A rapid and scalable system for studying gene function in mice using conditional RNA interference.

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:

Activated granulocytes and inflammatory cytokine signaling drive T-cell lymphoma progression and disease symptoms.

Peripheral T-cell lymphomas (PTCLs), especially angioimmunoblastic and follicular TCLs, have a dismal prognosis because of the lack of efficient therapies, and patients' symptoms are often dominated by an inflammatory phenotype, including fever, night sweats, weight loss, and skin rash. In this study, we investigated the role of inflammatory granulocytes and activated cytokine signaling on T-cell follicular helper-type PTCL (TFH-PTCL) disease progression and symptoms. We showed that ITK-SYK-driven murine PTCLs and primary human TFH-PTCL xenografts both induced inflammation in mice, including murine neutrophil expansion and massive cytokine release. Granulocyte/lymphoma interactions were mediated by positive autoregulatory cytokine loops involving interferon gamma (CD4+ malignant T cells) and interleukin 6 (IL-6; activated granulocytes), ultimately inducing broad JAK activation (JAK1/2/3 and TYK2) in both cell types. Inflammatory granulocyte depletion via antibodies (Ly6G), genetic granulocyte depletion (LyzM-Cre/MCL1flox/flox), or IL-6 deletion within microenvironmental cells blocked inflammatory symptoms, reduced lymphoma infiltration, and enhanced mouse survival. Furthermore, unselective JAK inhibitors (ruxolitinib) inhibited both TCL progression and granulocyte activation in various PTCL mouse models. Our results support the important role of granulocyte-driven inflammation, cytokine-induced granulocyte/CD4+ TCL interactions, and an intact JAK/STAT signaling pathway for TFH-PTCL development and also support broad JAK inhibition as an effective treatment strategy in early disease stages.

Targeting MDM2 enhances antileukemia immunity after allogeneic transplantation via MHC-II and TRAIL-R1/2 upregulation.

Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2 inhibition.

Primary Results of Patients with Genitourinary Malignancies Presented at a Molecular Tumor Board.

INTRODUCTION: Personalized medicine poses great opportunities and challenges. While the therapeutic landscape markedly expands, descriptions about status, clinical implementation and real-world benefits of precision oncology and molecular tumor boards (MTB) remain sparse, particularly in the field of genitourinary (GU) cancer. Hence, this study characterized urological MTB cases to better understand the potential role of MTB in uro-oncology. METHODS: We analyzed patients with complete data sets being reviewed at an MTB from January 2019 to October 2022, focusing on results of molecular analysis and treatment recommendations. RESULTS: We evaluated 102 patients with GU cancer with a mean patient age of 61.7 years. Prostate cancer (PCa) was the most frequent entity with 52.9% (54/102), followed by bladder cancer (18.6%, 19/102) and renal cell carcinoma (14.7%, 15/102). On average, case presentation at MTB took place 54.9 months after initial diagnosis and after 2.7 previous lines of therapy. During the study period, 49.0% (50/102) of patients deceased. Additional MTB-based treatment recommendations were achieved in a majority of 68.6% (70/102) of patients, with a recommendation for targeted therapy in 64.3% (45/70) of these patients. Only 6.7% (3/45) of patients - due to different reasons - received the recommended MTB-based therapy though, with 33% (1/3) of patients reaching disease control. Throughout the MTB study period, GU cancer case presentations and treatment recommendations increased, while the time interval between initial presentation and final therapy recommendation were decreasing over time. CONCLUSION: Presentation of uro-oncological patients at the MTB is a highly valuable measure for clinical decision-making. Prospectively, earlier presentation of patients at the MTB and changing legislative issues regarding comprehensive molecular testing and targeted treatment approval might further improve patients' benefits from comprehensive molecular diagnostics.

Multidisciplinary tumor board analysis: validation study of a central tool in tumor centers.

The established standard to ensure state-of-the-art cancer treatment is through multidisciplinary tumor boards (TBs), although resource- and time-intensive. In this validation study, the multiple myeloma (MM)-TB was reexamined, aiming to validate our previous (2012-2014) results, now using the TB data from March 2020 to February 2021. We assessed MM-TB protocols, physicians' documentation, patient, disease, remission status, progression-free survival (PFS), and overall survival (OS) as left-truncated survival times. Moreover, TB-adherence, level of evidence according to grade criteria, time requirements, study inclusion rates, and referral satisfaction were determined. Within a 1-year period, 312 discussed patients were documented in 439 TB protocols. Patient and disease characteristics were typical for comprehensive cancer centers. The percentages of patients discussed at initial diagnosis (ID), with disease recurrence or in need of interdisciplinary advice, were 39%, 28%, and 33%, respectively. Reasons for the MM-TB presentation were therapeutic challenges in 80% or staging/ID-defining questions in 20%. The numbers of presentations were mostly one in 73%, two in 20%, and three or more in 7%. The TB adherence rate was 93%. Reasons for non-adherence were related to patients' decisions or challenging inclusion criteria for clinical trials. Additionally, we demonstrate that with the initiation of TBs, that the number of interdisciplinarily discussed patients increased, that TB-questions involve advice on the best treatment, and that levels of compliance and evidence can be as high as ≥ 90%. Advantages of TBs are that they may also improve patients', referrers', and physicians' satisfaction, inclusion into clinical trials, and advance interdisciplinary projects, thereby encouraging cancer specialists to engage in them.

Senescence of activated stellate cells limits liver fibrosis.

Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to noncancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell-cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix-degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage.

Murine Oncostatin M Has Opposing Effects on the Proliferation of OP9 Bone Marrow Stromal Cells and NIH/3T3 Fibroblasts Signaling through the OSMR.

The IL-6 family cytokine Oncostatin M (OSM) is involved in cell development, growth, hematopoiesis, inflammation, and cancer. Intriguingly, OSM has proliferative and antiproliferative effects depending on the target cell. The molecular mechanisms underlying these opposing effects are not fully understood. Previously, we found OSM upregulation in different myeloproliferative syndromes. However, OSM receptor (OSMR) expression was detected on stromal cells but not the malignant cells themselves. In the present study, we, therefore, investigated the effect of murine OSM (mOSM) on proliferation in stromal and fibroblast cell lines. We found that mOSM impairs the proliferation of bone marrow (BM) stromal cells, whereas fibroblasts responded to mOSM with increased proliferation. When we set out to reveal the mechanisms underlying these opposing effects, we detected increased expression of the OSM receptors OSMR and LIFR in stromal cells. Interestingly, Osmr knockdown and Lifr overexpression attenuated the OSM-mediated effect on proliferation in both cell lines indicating that mOSM affected the proliferation signaling mainly through the OSMR. Furthermore, mOSM induced activation of the JAK-STAT, PI3K-AKT, and MAPK-ERK pathways in OP9 and NIH/3T3 cells with differences in total protein levels between the two cell lines. Our findings offer new insights into the regulation of proliferation by mOSM.

CDK9-mediated phosphorylation controls the interaction of TIP60 with the transcriptional machinery.

The acetyltransferase TIP60 is regulated by phosphorylation, and we have previously shown that phosphorylation of TIP60 on S86 by GSK-3 promotes p53-mediated induction of the BCL-2 protein PUMA. TIP60 phosphorylation by GSK-3 requires a priming phosphorylation on S90, and here, we identify CDK9 as a TIP60S90 kinase. We demonstrate that a phosphorylation-deficient mutant, TIP60S90A, exhibits reduced interaction with chromatin, histone 3 and RNA Pol II, while its association with the TIP60 complex subunit EPC1 is not affected. Consistently, we find a diminished association of TIP60S90A with the MYC gene. We show that cells expressing TIP60S90A, but also TIP60S86A, which retains S90 phosphorylation, exhibit reduced histone 4 acetylation and proliferation. Thus, our data indicate that, during transcription, phosphorylation of TIP60 at two sites has different regulatory effects on TIP60, whereby S90 phosphorylation controls association with the transcription machinery, and S86 phosphorylation is regulating TIP60 HAT activity.

PTEN action in leukaemia dictated by the tissue microenvironment.

PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner. Postnatal Pten knockdown in the haematopoietic compartment produced highly disseminated T-cell acute lymphoblastic leukaemia. Notably, reactivation of PTEN mainly reduced T-cell leukaemia dissemination but had little effect on tumour load in haematopoietic organs. Leukaemia infiltration into the intestine was dependent on CCR9 G-protein-coupled receptor signalling, which was amplified by PTEN loss. Our results suggest that in the absence of PTEN, G-protein-coupled receptors may have an unanticipated role in driving tumour growth and invasion in an unsupportive environment. They further reveal that the role of PTEN loss in tumour maintenance is not invariant and can be influenced by the tissue microenvironment, thereby producing a form of intratumoral heterogeneity that is independent of cancer genotype.

A tumour suppressor network relying on the polyamine-hypusine axis.

Tumour suppressor genes encode a broad class of molecules whose mutational attenuation contributes to malignant progression. In the canonical situation, the tumour suppressor is completely inactivated through a two-hit process involving a point mutation in one allele and chromosomal deletion of the other. Here, to identify tumour suppressor genes in lymphoma, we screen a short hairpin RNA library targeting genes deleted in human lymphomas. We functionally identify those genes whose suppression promotes tumorigenesis in a mouse lymphoma model. Of the nine tumour suppressors we identified, eight correspond to genes occurring in three physically linked 'clusters', suggesting that the common occurrence of large chromosomal deletions in human tumours reflects selective pressure to attenuate multiple genes. Among the new tumour suppressors are adenosylmethionine decarboxylase 1 (AMD1) and eukaryotic translation initiation factor 5A (eIF5A), two genes associated with hypusine, a unique amino acid produced as a product of polyamine metabolism through a highly conserved pathway. Through a secondary screen surveying the impact of all polyamine enzymes on tumorigenesis, we establish the polyamine-hypusine axis as a new tumour suppressor network regulating apoptosis. Unexpectedly, heterozygous deletions encompassing AMD1 and eIF5A often occur together in human lymphomas and co-suppression of both genes promotes lymphomagenesis in mice. Thus, some tumour suppressor functions can be disabled through a two-step process targeting different genes acting in the same pathway.

RNAi screening uncovers Dhx9 as a modifier of ABT-737 resistance in an Eµ-myc/Bcl-2 mouse model.

ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3 mimetic to neutralize Bcl-2-like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and thus is a major cause of resistance. We performed a short hairpin RNA (shRNA)-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eµ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival. Several drug-sensitive shRNAs were identified that were selectively depleted in the presence of ABT-737. Of these, 2 independent shRNAs targeting the RNA/DNA helicase Dhx9 were found to sensitize lymphomas to ABT-737 to an extent comparable to control Mcl-1 shRNAs. Although Dhx9 suppression sensitized both mouse and human cells to ABT-737 treatment, it did so without altering MCL-1 levels. Rather, loss of Dhx9 appeared to activate a p53-dependent apoptotic program, through aggravation of replicative stress, which was found to be both necessary and sufficient for the ABT-737-shDhx9 synthetic lethal relationship.

ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease.

Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1ß, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings.

An RNAi-based system for loss-of-function analysis identifies Raf1 as a crucial mediator of BCR-ABL-driven leukemogenesis.

Genetic loss-of-function studies in murine tumor models have been essential in the analysis of downstream mediators of oncogenic transformation. Unfortunately, these studies are frequently limited by the availability of genetically modified mouse strains. Here we describe a versatile method allowing the efficient expression of an oncogene and simultaneous knockdown of targets of interest (TOI) from a single retroviral vector. Both oncogene and TOI-specific miR30-based shRNA are under the control of the strong viral long terminal repeat promoter, resulting in a single shared RNA transcript. Using this vector in a murine syngeneic BM transplantation model for BCR-ABL-induced chronic myeloid leukemia, we find that oncogene expression and target knockdown in primary hematopoietic cells with this vector is efficient both in vitro and in vivo, and demonstrate that Raf1, but not BRAF, modulates BCR-ABL-dependent ERK activation and transformation of hematopoietic cells. This expression system could facilitate genetic loss-of-function studies and allow the rapid validation of potential drug targets in a broad range of oncogene-driven murine tumor models.

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas.

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.

Degradome-focused RNA interference screens to identify proteases important for breast cancer cell growth.

Proteases are known to promote or impair breast cancer progression and metastasis. However, while a small number of the 588 human and 672 murine protease genes have been extensively studied, others were neglected. For an unbiased functional analysis of all genome-encoded proteases, i.e., the degradome, in breast cancer cell growth, we applied an inducible RNA interference library for protease-focused genetic screens. Importantly, these functional screens were performed in two phenotypically different murine breast cancer cell lines, including one stem cell-like cell line that showed phenotypic plasticity under changed nutrient and oxygen availability. Our unbiased genetic screens identified 252 protease genes involved in breast cancer cell growth that were further restricted to 100 hits by a selection process. Many of those hits were supported by literature, but some proteases were novel in their functional link to breast cancer. Interestingly, we discovered that the environmental conditions influence the degree of breast cancer cell dependency on certain proteases. For example, breast cancer stem cell-like cells were less susceptible to depletion of several mitochondrial proteases in hypoxic conditions. From the 100 hits, nine proteases were functionally validated in murine breast cancer cell lines using individual knockdown constructs, highlighting the high reliability of our screens. Specifically, we focused on mitochondrial processing peptidase (MPP) subunits alpha (Pmpca) and beta (Pmpcb) and discovered that MPP depletion led to a disadvantage in cell growth, which was linked to mitochondrial dysfunction.

RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells.

RATIONALE: PI3K/mTOR signaling is frequently upregulated in breast cancer making inhibitors of this pathway highly promising anticancer drugs. However, PI3K-inhibitors have a low therapeutic index. Therefore, finding novel combinatory treatment options represents an important step towards clinical implementation of PI3K pathway inhibition in breast cancer therapy. Here, we propose proteases as potential synergistic partners with simultaneous PI3K inhibition in breast cancer cells. METHODS: We performed mRNA expression studies and unbiased functional genetic synthetic lethality screens by a miR-E based knockdown system targeting all genome-encoded proteases, i.e. the degradome of breast cancer cells. Importantly theses RNA interference screens were done in combination with two PI3K pathway inhibitors. Protease hits were validated in human and murine breast cancer cell lines as well as in non-cancerous cells by viability and growth assays. RESULTS: The degradome-wide genetic screens identified 181 proteases that influenced susceptibility of murine breast cancer cells to low dose PI3K inhibition. Employing independently generated inducible knockdown cell lines we validated 12 protease hits in breast cancer cells. In line with the known tumor promoting function of these proteases we demonstrated Usp7 and Metap2 to be important for murine and human breast cancer cell growth and discovered a role for Metap1 in this context. Most importantly, we demonstrated that Usp7, Metap1 or Metap2 knockdown combined with simultaneous PI3K inhibition resulted in synergistic impairment of murine and human breast cancer cell growth Conclusion: We successfully established proteases as combinatory targets with PI3K inhibition in human and murine breast cancer cells. Usp7, Metap1 and Metap2 are synthetic lethal partners of simultaneous protease/PI3K inhibition, which may refine future breast cancer therapy.

NIPA (Nuclear Interaction Partner of ALK) Is Crucial for Effective NPM-ALK Mediated Lymphomagenesis.

The NPM-ALK fusion kinase is expressed in 60% of systemic anaplastic large-cell lymphomas (ALCL). A Nuclear Interaction Partner of ALK (NIPA) was identified as a binding partner of NPM-ALK. To identify the precise role of NIPA for NPM-ALK-driven lymphomagenesis, we investigated various NPM-ALK+ cell lines and mouse models. Nipa deletion in primary mouse embryonic fibroblasts resulted in reduced transformation ability and colony formation upon NPM-ALK expression. Downregulating NIPA in murine NPM-ALK+ Ba/F3 and human ALCL cells decreased their proliferation ability and demonstrated synergistic effects of ALK inhibition and NIPA knockdown. Comprehensive in vivo analyses using short- and long-latency transplantation mouse models with NPM-ALK+ bone marrow (BM) revealed that Nipa deletion inhibited NPM-ALK-induced tumorigenesis with prolonged survival and reduced spleen colonies. To avoid off-target effects, we combined Nipa deletion and NPM-ALK expression exclusively in T cells using a lineage-restricted murine ALCL-like model resembling human disease: control mice died from neoplastic T-cell infiltration, whereas mice transplanted with Lck-CreTG/wtNipaflox/flox NPM-ALK+ BM showed significantly prolonged survival. Immunophenotypic analyses indicated a characteristic ALCL-like phenotype in all recipients but revealed fewer "stem-cell-like" features of Nipa-deficient lymphomas compared to controls. Our results identify NIPA as a crucial player in effective NPM-ALK-driven ALCL-like disease in clinically relevant murine and cell-based models.