CD8 T cell-mediated depletion of HBV surface-antigen-expressing, bilineal-differentiated liver carcinoma cells generates highly aggressive escape variants.

The expression of viral antigens in chronic hepatitis B virus (HBV) infection drives continuous liver inflammation, one of the main risk factors to develop liver cancer. HBV developed immune-suppressive functions to escape from the host immune system, but their link to liver tumor development is not well understood. Here, we analyzed if and how HBV surface antigen (HBs) expression in combined hepatocellular-cholangiocarcinoma (cHCC/iCCA) cells influences their antigenicity for CD8 T cells. We randomly isolated liver tumor tissues from AlfpCre+-Trp53fl/fl/Alb-HBs+ tg mice and established primary carcinoma cell lines (pCCL) that showed a bilineal (CK7+/HNF4α+) cHCC/iCCA phenotype. These pCCL uniformly expressed HBs (HBshi), and low levels of MHC-I (MHC-Ilo), and were transiently convertible to a high antigenicity (MHC-Ihi) phenotype by IFN-γ treatment. HBshi/pCCL induced HBs/(Kb/S190-197)-specific CD8 T cells and developed slow-growing tumors in subcutaneously transplanted C57Bl/6J (B6) mice. Interestingly, pCCL-ex cells, established from HBshi/pCCL-induced and re-explanted tumors in B6 but not those in immune-deficient Rag1-/- mice showed major alterations, like an MHC-Ihi phenotype, a prominent growth-biased gene expression signature, a significantly decreased HBs expression (HBslo) and a switch to fast-growing tumors in re-transplanted B6 or PD-1-/- hosts with an unlocked PD-1/PD-L1 control system. CD8 T cell-mediated elimination of HBshi/pCCL, together with the attenuation of the negative restraints of HBs in the tumor cells, like ER-stress, reveals a novel mechanism to unleash highly aggressive HBslo/pCCL-ex immune-escape variants. Under certain conditions, HBs-specific CD8 T-cell responses thus potentiate tumor growth, an aspect that should be considered for therapeutic vaccination strategies against chronic HBV infection and liver tumors.

Loss of SUV420H2-Dependent Chromatin Compaction Drives Right-Sided Colon Cancer Progression.

BACKGROUND & AIMS: Epigenetic processes regulating gene expression contribute markedly to epithelial cell plasticity in colorectal carcinogenesis. The lysine methyltransferase SUV420H2 comprises an important regulator of epithelial plasticity and is primarily responsible for trimethylation of H4K20 (H4K20me3). Loss of H4K20me3 has been suggested as a hallmark of human cancer due to its interaction with DNMT1. However, the role of Suv4-20h2 in colorectal cancer is unknown. METHODS: We examined the alterations in histone modifications in patient-derived colorectal cancer organoids. Patient-derived colorectal cancer organoids and mouse intestinal organoids were genetically manipulated for functional studies in patient-derived xenograft and orthotopic transplantation. Gene expression profiling, micrococcal nuclease assay, and chromatin immunoprecipitation were performed to understand epigenetic regulation of chromatin states and gene expression in patient-derived and mouse intestinal organoids. RESULTS: We found that reduced H4K20me3 levels occurred predominantly in right-sided patient-derived colorectal cancer organoids, which were associated with increased chromatin accessibility. Re-compaction of chromatin by methylstat, a histone demethylase inhibitor, resulted in reduced growth selectively in subcutaneously grown tumors derived from right-sided cancers. Using mouse intestinal organoids, we confirmed that Suv4-20h2-mediated H4K20me3 is required for maintaining heterochromatin compaction and to prevent R-loop formation. Cross-species comparison of Suv4-20h2-depleted murine organoids with right-sided colorectal cancer organoids revealed a large overlap of gene signatures involved in chromatin silencing, DNA methylation, and stemness/Wnt signaling. CONCLUSIONS: Loss of Suv4-20h2-mediated H4K20me3 drives right-sided colorectal tumorigenesis through an epigenetically controlled mechanism of chromatin compaction. Our findings unravel a conceptually novel approach for subtype-specific therapy of this aggressive form of colorectal cancer.

CARD9 Forms an Alternative CBM Complex in Richter Syndrome.

Richter syndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, mostly diffuse large B-cell lymphoma (DLBCL). Despite intensive therapy, patients with RS have an unfavorable clinical outcome. The detailed pathobiology of Richter transformation still needs to be elucidated. Here, we report high mRNA and protein levels of CARD9 in the RS cell line U-RT1. Co-immunoprecipitation revealed the assembly of a CBM complex using CARD9 instead of CARD11. CARD9 is known to be an activator of NF-кB signaling in myeloid cells. U-RT1 Western blot analyses showed phosphorylation of IκB as well as IKK, indicating a constitutively active canonical NF-кB pathway. This was further supported by the significant reduction in cell viability and CYLD cleavage products after CARD9 siRNA knockdown. We also showed immunostaining for CARD9 in 53% of cases analyzed in a series of RS tissue specimens, whereas other lymphomas rarely show CARD9 expression. This is the first report on ectopic expression and function of CARD9 in an aggressive B-cell lymphoma. Our findings suggest that CARD9 may contribute to the pathogenesis of RS.

Small Extracellular Vesicles Propagate the Inflammatory Response After Trauma.

Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma (TxT) is strongly associated with trauma-related death, an unbalanced innate immune response, sepsis, acute respiratory distress syndrome, and multiple organ dysfunction. It is shown that different in vivo traumata, such as TxT or an in vitro polytrauma cytokine cocktail trigger secretion of small extracellular nanovesicles (sEVs) from endothelial cells with pro-inflammatory cargo. These sEVs transfer transcripts for ICAM-1, VCAM-1, E-selectin, and cytokines to systemically activate the endothelium, facilitate neutrophil-endothelium interactions, and destabilize barrier integrity. Inhibition of sEV-release after TxT in mice ameliorates local as well as systemic inflammation, neutrophil infiltration, and distant organ damage in kidneys (acute kidney injury, AKI). Vice versa, injection of TxT-plasma-sEVs into healthy animals is sufficient to trigger pulmonary and systemic inflammation as well as AKI. Accordingly, increased sEV concentrations and transfer of similar cargos are observed in polytrauma patients, suggesting a fundamental pathophysiological mechanism.

Telomerase and Pluripotency Factors Jointly Regulate Stemness in Pancreatic Cancer Stem Cells.

To assess the role of telomerase activity and telomere length in pancreatic CSCs we used different CSC enrichment methods (CD133, ALDH, sphere formation) in primary patient-derived pancreatic cancer cells. We show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic knockdown or pharmacological inhibitor (BIBR1532), resulted in CSC marker depletion, abrogation of sphere formation in vitro and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (NANOG, OCT3/4, SOX2, KLF4) and telomerase, which is essential for the self-renewal of CSCs. Disruption of the balance between telomerase activity and stemness factors eliminates CSCs via induction of DNA damage and apoptosis in primary patient-derived pancreatic cancer samples, opening future perspectives to avoid CSC-driven tumor relapse. In the present study, we demonstrate that telomerase regulation is critical for the "stemness" maintenance in pancreatic CSCs and examine the effects of telomerase inhibition as a potential treatment option of pancreatic cancer. This may significantly promote our understanding of PDAC tumor biology and may result in improved treatment for pancreatic cancer patients.

Functional Genomic Screening During Somatic Cell Reprogramming Identifies DKK3 as a Roadblock of Organ Regeneration.

Somatic cell reprogramming and tissue repair share relevant factors and molecular programs. Here, Dickkopf-3 (DKK3) is identified as novel factor for organ regeneration using combined transcription-factor-induced reprogramming and RNA-interference techniques. Loss of Dkk3 enhances the generation of induced pluripotent stem cells but does not affect de novo derivation of embryonic stem cells, three-germ-layer differentiation or colony formation capacity of liver and pancreatic organoids. However, DKK3 expression levels in wildtype animals and serum levels in human patients are elevated upon injury. Accordingly, Dkk3-null mice display less liver damage upon acute and chronic failure mediated by increased proliferation in hepatocytes and LGR5+ liver progenitor cell population, respectively. Similarly, recovery from experimental pancreatitis is accelerated. Regeneration onset occurs in the acinar compartment accompanied by virtually abolished canonical-Wnt-signaling in Dkk3-null animals. This results in reduced expression of the Hedgehog repressor Gli3 and increased Hedgehog-signaling activity upon Dkk3 loss. Collectively, these data reveal Dkk3 as a key regulator of organ regeneration via a direct, previously unacknowledged link between DKK3, canonical-Wnt-, and Hedgehog-signaling.

Molecular features and vulnerabilities of recurrent chordomas.

BACKGROUND: Tumor recurrence is one of the major challenges in clinical management of chordoma. Despite R0-resection, approximately 50% of chordomas recur within ten years after initial surgery. The underlying molecular processes are poorly understood resulting in the lack of associated therapeutic options. This is not least due to the absence of appropriate cell culture models of this orphan disease. METHODS: The intra-personal progression model cell lines U-CH11 and U-CH11R were compared using array comparative genomic hybridization, expression arrays, RNA-seq, and immunocytochemistry. Cell line origin was confirmed by short tandem repeat analysis. Inter-personal cell culture models (n = 6) were examined to validate whether the new model is representative. Cell viability after HOX/PBX complex inhibition with small peptides was determined by MTS assays. RESULTS: Using whole genome microarray analyses, striking differences in gene expression between primary and recurrent chordomas were identified. These expression differences were confirmed in the world's first intra-personal model of chordoma relapse consisting of cell lines established from a primary (U-CH11) and the corresponding recurrent tumor (U-CH11R). Array comparative genomic hybridization and RNA-sequencing analyses revealed profound genetic similarities between both cell lines pointing to transcriptomic reprogramming as a key mechanism of chordoma progression. Network analysis of the recurrence specific genes highlighted HOX/PBX signaling as a common dysregulated event. Hence, HOX/PBX complexes were used as so far unknown therapeutic targets in recurrent chordomas. Treating chordoma cell lines with the complex formation inhibiting peptide HXR9 induced cFOS mediated apoptosis in all chordoma cell lines tested. This effect was significantly stronger in cell lines established from chordoma relapses. CONCLUSION: Clearly differing gene expression patterns and vulnerabilities to HOX/PBX complex inhibition in highly therapy resistant chordoma relapses were identified using the first intra-personal loco-regional and further inter-personal chordoma progression models. For the first time, HOX/PBX interference was used to induce cell death in chordoma and might serve as the basic concept of an upcoming targeted therapy for chordomas of all progression stages.

Iron at the Interface of Hepatocellular Carcinoma.

Cancer incidence and mortality are rapidly growing, with liver cancer being the sixth most diagnosed cancer worldwide and the third leading cause of cancer death in 2020. A number of risk factors have been identified that trigger the progression to hepatocellular carcinoma. In this review, we focus on iron as a potential risk factor for liver carcinogenesis. Molecules involved in the regulation of iron metabolism are often upregulated in cancer cells, in order to provide a supply of this essential trace element for all stages of tumor development, survival, proliferation, and metastasis. Thus, cellular and systemic iron levels must be tightly regulated to prevent or delay liver cancer progression. Disorders associated with dysregulated iron metabolism are characterized with increased susceptibility to hepatocellular carcinoma. This review discusses the association of iron with metabolic disorders such as hereditary hemochromatosis, non-alcoholic fatty liver disease, obesity, and type 2 diabetes, in the background of hepatocellular carcinoma.

RINT1 Regulates SUMOylation and the DNA Damage Response to Preserve Cellular Homeostasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) still presents with a dismal prognosis despite intense research. Better understanding of cellular homeostasis could identify druggable targets to improve therapy. Here we propose RAD50-interacting protein 1 (RINT1) as an essential mediator of cellular homeostasis in PDAC. In a cohort of resected PDAC, low RINT1 protein expression correlated significantly with better survival. Accordingly, RINT1 depletion caused severe growth defects in vitro associated with accumulation of DNA double-strand breaks (DSB), G2 cell cycle arrest, disruption of Golgi-endoplasmic reticulum homeostasis, and cell death. Time-resolved transcriptomics corroborated by quantitative proteome and interactome analyses pointed toward defective SUMOylation after RINT1 loss, impairing nucleocytoplasmic transport and DSB response. Subcutaneous xenografts confirmed tumor response by RINT1 depletion, also resulting in a survival benefit when transferred to an orthotopic model. Primary human PDAC organoids licensed RINT1 relevance for cell viability. Taken together, our data indicate that RINT1 loss affects PDAC cell fate by disturbing SUMOylation pathways. Therefore, a RINT1 interference strategy may represent a new putative therapeutic approach. SIGNIFICANCE: These findings provide new insights into the aggressive behavior of PDAC, showing that RINT1 directly correlates with survival in patients with PDAC by disturbing the SUMOylation process, a crucial modification in carcinogenesis.

p53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

BACKGROUND & AIMS: A coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury. METHODS: Nitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2. RESULTS: In a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response. CONCLUSIONS: Characteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.

Synergistic targeting and resistance to PARP inhibition in DNA damage repair-deficient pancreatic cancer.

OBJECTIVE: ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC). DESIGN: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy. RESULTS: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance. CONCLUSION: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition.

IFN-γ treatment protocol for MHC-Ilo/PD-L1+ pancreatic tumor cells selectively restores their TAP-mediated presentation competence and CD8 T-cell priming potential.

BACKGROUND: Many cancer cells express a major histocompatibility complex class I low/ programmed cell death 1 ligand 1 positive (MHC-Ilo/PD-L1+) cell surface profile. For immunotherapy, there is, thus, an urgent need to restore presentation competence of cancer cells with defects in MHC-I processing/presentation combined with immune interventions that tackle the tumor-initiated PD-L1/PD-1 signaling axis. Using pancreatic ductal adenocarcinoma cells (PDACCs) as a model, we here explored if (and how) expression/processing of tumor antigens via transporters associated with antigen processing (TAP) affects priming of CD8 T cells in PD-1/PD-L1-competent/-deficient mice. METHODS: We generated tumor antigen-expressing vectors, immunized TAP-competent/-deficient mice and determined de novo primed CD8 T-cell frequencies by flow cytometry. Similarly, we explored the antigenicity and PD-L1/PD-1 sensitivity of PDACCs versus interferon-γ (IFN-γ)-treated PDACCs in PD-1/PD-L1-competent/deficient mice. The IFN-γ-induced effects on gene and cell surface expression profiles were determined by microarrays and flow cytometry. RESULTS: We identified two antigens (cripto-1 and an endogenous leukemia virus-derived gp70) that were expressed in the Endoplasmic Reticulum (ER) of PDACCs and induced CD8 T-cell responses either independent (Cripto-1:Kb/Cr16-24) or dependent (gp70:Kb/p15E) on TAP by DNA immunization. IFN-γ-treatment of PDACCs in vitro upregulated MHC-I- and TAP- but also PD-L1-expression. Mechanistically, PD-L1/PD-1 signaling was superior to the reconstitution of MHC-I presentation competence, as subcutaneously transplanted IFN-γ-treated PDACCs developed tumors in C57BL/6J and PD-L1-/- but not in PD-1-/- mice. Using PDACCs, irradiated at day 3 post-IFN-γ-treatment or PD-L1 knockout PDACCs as vaccines, we could selectively bypass upregulation of PD-L1, preferentially induce TAP-dependent gp70:Kb/p15E-specific CD8 T cells associated with a weakened PD-1+ exhaustion phenotype and reject consecutively injected tumor transplants in C57BL/6J mice. CONCLUSIONS: The IFN-γ-treatment protocol is attractive for cell-based immunotherapies, because it restores TAP-dependent antigen processing in cancer cells, facilitates priming of TAP-dependent effector CD8 T-cell responses without additional check point inhibitors and could be combined with genetic vaccines that complement priming of TAP-independent CD8 T cells.

Telomeres and Telomerase in the Development of Liver Cancer.

Liver cancer is one of the most common cancer types worldwide and the fourth leading cause of cancer-related death. Liver carcinoma is distinguished by a high heterogeneity in pathogenesis, histopathology and biological behavior. Dysregulated signaling pathways and various gene mutations are frequent in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), which represent the two most common types of liver tumors. Both tumor types are characterized by telomere shortening and reactivation of telomerase during carcinogenesis. Continuous cell proliferation, e.g., by oncogenic mutations, can cause extensive telomere shortening in the absence of sufficient telomerase activity, leading to dysfunctional telomeres and genome instability by breakage-fusion-bridge cycles, which induce senescence or apoptosis as a tumor suppressor mechanism. Telomerase reactivation is required to stabilize telomere functionality and for tumor cell survival, representing a genetic risk factor for the development of liver cirrhosis and liver carcinoma. Therefore, telomeres and telomerase could be useful targets in hepatocarcinogenesis. Here, we review similarities and differences between HCC and iCCA in telomere biology.

Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice.

BACKGROUND & AIMS: Pancreatic tumor cells release small extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules that might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas. We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form premetastatic niches for pancreatic cancer cells in mice. METHODS: We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and nontumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1KO mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1KO-KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative polymerase chain reaction; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. RESULTS: Levels of PRKD1 were reduced in human pancreatic ductal adenocarcinoma tissues compared with nontumor tissues. PRKD1KO-KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1KO-KC mice had increased levels of sEVs compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6ß4 into sEVs-a process that required CD82. CONCLUSIONS: Human pancreatic ductal adenocarcinoma has reduced levels of PRKD1 compared with nontumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice.

Author Correction: Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pancreatic cancer-derived organoids - a disease modeling tool to predict drug response.

BACKGROUND: Organotypic cultures derived from pancreatic ductal adenocarcinoma (PDAC) termed pancreatic ductal cancer organoids (PDOs) recapitulate the primary cancer and can be derived from primary or metastatic biopsies. Although isolation and culture of patient-derived pancreatic organoids were established several years ago, pros and cons for individualized medicine have not been comprehensively investigated to date. METHODS: We conducted a feasibility study, systematically comparing head-to-head patient-derived xenograft tumor (PDX) and PDX-derived organoids by rigorous immunohistochemical and molecular characterization. Subsequently, a drug testing platform was set up and validated in vivo. Patient-derived organoids were investigated as well. RESULTS: First, PDOs faithfully recapitulated the morphology and marker protein expression patterns of the PDXs. Second, quantitative proteomes from the PDX as well as from corresponding organoid cultures showed high concordance. Third, genomic alterations, as assessed by array-based comparative genomic hybridization, revealed similar results in both groups. Fourth, we established a small-scale pharmacotyping platform adjusted to operate in parallel considering potential obstacles such as culture conditions, timing, drug dosing, and interpretation of the results. In vitro predictions were successfully validated in an in vivo xenograft trial. Translational proof-of-concept is exemplified in a patient with PDAC receiving palliative chemotherapy. CONCLUSION: Small-scale drug screening in organoids appears to be a feasible, robust and easy-to-handle disease modeling method to allow response predictions in parallel to daily clinical routine. Therefore, our fast and cost-efficient assay is a reasonable approach in a predictive clinical setting.

Aneuploidy-inducing gene knockdowns overlap with cancer mutations and identify Orp3 as a B-cell lymphoma suppressor.

Aneuploidy can instigate tumorigenesis. However, mutations in genes that control chromosome segregation are rare in human tumors as these mutations reduce cell fitness. Screening experiments indicate that the knockdown of multiple classes of genes that are not directly involved in chromosome segregation can lead to aneuploidy induction. The possible contribution of these genes to cancer formation remains yet to be defined. Here we identified gene knockdowns that lead to an increase in aneuploidy in checkpoint-deficient human cancer cells. Computational analysis revealed that the identified genes overlap with recurrent mutations in human cancers. The knockdown of the three strongest selected candidate genes (ORP3, GJB3, and RXFP1) enhances the malignant transformation of human fibroblasts in culture. Furthermore, the knockout of Orp3 results in an aberrant expansion of lymphoid progenitor cells and a high penetrance formation of chromosomal instable, pauci-clonal B-cell lymphoma in aging mice. At pre-tumorous stages, lymphoid cells from the animals exhibit deregulated phospholipid metabolism and an aberrant induction of proliferation regulating pathways associating with increased aneuploidy in hematopoietic progenitor cells. Together, these results support the concept that aneuploidy-inducing gene deficiencies contribute to cellular transformation and carcinogenesis involving the deregulation of various molecular processes such as lipid metabolism, proliferation, and cell survival.