PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD-1 blockade.

BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

Humanized mouse models for anti-cancer therapy.

Patient-derived xenograft (PDX) models are the golden standard for preclinical oncology as they can recapitulate the genotypic and phenotypic complexity of human tumors, thus enabling the development of effective therapeutic strategies. PDX models are typically established in immunocompromised animals that allow efficient growth of the xenografted tumor. Given the recent success of immune therapies in different tumors however, the establishment of humanized PDX models is critical to evaluate immune oncology drugs and/or combinations thereof. Here, we describe the detailed methods to obtain humanized PDX models for anti-cancer therapy testing.

Novel Therapeutic Approaches Targeting Post-Translational Modifications in Lung Cancer.

Lung cancer is one of the most common cancers worldwide. It consists of two different subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Despite novel therapeutic options such as immunotherapy, only 20% of lung cancer patients survive the disease after five years. This low survival rate is due to acquired drug resistance and severe off-target effects caused by currently used therapies. Identification and development of novel and targeted therapeutic approaches are urgently required to improve the standard of care for lung cancer patients. Here, we describe the recent development of novel drug-delivery approaches, such as adenovirus, lipid nanoparticles, and PROTACs, that have been tested in clinical trials and experimentally in the context of fundamental research. These different options show that it is now possible to target protein kinases, phosphatases, ubiquitin ligases, or protein modifications directly in lung cancer to block disease progression. Furthermore, the recent acceptance of RNA vaccines using lipid nanoparticles has further revealed therapeutic options that could be combined with chemo-/immunotherapies to improve current lung cancer therapies. This review aims to compare recent advances in the pharmaceutical research field for the development of technologies targeting post-translational modifications or protein modifiers involved in the tumorigenesis of lung cancer.

Whole transcriptome profiling of liquid biopsies from tumour xenografted mouse models enables specific monitoring of tumour-derived extracellular RNA.

While cell-free DNA (cfDNA) is widely being investigated, free circulating RNA (extracellular RNA, exRNA) has the potential to improve cancer therapy response monitoring and detection due to its dynamic nature. However, it remains unclear in which blood subcompartment tumour-derived exRNAs primarily reside. We developed a host-xenograft deconvolution framework, exRNAxeno, with mapping strategies to either a combined human-mouse reference genome or both species genomes in parallel, applicable to exRNA sequencing data from liquid biopsies of human xenograft mouse models. The tool enables to distinguish (human) tumoural RNA from (murine) host RNA, to specifically analyse tumour-derived exRNA. We applied the combined pipeline to total exRNA sequencing data from 95 blood-derived liquid biopsy samples from 30 mice, xenografted with 11 different tumours. Tumoural exRNA concentrations are not determined by plasma platelet levels, while host exRNA concentrations increase with platelet content. Furthermore, a large variability in exRNA abundance and transcript content across individual mice is observed. The tumoural gene detectability in plasma is largely correlated with the RNA expression levels in the tumour tissue or cell line. These findings unravel new aspects of tumour-derived exRNA biology in xenograft models and open new avenues to further investigate the role of exRNA in cancer.

Establishment and Characterization of Advanced Penile Cancer Patient-derived Tumor Xenografts: Paving the Way for Personalized Treatments.

BACKGROUND: Systemic treatments for penile squamous cell carcinoma (pSCC) are toxic and inefficient. Patient-based preclinical models are essential to study novel treatments. OBJECTIVE: To establish a library of patient-derived tumor xenograft (PDX) models of human papillomavirus-positive (HPV+) and -negative (HPV-) pSCC and characterize these at the genomic and histological levels. DESIGN, SETTING, AND PARTICIPANTS: Eighteen tumor samples from 14 patients with recurrent or metastatic pSCC were implanted in nude mice. A biobank of PDX tumors was established after passaging of patient samples (F0) for three generations (F1, F2, F3) and was characterized using histopathology and targeted next-generation sequencing (tNGS). Single-nucleotide polymorphism fingerprinting was used to confirm PDX genealogy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The engraftment rate, overall growth rate, and pSCC histomorphology were checked for each PDX generation. Staining for p40 (a pSCC marker) and p16 (a surrogate for HPV infection) was performed for F0 samples. The mutational profile according to a validated panel of 96 cancer genes was determined for F0 and F3 samples and compared to a larger tNGS database. RESULTS AND LIMITATIONS: Including a previously established pilot model, 11 out of 18 tumor samples (61%) successfully engrafted in F1. The mean time from implantation in F1 to completion of F3 was 36 wk (standard deviation 18). Histological fidelity was demonstrated across generations. The patient mutational profiles were preserved in F3 and were representative of 277 pSCC samples in the Foundation Medicine database. The rapid progression of pSCC in patients from our selected high-risk cohort impeded the use of PDXs as avatars. CONCLUSIONS: We successfully established the first library of 11 PDX models of HPV- and HPV+ pSCC. Our PDX models showed high engraftment rates and histological and genomic fidelity to the tumor tissue of origin. These models may help in paving the way towards the development of novel treatments. PATIENT SUMMARY: We established 11 animal models based on tumor tissue from patients with penile cancer. These models could play a vital role in selection of novel treatments according to genetic mutations. In the future, therapies with confirmed preclinical effects may have a profound impact on the development of personalized treatments in penile cancer.

Wnt/ß-Catenin Inhibition Disrupts Carboplatin Resistance in Isogenic Models of Triple-Negative Breast Cancer.

Triple-Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype, characterized by limited treatment options and higher relapse rates than hormone-receptor-positive breast cancers. Chemotherapy remains the mainstay treatment for TNBC, and platinum salts have been explored as a therapeutic alternative in neo-adjuvant and metastatic settings. However, primary and acquired resistance to chemotherapy in general and platinum-based regimens specifically strongly hampers TNBC management. In this study, we used carboplatin-resistant in vivo patient-derived xenograft and isogenic TNBC cell-line models and detected enhanced Wnt/ß-catenin activity correlating with an induced expression of stem cell markers in both resistant models. In accordance, the activation of canonical Wnt signaling in parental TNBC cell lines increases stem cell markers' expression, formation of tumorspheres and promotes carboplatin resistance. Finally, we prove that Wnt signaling inhibition resensitizes resistant models to carboplatin both in vitro and in vivo, suggesting the synergistic use of Wnt inhibitors and carboplatin as a therapeutic option in TNBC. Here we provide evidence for a prominent role of Wnt signaling in mediating resistance to carboplatin, and we establish that combinatorial targeting of Wnt signaling overcomes carboplatin resistance enhancing chemotherapeutic drug efficacy.

Loss-of-Function Mutations in TRAF7 and KLF4 Cooperatively Activate RAS-Like GTPase Signaling and Promote Meningioma Development.

Meningiomas are the most common benign brain tumors. Mutations of the E3 ubiquitin ligase TRAF7 occur in 25% of meningiomas and commonly cooccur with mutations in KLF4, yet the functional link between TRAF7 and KLF4 mutations remains unclear. By generating an in vitro meningioma model derived from primary meningeal cells, we elucidated the cooperative interactions that promote meningioma development. By integrating TRAF7-driven ubiquitinome and proteome alterations in meningeal cells and the TRAF7 interactome, we identified TRAF7 as a proteostatic regulator of RAS-related small GTPases. Meningioma-associated TRAF7 mutations disrupted either its catalytic activity or its interaction with RAS GTPases. TRAF7 loss in meningeal cells altered actin dynamics and promoted anchorage-independent growth by inducing CDC42 and RAS signaling. TRAF deficiency-driven activation of the RAS/MAPK pathway promoted KLF4-dependent transcription that led to upregulation of the tumor-suppressive Semaphorin pathway, a negative regulator of small GTPases. KLF4 loss of function disrupted this negative feedback loop and enhanced mutant TRAF7-mediated cell transformation. Overall, this study provides new mechanistic insights into meningioma development, which could lead to novel treatment strategies. SIGNIFICANCE: The intricate molecular cross-talk between the ubiquitin ligase TRAF7 and the transcription factor KLF4 provides a first step toward the identification of new therapies for patients with meningioma.

The mitotic checkpoint is a targetable vulnerability of carboplatin-resistant triple negative breast cancers.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, lacking effective therapy. Many TNBCs show remarkable response to carboplatin-based chemotherapy, but often develop resistance over time. With increasing use of carboplatin in the clinic, there is a pressing need to identify vulnerabilities of carboplatin-resistant tumors. In this study, we generated carboplatin-resistant TNBC MDA-MB-468 cell line and patient derived TNBC xenograft models. Mass spectrometry-based proteome profiling demonstrated that carboplatin resistance in TNBC is linked to drastic metabolism rewiring and upregulation of anti-oxidative response that supports cell replication by maintaining low levels of DNA damage in the presence of carboplatin. Carboplatin-resistant cells also exhibited dysregulation of the mitotic checkpoint. A kinome shRNA screen revealed that carboplatin-resistant cells are vulnerable to the depletion of the mitotic checkpoint regulators, whereas the checkpoint kinases CHEK1 and WEE1 are indispensable for the survival of carboplatin-resistant cells in the presence of carboplatin. We confirmed that pharmacological inhibition of CHEK1 by prexasertib in the presence of carboplatin is well tolerated by mice and suppresses the growth of carboplatin-resistant TNBC xenografts. Thus, abrogation of the mitotic checkpoint by CHEK1 inhibition re-sensitizes carboplatin-resistant TNBCs to carboplatin and represents a potential strategy for the treatment of carboplatin-resistant TNBCs.

Loss of 9p21 Regulatory Hub Promotes Kidney Cancer Progression by Upregulating HOXB13.

Loss of chromosome 9p21 is observed in one-thirds of clear-cell renal cell carcinoma (ccRCC) and is associated with poorer patient survival. Unexpectedly, 9p21 LOH does not lead to decreased expression of the 9p21 tumor suppressor genes, CDKN2A and CDKN2B, suggesting alternative mechanisms of 9p-mediated tumorigenesis. Concordantly, CRISPR-mediated 9p21 deletion promotes growth of immortalized human embryonic kidney epithelial cells independently of the CDKN2A/B pathway inactivation. The 9p21 locus has a highly accessible chromatin structure, suggesting that 9p21 loss might contribute to kidney cancer progression by dysregulating genes distal to the 9p21 locus. We identified several 9p21 regulatory hubs by assessing which of the 9p21-interacting genes are dysregulated in 9p21-deleted kidney cells and ccRCCs. By focusing on the analysis of the homeobox gene 13 (HOXB13) locus, we found that 9p21 loss relieves the HOXB13 locus, decreasing HOXB13 methylation and promoting its expression. Upregulation of HOXB13 facilitates cell growth and is associated with poorer survival of patients with ccRCC. IMPLICATIONS: The results of our study propose a novel tumor suppressive mechanism on the basis of coordinated expression of physically associated genes, providing a better understanding of the role of chromosomal deletions in cancer.

Cracking the Monoubiquitin Code of Genetic Diseases.

Ubiquitination is a versatile and dynamic post-translational modification in which single ubiquitin molecules or polyubiquitin chains are attached to target proteins, giving rise to mono- or poly-ubiquitination, respectively. The majority of research in the ubiquitin field focused on degradative polyubiquitination, whereas more recent studies uncovered the role of single ubiquitin modification in important physiological processes. Monoubiquitination can modulate the stability, subcellular localization, binding properties, and activity of the target proteins. Understanding the function of monoubiquitination in normal physiology and pathology has important therapeutic implications, as alterations in the monoubiquitin pathway are found in a broad range of genetic diseases. This review highlights a link between monoubiquitin signaling and the pathogenesis of genetic disorders.

Stress-induced lncRNA LASTR fosters cancer cell fitness by regulating the activity of the U4/U6 recycling factor SART3.

Dysregulated splicing is a common event in cancer even in the absence of mutations in the core splicing machinery. The aberrant long non-coding transcriptome constitutes an uncharacterized level of regulation of post-transcriptional events in cancer. Here, we found that the stress-induced long non-coding RNA (lncRNA), LINC02657 or LASTR (lncRNA associated with SART3 regulation of splicing), is upregulated in hypoxic breast cancer and is essential for the growth of LASTR-positive triple-negative breast tumors. LASTR is upregulated in several types of epithelial cancers due to the activation of the stress-induced JNK/c-JUN pathway. Using a mass-spectrometry based approach, we identified the RNA-splicing factor SART3 as a LASTR-interacting partner. We found that LASTR promotes splicing efficiency by controlling SART3 association with the U4 and U6 small nuclear ribonucleoproteins (snRNP) during spliceosome recycling. Intron retention induced by LASTR depletion downregulates expression of essential genes, ultimately decreasing the fitness of cancer cells.

Loss of Chromosome 8p Governs Tumor Progression and Drug Response by Altering Lipid Metabolism.

Large-scale heterozygous deletions are a hallmark of cancer genomes. The concomitant loss of multiple genes creates vulnerabilities that are impossible to reveal through the study of individual genes. To delineate the functional outcome of chromosome 8p loss of heterozygosity (LOH), a common aberration in breast cancer, we modeled 8p LOH using TALEN-based genomic engineering. 8p LOH alters fatty acid and ceramide metabolism. The shift in lipid metabolism triggers invasiveness and confers tumor growth under stress conditions due to increased autophagy. The resistance of 8p-deleted cells to chemotherapeutic drugs concurs with poorer survival rates of breast cancer patients harboring an 8p LOH. The autophagy dependency of 8p-deleted cells provides the rational basis for treatment of 8p LOH tumors with autophagy inhibitors.

OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination.

Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development.

The tyrosine phosphatase PTPRO sensitizes colon cancer cells to anti-EGFR therapy through activation of SRC-mediated EGFR signaling.

Inappropriate activation of epidermal growth factor receptor (EGFR) plays a causal role in many cancers including colon cancer. The activation of EGFR by phosphorylation is balanced by receptor kinase and protein tyrosine phosphatase activities. However, the mechanisms of negative EGFR regulation by tyrosine phosphatases remain largely unexplored. Our previous results indicate that protein tyrosine phosphatase receptor type O (PTPRO) is down-regulated in a subset of colorectal cancer (CRC) patients with a poor prognosis. Here we identified PTPRO as a phosphatase that negatively regulates SRC by directly dephosphorylating Y416 phosphorylation site. SRC activation triggered by PTPRO down-regulation induces phosphorylation of both EGFR at Y845 and the c-CBL ubiquitin ligase at Y731. Increased EGFR phosphorylation at Y845 promotes its receptor activity, whereas enhanced phosphorylation of c-CBL triggers its degradation promoting EGFR stability. Importantly, hyperactivation of SRC/EGFR signaling triggered by loss of PTPRO leads to high resistance of colon cancer to EGFR inhibitors. Our results not only highlight the PTPRO contribution in negative regulation of SRC/EGFR signaling but also suggest that tumors with low PTPRO expression may be therapeutically targetable by anti-SRC therapies.

The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response.

The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively engaging two components of the exocyst complex, EXO84 and SEC5. RALB employs SEC5 to trigger innate immunity signalling, whereas RALB-EXO84 interaction induces autophagocytosis. How this differential interaction is achieved molecularly by the RAL GTPase remains unknown. We found that whereas GTP binding turns on RALB activity, ubiquitylation of RALB at Lys 47 tunes its activity towards a particular effector. Specifically, ubiquitylation at Lys 47 sterically inhibits RALB binding to EXO84, while facilitating its interaction with SEC5. Double-stranded RNA promotes RALB ubiquitylation and SEC5-TBK1 complex formation. In contrast, nutrient starvation induces RALB deubiquitylation by accumulation and relocalization of the deubiquitylase USP33 to RALB-positive vesicles. Deubiquitylated RALB promotes the assembly of the RALB-EXO84-beclin-1 complexes driving autophagosome formation. Thus, ubiquitylation within the effector-binding domain provides the switch for the dual functions of RALB in autophagy and innate immune responses.

Syndecan-syntenin-ALIX regulates the biogenesis of exosomes.

The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes.