anti-tumor immunity. However, the pleiotropic activity of QPCTL makes it difficult to predict the effects of QPCTL inhibition on the tumor microenvironment (TME). Using a syngeneic mouse melanoma model, we demonstrate that QPCTL deficiency alters the intra-tumoral monocyte-to-macrophage ratio, results in a profound increase in the presence of pro-inflammatory cancer-associated fibroblasts (CAFs) relative to immunosuppressive TGF-ß1-driven CAFs, and leads to an increased IFN and decreased TGF-ß transcriptional response signature in tumor cells. Importantly, the functional relevance of the observed TME remodeling is demonstrated by the synergy between QPCTL deletion and anti PD-L1 therapy, sensitizing an otherwise refractory melanoma model to anti-checkpoint therapy. Collectively, these data provide support for the development of strategies to interfere with QPCTL activity as a means to promote tumor-specific immunity.

Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer.

Cervical cancer is a common gynecological malignancy often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study the cervical epithelium and the cancers derived thereof. Here we describe a long-term culturing protocol for ecto- and endocervical epithelia that generates 3D organoids that stably recapitulate the two tissues of origin. As evidenced for HSV-1, organoid-based cervical models may serve to study sexually transmitted infections. Starting from Pap brush material, a small biobank of tumoroids derived from affected individuals was established that retained the causative human papillomavirus (HPV) genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutic agents and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches.

SLC1A3 contributes to L-asparaginase resistance in solid tumors.

L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.

An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.

Inhibition of the Replication Stress Response Is a Synthetic Vulnerability in SCLC That Acts Synergistically in Combination with Cisplatin.

Small cell lung cancer (SCLC) is generally regarded as very difficult to treat, mostly due to the development of metastases early in the disease and a quick relapse with resistant disease. SCLC patients initially show a good response to treatment with the DNA damaging agents cisplatin and etoposide. This is, however, quickly followed by the development of resistant disease, which urges the development of novel therapies for this type of cancer. In this study, we set out to compile a comprehensive overview of the vulnerabilities of SCLC. A functional genome-wide screen where all individual genes were knocked out was performed to identify novel vulnerabilities of SCLC. By analysis of the knockouts that were lethal to these cancer cells, we identified several processes to be synthetic vulnerabilities in SCLC. We were able to validate the vulnerability to inhibition of the replication stress response machinery by use of Chk1 and ATR inhibitors. Strikingly, SCLC cells were more sensitive to these inhibitors than nontransformed cells. In addition, these inhibitors work synergistically with either etoposide and cisplatin, where the interaction is largest with the latter. ATR inhibition by VE-822 treatment in combination with cisplatin also outperforms the combination of cisplatin with etoposide in vivo Altogether, our study uncovered a critical dependence of SCLC on the replication stress response and urges the validation of ATR inhibitors in combination with cisplatin in a clinical setting.

Long-term expanding human airway organoids for disease modeling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Radiosensitivity Is an Acquired Vulnerability of PARPi-Resistant BRCA1-Deficient Tumors.

The defect in homologous recombination (HR) found in BRCA1-associated cancers can be therapeutically exploited by treatment with DNA-damaging agents and PARP inhibitors. We and others previously reported that BRCA1-deficient tumors are initially hypersensitive to the inhibition of topoisomerase I/II and PARP, but acquire drug resistance through restoration of HR activity by the loss of end-resection antagonists of the 53BP1/RIF1/REV7/Shieldin/CST pathway. Here, we identify radiotherapy as an acquired vulnerability of 53BP1;BRCA1-deficient cells in vitro and in vivo. In contrast to the radioresistance caused by HR restoration through BRCA1 reconstitution, HR restoration by 53BP1 pathway inactivation further increases radiosensitivity. This highlights the relevance of this pathway for the repair of radiotherapy-induced damage. Moreover, our data show that BRCA1-mutated tumors that acquire drug resistance due to BRCA1-independent HR restoration can be targeted by radiotherapy. SIGNIFICANCE: These findings uncover radiosensitivity as a novel, therapeutically viable vulnerability of BRCA1-deficient mouse mammary cells that have acquired drug resistance due to the loss of the 53BP1 pathway.

Ezh2 inhibition in Kras-driven lung cancer amplifies inflammation and associated vulnerabilities.

Kras-driven non-small-cell lung cancers (NSCLCs) are a leading cause of death with limited therapeutic options. Many NSCLCs exhibit high levels of Ezh2, the enzymatic subunit of polycomb repressive complex 2 (PRC2). We tested Ezh2 inhibitors as single agents or before chemotherapy in mice with orthotopic Kras-driven NSCLC grafts, which homogeneously express Ezh2. These tumors display sensitivity to EZH2 inhibition by GSK126 but also amplify an inflammatory program involving signaling through NF-κB and genes residing in PRC2-regulated chromatin. During this process, tumor cells overcome GSK126 antiproliferative effects. We identified oncogenes that may mediate progression through an in vivo RNAi screen aimed at targets of PRC2/NF-κB. An in vitro compound screening linked GSK126-driven inflammation and therapeutic vulnerability in human cells to regulation of RNA synthesis and proteostasis. Interestingly, GSK126-treated NSCLCs in vivo also showed an enhanced response to a combination of nimesulide and bortezomib. Thus, Ezh2 inhibition may restrict cell proliferation and promote defined adaptive responses. Targeting these responses potentially improves outcomes in Kras-driven NSCLCs.

SOX2 Is the Determining Oncogenic Switch in Promoting Lung Squamous Cell Carcinoma from Different Cells of Origin.

Lung squamous cell carcinoma (LSCC) is a devastating malignancy with no effective treatments, due to its complex genomic profile. Therefore, preclinical models mimicking its salient features are urgently needed. Here we describe mouse models bearing various combinations of genetic lesions predominantly found in human LSCC. We show that SOX2 but not FGFR1 overexpression in tracheobronchial basal cells combined with Cdkn2ab and Pten loss results in LSCC closely resembling the human counterpart. Interestingly, Sox2;Pten;Cdkn2ab mice develop LSCC with a more peripheral location when Club or Alveolar type 2 (AT2) cells are targeted. Our model highlights the essential role of SOX2 in commanding the squamous cell fate from different cells of origin and represents an invaluable tool for developing better intervention strategies.

Transcription Factor NFIB Is a Driver of Small Cell Lung Cancer Progression in Mice and Marks Metastatic Disease in Patients.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor, and no effective treatment is available to date. Mouse models of SCLC based on the inactivation of Rb1 and Trp53 show frequent amplifications of the Nfib and Mycl genes. Here, we report that, although overexpression of either transcription factor accelerates tumor growth, NFIB specifically promotes metastatic spread. High NFIB levels are associated with expansive growth of a poorly differentiated and almost exclusively E-cadherin (CDH1)-negative invasive tumor cell population. Consistent with the mouse data, we find that NFIB is overexpressed in almost all tested human metastatic high-grade neuroendocrine lung tumors, warranting further assessment of NFIB as a tumor progression marker in a clinical setting.

Polycomb Repressive Complex 2 Is a Barrier to KRAS-Driven Inflammation and Epithelial-Mesenchymal Transition in Non-Small-Cell Lung Cancer.

Polycomb repressive complexes (PRC) are frequently implicated in human cancer, acting either as oncogenes or tumor suppressors. Here, we show that PRC2 is a critical regulator of KRAS-driven non-small cell lung cancer progression. Modulation of PRC2 by either Ezh2 overexpression or Eed deletion enhances KRAS-driven adenomagenesis and inflammation, respectively. Eed-loss-driven inflammation leads to massive macrophage recruitment and marked decline in tissue function. Additional Trp53 inactivation activates a cell-autonomous epithelial-to-mesenchymal transition program leading to an invasive mucinous adenocarcinoma. A switch between methylated/acetylated chromatin underlies the tumor phenotypic evolution, prominently involving genes controlled by Hippo/Wnt signaling. Our observations in the mouse models were conserved in human cells. Importantly, PRC2 inactivation results in context-dependent phenotypic alterations, with implications for its therapeutic application.

Paracrine signaling between tumor subclones of mouse SCLC: a critical role of ETS transcription factor Pea3 in facilitating metastasis.

Tumor heterogeneity can create a unique symbiotic tumor microenvironment. Earlier, we showed that clonal evolution in mouse small cell lung cancer (SCLC) can result in subclones that, upon cografting, endow the neuroendocrine tumor cells with metastatic potential. We now show that paracrine signaling between SCLC subclones is a critical requirement in the early steps of the metastatic process, such as local invasion and intravasation. We further show evidence that paracrine signaling via fibroblast growth factor 2 (Fgf2) and Mapk between these diverged tumor subclones causes enhanced expression of the Pea3 (polyomavirus enhancer activator 3) transcription factor, resulting in metastatic dissemination of the neuroendocrine tumor subclones. Our data reveal for the first time paracrine signaling between tumor cell subclones in SCLC that results in metastatic spread of SCLC.