A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.

DNA repair, gap suppression, or fork protection: BRCA2 needs a break!

In this issue of Molecular Cell, Lim et al.1 reveal new insights into the distinct roles of BRCA2 in coping with DNA breaks, highlighting homologous recombination as the pivotal function that affects tumorigenesis and therapy response.

DNA-PKcs promotes fork reversal and chemoresistance.

The DNA-PKcs kinase mediates the repair of DNA double-strand breaks via classical non-homologous end joining (NHEJ). DNA-PKcs is also recruited to active replication forks, although a role for DNA-PKcs in the control of fork dynamics is unclear. Here, we identify a crucial role for DNA-PKcs in promoting fork reversal, a process that stabilizes stressed replication forks and protects genome integrity. DNA-PKcs promotes fork reversal and slowing in response to several replication stress-inducing agents in a manner independent of its role in NHEJ. Cells lacking DNA-PKcs activity show increased DNA damage during S-phase and cellular sensitivity to replication stress. Notably, prevention of fork slowing and reversal via DNA-PKcs inhibition efficiently restores chemotherapy sensitivity in BRCA2-deficient mammary tumors with acquired PARPi resistance. Together, our data uncover a new key regulator of fork reversal and show how DNA-PKcs signaling can be manipulated to alter fork dynamics and drug resistance in cancer.

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.

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.

Tumour microenvironment characterisation to stratify patients for hyperthermic intraperitoneal chemotherapy in high-grade serous ovarian cancer (OVHIPEC-1).

BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) improves survival in patients with Stage III ovarian cancer following interval cytoreductive surgery (CRS). Optimising patient selection is essential to maximise treatment efficacy and avoid overtreatment. This study aimed to identify biomarkers that predict HIPEC benefit by analysing gene signatures and cellular composition of tumours from participants in the OVHIPEC-1 trial. METHODS: Whole-transcriptome RNA sequencing data were retrieved from high-grade serous ovarian cancer (HGSOC) samples from 147 patients obtained during interval CRS. We performed differential gene expression analysis and applied deconvolution methods to estimate cell-type proportions in bulk mRNA data, validated by histological assessment. We tested the interaction between treatment and potential predictors on progression-free survival using Cox proportional hazards models. RESULTS: While differential gene expression analysis did not yield any predictive biomarkers, the cellular composition, as characterised by deconvolution, indicated that the absence of macrophages and the presence of B cells in the tumour microenvironment are potential predictors of HIPEC benefit. The histological assessment confirmed the predictive value of macrophage absence. CONCLUSION: Immune cell composition, in particular macrophages absence, may predict response to HIPEC in HGSOC and these hypothesis-generating findings warrant further investigation. CLINICAL TRIAL REGISTRATION: NCT00426257.

DNA Damage Response and Mismatch Repair Gene Defects in Advanced and Metastatic Prostate Cancer.

Alterations in DNA damage response (DDR) and related genes are present in up to 25% of advanced prostate cancers (PCa). Most frequently altered genes are involved in the homologous recombination repair, the Fanconi anemia, and the mismatch repair pathways, and their deficiencies lead to a highly heterogeneous spectrum of DDR-deficient phenotypes. More than half of these alterations concern non- BRCA DDR genes. From a therapeutic perspective, poly-ADP-ribose polymerase inhibitors have demonstrated robust clinical efficacy in tumors with BRCA2 and BRCA1 alterations. Mismatch repair-deficient PCa, and a subset of CDK12-deficient PCa, are vulnerable to immune checkpoint inhibitors. Emerging data point to the efficacy of ATR inhibitors in PCa with ATM deficiencies. Still, therapeutic implications are insufficiently clarified for most of the non- BRCA DDR alterations, and no successful targeted treatment options have been established.

53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ.

53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination1,2, and reduced fidelity of long-range V(D)J recombination3. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres4, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis5-7. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive8, unlike 53BP1-deficient cells9,10, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.

The shieldin complex mediates 53BP1-dependent DNA repair.

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

HELB Is a Feedback Inhibitor of DNA End Resection.

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.

Canine follicular cell and medullary thyroid carcinomas: Immunohistochemical characterization.

Research on modulation of iodine uptake by thyroid cells could help improve radioiodine treatment of dogs with thyroid tumors. The aim of this study was to characterize the immunohistochemical expression of thyroid transcription factor-1 (TTF-1), thyroglobulin, thyrotropin receptor (TSHR), sodium iodide symporter (NIS), pendrin, thyroid peroxidase (TPO), vimentin, and Ki-67 in follicular cell thyroid carcinomas (FTCs) and medullary thyroid carcinomas (MTCs), and to compare protein expression between FTC causing hyperthyroidism and FTC of euthyroid dogs. Immunohistochemistry was performed in 25 FTCs (9 follicular, 8 follicular-compact, and 8 compact) and 8 MTCs. FTCs and MTCs were positive for TTF-1, and expression was higher in FTCs of euthyroid dogs compared with FTCs of hyperthyroid dogs (P= .041). Immunolabeling for thyroglobulin was higher in follicular and follicular-compact FTCs compared with compact FTCs (P = .001), while vimentin expression was higher in follicular-compact FTCs compared with follicular FTCs (P = .011). The expression of TSHR, NIS, pendrin, and TPO was not significantly different among the different subtypes of FTCs or between FTCs causing hyperthyroidism and FTCs in euthyroid dogs. TSHR, NIS, pendrin, and TPO were also expressed in MTCs. Ki-67 labeling index was comparable between FTCs and MTCs, and between FTCs causing hyperthyroidism and FTCs in euthyroid dogs. Proteins of iodine transport were also expressed in canine MTCs, which could have implications for diagnosis and treatment. The different expression of thyroglobulin and vimentin between FTC histological subtypes could reflect variations in tumor differentiation.

Replication fork stability confers chemoresistance in BRCA-deficient cells.

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.

BRCA-deficient mouse mammary tumor organoids to study cancer-drug resistance.

Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.

REV7 counteracts DNA double-strand break resection and affects PARP inhibition.

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Validation of Immunohistochemistry for Canine Proteins Involved in Thyroid Iodine Uptake and Their Expression in Canine Follicular Cell Thyroid Carcinomas (FTCs) and FTC-Derived Organoids.

Thyrotropin receptor (TSHR), sodium iodide symporter (NIS), pendrin, and thyroid peroxidase (TPO) are essential for the uptake of iodine by follicular thyroid cells. The aim of this study was to establish immunohistochemistry (IHC) protocols for TSHR, NIS, pendrin, and TPO in canine tissues and characterize their expression in organoids derived from canine follicular cell thyroid carcinoma (FTC) and in the respective primary tumors. This constitutes a fundamental step to establish organoids as a model to study the uptake of iodine in canine FTC. Commercially available antibodies directed against human proteins were selected. Antibody specificity was confirmed by western blot using lysates of the HTori-3 human thyroid cell line and healthy canine thyroid gland. IHC was validated using HTori-3 cells and a set of canine normal tissues including healthy thyroid gland. The expression of TSHR, NIS, pendrin, and TPO was evaluated in 3 organoid lines derived from FTC and respective primary tumors. All 4 antibodies produced specific bands by western blot and cytoplasmic labeling in follicular cells by IHC in both human HTori-3 cells and canine thyroid gland. NIS also showed basolateral membrane immunolabeling in follicular cells. All 4 proteins were highly expressed in organoids derived from FTC. The expression was similar or higher compared to the primary tumors. The results of this study characterize organoids derived from canine FTC as a suitable in vitro model to investigate iodine uptake, opening new research possibilities in the field of canine thyroid cancer therapy.

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs.

Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.

Identification and characterisation of a Theileria annulata proline-rich microtubule and SH3 domain-interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface.

Theileria annulata is an apicomplexan parasite that modifies the phenotype of its host cell completely, inducing uncontrolled proliferation, resistance to apoptosis, and increased invasiveness. The infected cell thus resembles a cancer cell, and changes to various host cell signalling pathways accompany transformation. Most of the molecular mechanisms leading to Theileria-induced immortalization of leukocytes remain unknown. The parasite dissolves the surrounding host cell membrane soon after invasion and starts interacting with host proteins, ensuring its propagation by stably associating with the host cell microtubule network. By using BioID technology together with fluorescence microscopy and co-immunoprecipitation, we identified a CLASP1/CD2AP/EB1-containing protein complex that surrounds the schizont throughout the host cell cycle and integrates bovine adaptor proteins (CIN85, 14-3-3 epsilon, and ASAP1). This complex also includes the schizont membrane protein Ta-p104 together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain-interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface and contains a functional EB1-binding SxIP motif, as well as functional SH3 domain-binding Px(P/A)xPR motifs that mediate its interaction with CD2AP. Upon overexpression in non-infected bovine macrophages, TaMISHIP causes binucleation, potentially indicative of a role in cytokinesis.

New tools for old drugs: Functional genetic screens to optimize current chemotherapy.

Despite substantial advances in the treatment of various cancers, many patients still receive anti-cancer therapies that hardly eradicate tumor cells but inflict considerable side effects. To provide the best treatment regimen for an individual patient, a major goal in molecular oncology is to identify predictive markers for a personalized therapeutic strategy. Regarding novel targeted anti-cancer therapies, there are usually good markers available. Unfortunately, however, targeted therapies alone often result in rather short remissions and little cytotoxic effect on the cancer cells. Therefore, classical chemotherapy with frequent long remissions, cures, and a clear effect on cancer cell eradication remains a corner stone in current anti-cancer therapy. Reliable biomarkers which predict the response of tumors to classical chemotherapy are rare, in contrast to the situation for targeted therapy. For the bulk of cytotoxic therapeutic agents, including DNA-damaging drugs, drugs targeting microtubules or antimetabolites, there are still no reliable biomarkers used in the clinic to predict tumor response. To make progress in this direction, meticulous studies of classical chemotherapeutic drug action and resistance mechanisms are required. For this purpose, novel functional screening technologies have emerged as successful technologies to study chemotherapeutic drug response in a variety of models. They allow a systematic analysis of genetic contributions to a drug-responsive or -sensitive phenotype and facilitate a better understanding of the mode of action of these drugs. These functional genomic approaches are not only useful for the development of novel targeted anti-cancer drugs but may also guide the use of classical chemotherapeutic drugs by deciphering novel mechanisms influencing a tumor's drug response. Moreover, due to the advances of 3D organoid cultures from patient tumors and in vivo screens in mice, these genetic screens can be applied using conditions that are more representative of the clinical setting. Patient-derived 3D organoid lines furthermore allow the characterization of the "essentialome", the specific set of genes required for survival of these cells, of an individual tumor, which could be monitored over the course of treatment and help understanding how drug resistance evolves in clinical tumors. Thus, we expect that these functional screens will enable the discovery of novel cancer-specific vulnerabilities, and through clinical validation, move the field of predictive biomarkers forward. This review focuses on novel advanced techniques to decipher the interplay between genetic alterations and drug response.