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High expression of the receptor tyrosine kinase AXL is implicated in epithelial-to-mesenchymal transition, cancer progression, and therapy resistance. For example, AXL is abundant in BRAF mutant melanomas progressing on targeted BRAF/MEK inhibition. Therefore, AXL is thought to represent an attractive therapeutic target. This notwithstanding, little is known about the mechanisms governing expression of AXL. Here, we describe a FACS-based whole-genome-wide CRISPR-Cas9 screen to uncover regulators of AXL expression. We identified several genes, inactivation of which led to increased AXL expression. Most remarkable was the identification of five components that associate with the Elongin BC heterodimer. Elongin B/C engage in multiple protein–protein interactions, including the transcription factor complex subunit Elongin A, the von Hippel-Lindau (VHL) tumor suppressor protein, and members of the SOCS-box protein family. The screen identified ELOB, ELOC, SOCS5, UBE2F, and RNF7, each of which we demonstrate to serve as an inhibitor of AXL expression. Although the AXL promoter contains hypoxia response elements and Elongin B/C are found in the VHL complex, Elongin B/C unexpectedly regulate AXL independently of hypoxia. Instead, we demonstrate that the Elongin BC complex interacts with AXL through ELOB, and contributes to proteasomal AXL turnover. RNA-sequencing and IHC analyses of melanoma patient-derived xenografts and clinical samples revealed a negative association between Elongin B/C and dedifferentiation. Together, the Elongin BC complex regulates AXL and marks a differentiated melanoma phenotype. Implications: This study identifies the Elongin BC complex as a key regulator of AXL expression and marker of melanoma differentiation.

Extended data on Figure 5: Low expression of ELOB/C is associated with phenotype switching and EMT.

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High expression of the receptor tyrosine kinase AXL is implicated in epithelial-to-mesenchymal transition, cancer progression, and therapy resistance. For example, AXL is abundant in BRAF mutant melanomas progressing on targeted BRAF/MEK inhibition. Therefore, AXL is thought to represent an attractive therapeutic target. This notwithstanding, little is known about the mechanisms governing expression of AXL. Here, we describe a FACS-based whole-genome-wide CRISPR-Cas9 screen to uncover regulators of AXL expression. We identified several genes, inactivation of which led to increased AXL expression. Most remarkable was the identification of five components that associate with the Elongin B/C heterodimer. Elongin B/C engage in multiple protein–protein interactions, including the transcription factor complex subunit Elongin A, the von Hippel-Lindau (VHL) tumor suppressor protein, and members of the SOCS-box protein family. The screen identified ELOB, ELOC, SOCS5, UBE2F, and RNF7, each of which we demonstrate to serve as an inhibitor of AXL expression. Although the AXL promoter contains hypoxia response elements and Elongin B/C are found in the VHL complex, Elongin B/C unexpectedly regulate AXL independently of hypoxia. Instead, we demonstrate that the Elongin BC complex interacts with AXL through ELOB, and contributes to proteasomal AXL turnover. RNA-sequencing and IHC analyses of melanoma patient-derived xenografts and clinical samples revealed a negative association between Elongin B/C and dedifferentiation. Together, the Elongin BC complex regulates AXL and marks a differentiated melanoma phenotype.Implications:This study identifies the Elongin BC complex as a key regulator of AXL expression and marker of melanoma differentiation.

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Purpose:Combining anti–PD-1 + anti–CTLA-4 immune-checkpoint blockade (ICB) shows improved patient benefit, but it is associated with severe immune-related adverse events and exceedingly high cost. Therefore, there is a dire need to predict which patients respond to monotherapy and which require combination ICB treatment.Experimental Design:In patient-derived melanoma xenografts (PDX), human tumor microenvironment (TME) cells were swiftly replaced by murine cells upon transplantation. Using our XenofilteR deconvolution algorithm we curated human tumor cell RNA reads, which were subsequently subtracted in silico from bulk (tumor cell + TME) patients' melanoma RNA. This produced a purely tumor cell–intrinsic signature (“InTumor”) and a signature comprising tumor cell–extrinsic RNA reads (“ExTumor”).Results:We show that whereas the InTumor signature predicts response to anti–PD-1, the ExTumor predicts anti–CTLA-4 benefit. In PDX, InTumorLO, but not InTumorHI, tumors are effectively eliminated by cytotoxic T cells. When used in conjunction, the InTumor and ExTumor signatures identify not only patients who have a substantially higher chance of responding to combination treatment than to either monotherapy, but also those who are likely to benefit little from anti–CTLA-4 on top of anti–PD-1.Conclusions:These signatures may be exploited to distinguish melanoma patients who need combination ICB blockade from those who likely benefit from either monotherapy.

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Purpose:Combining anti–PD-1 + anti–CTLA-4 immune-checkpoint blockade (ICB) shows improved patient benefit, but it is associated with severe immune-related adverse events and exceedingly high cost. Therefore, there is a dire need to predict which patients respond to monotherapy and which require combination ICB treatment.Experimental Design:In patient-derived melanoma xenografts (PDX), human tumor microenvironment (TME) cells were swiftly replaced by murine cells upon transplantation. Using our XenofilteR deconvolution algorithm we curated human tumor cell RNA reads, which were subsequently subtracted in silico from bulk (tumor cell + TME) patients' melanoma RNA. This produced a purely tumor cell–intrinsic signature (“InTumor”) and a signature comprising tumor cell–extrinsic RNA reads (“ExTumor”).Results:We show that whereas the InTumor signature predicts response to anti–PD-1, the ExTumor predicts anti–CTLA-4 benefit. In PDX, InTumorLO, but not InTumorHI, tumors are effectively eliminated by cytotoxic T cells. When used in conjunction, the InTumor and ExTumor signatures identify not only patients who have a substantially higher chance of responding to combination treatment than to either monotherapy, but also those who are likely to benefit little from anti–CTLA-4 on top of anti–PD-1.Conclusions:These signatures may be exploited to distinguish melanoma patients who need combination ICB blockade from those who likely benefit from either monotherapy.

High expression of the receptor tyrosine kinase AXL is implicated in epithelial-to-mesenchymal transition, cancer progression and therapy resistance. For example, AXL is abundant in BRAF mutant melanomas progressing on targeted BRAF/MEK inhibition. Therefore, AXL is thought to represent an attractive therapeutic target. This notwithstanding, little is known about the mechanisms governing expression of AXL. Here, we describe a fluorescence-activated cell sorting (FACS)-based whole genome-wide CRISPR-Cas9 screen to uncover regulators of AXL expression. We identified several genes, inactivation of which led to increased AXL expression. Most remarkable was the identification of five components that associate with the Elongin B/C heterodimer. The Elongin B/C heterodimer engages in multiple protein-protein interactions, including the transcription factor complex subunit Elongin A, the von Hippel-Lindau (VHL) tumor suppressor protein, and members of the SOCS-box protein family. The screen identified ELOB, ELOC, SOCS5, UBE2F and RNF7, each of which we demonstrate to serve as an inhibitor of AXL expression. Although the AXL promoter contains hypoxia response elements and Elongin B/C are found in the VHL complex, Elongin B/C unexpectedly regulate AXL independently of hypoxia. Instead, we demonstrate that the Elongin BC complex interacts with AXL through ELOB, and contributes to proteasomal AXL turnover. RNA-sequencing analysis of several melanoma datasets revealed a negative association between Elongin B/C and dedifferentiation. This was confirmed by immunohistochemistry in patient-derived melanoma xenografts. These results uncover the Elongin BC complex as a key determinant of AXL expression in melanoma, and that this correlates with a differentiated state.

Although high clinical response rates are seen for immune checkpoint blockade (ICB) treatment of metastatic melanomas, both intrinsic and acquired ICB resistance remain considerable clinical challenges1. Combination ICB (anti-PD-1 + anti-CTLA-4) shows improved patient benefit2–5, but is associated with severe adverse events and exceedingly high cost. Therefore, there is a dire need to stratify individual patients for their likelihood of responding to either anti-PD-1 or anti-CTLA-4 monotherapy, or the combination. Since it is conceivable that ICB responses are influenced by both tumor cell-intrinsic and -extrinsic factors6–9, we hypothesized that a predictive genetic classifier ought to mirror both these features. In a panel of patient-derived melanoma xenografts10(PDX), we noted that cells derived from the human tumor microenvironment (TME) that were co-grafted with the tumor cells were naturally replaced by murine cells after the first passage. Taking advantage of the XenofilteR11algorithm we recently developed to deconvolute human from murine RNA sequence reads from PDX10, we obtained curated human melanoma tumor cell RNA reads. These expression signals were computationally subtracted from the total RNA profiles in bulk (tumor cell + TME) melanomas from patients. We thus derived one genetic signature that is purely tumor cell-intrinsic (“InTumor”), and one that comprises tumor cell-extrinsic RNA profiles (“ExTumor”). Here we report that the InTumor signature predicts patient response to anti-PD-1, but not anti-CTLA-4 treatment. This was validated in melanoma PDX and cell lines, which confirmed that InTumorLOtumors were effectively eliminated by adoptive cell transfer of T-Cell Receptor (TCR)-matched cytotoxic T cells, whereas InTumorHImelanomas were refractory and grew out as fast as tumors challenged with unmatched T cells. In contrast, the ExTumor signature predicts patient response to anti-CTLA-4 but not anti-PD-1. Most importantly, we used the InTumor and ExTumor signatures in conjunction to generate an ICB response quadrant, which predicts clinical benefit for five independent melanoma patient cohorts treated with either mono- or combination ICB. Specifically, these signatures enable identification of patients who have a much higher chance of responding to the combination treatment than to either monotherapy (p < 0.05), as well as patients who are likely to experience little benefit from receiving anti-CTLA-4 on top of anti-PD-1 (p < 0.05). These signatures may be clinically exploited to distinguish patients who need combined PD-1 + CTLA-4 blockade from those who are likely to benefit from either anti-CTLA-4 or anti-PD-1 monotherapy.

Background The tumor immune microenvironment is a heterogeneous entity. Gene expression analysis allows us to perform comprehensive immunoprofiling and may assist in dissecting the different components of the immune infiltrate. As gene expression analysis also provides information regarding tumor cells, differences in interactions between the immune system and specific tumor characteristics can also be explored. This study aims to gain further insights in the composition of the tumor immune infiltrate and to correlate these components to histology and overall survival in non-small cell lung cancer (NSCLC). Methods Archival tissues from 530 early stage, resected NSCLC patients with annotated tumor and patient characteristics were analyzed using the NanoString nCounter Analysis system. Results Unsupervised clustering of the samples was mainly driven by the overall level of inflammation, which was not correlated with survival in this patient set. Adenocarcinoma (AD) showed a significantly higher degree of immune infiltration compared to squamous cell carcinoma (SCC). A 34-gene signature, which did not correlate with the overall level of immune infiltration, was identified and showed an OS benefit in SCC. Strikingly, this benefit was not observed in AD. This difference in OS in SCC specifically was confirmed in two independent NSCLC cohorts. The highest correlation between expression of the 34-gene signature and specific immune cell populations was observed for NK cells, but although a plausible mechanism for NK cell intervention in tumor growth could be established in SCC over AD, this could not be translated back to immunohistochemistry, which showed that NK cell infiltration is scarce irrespective of histology. Conclusions These findings suggest that the ability of immune cell infiltration and the interaction between tumor and immune cells may be different between AD and SCC histology and that a subgroup of SCC tumors seems more susceptible to Natural Killer cell recognition and killing, whereas this may not occur in AD tumors. A highly sensitive technique like NanoString was able to detect this subgroup based on a 34-gene signature, but further research will be needed to assist in explaining the biological rationale of such low-level expression signatures.

Observations from cultured cells 1-3, animal models4 and patients5-7raise the possibility that the dependency of tumours on the therapeutic drugs to which they have acquired resistance represents a vulnerability with potential applications in cancer treatment. However, for this drug addiction trait to become of clinical interest, we must first define the mechanism that underlies it. We performed an unbiased CRISPR-Cas9 knockout screen on melanoma cells that were both resistant and addicted to inhibition of the serine/ threonine-protein kinase BRAF, in order to functionally mine their genome for 'addiction genes'. Here we describe a signalling pathway comprising ERK2 kinase and JUNB and FRA1 transcription factors, disruption of which allowed addicted tumour cells to survive on treatment discontinuation. This occurred in both cultured cells and mice and was irrespective of the acquired drug resistance mechanism. In melanoma and lung cancer cells, death induced by drug withdrawal was preceded by a specific ERK2-dependent phenotype switch, alongside transcriptional reprogramming reminiscent of the epithelial-mesenchymal transition. In melanoma cells, this reprogramming caused the shutdown of microphthalmia-associated transcription factor (MITF), a lineage survival oncoprotein; restoring this protein reversed phenotype switching and prevented the lethality associated with drug addiction. In patients with melanoma that had progressed during treatment with a BRAF inhibitor, treatment cessation was followed by increased expression of the receptor tyrosine kinase AXL, which is associated with the phenotype switch. Drug discontinuation synergized with the melanoma chemotherapeutic agent dacarbazine by further suppressing MITF and its prosurvival target, B-cell lymphoma 2 (BCL-2), and by inducing DNA damage in cancer cells. Our results uncover a pathway that underpins drug addiction in cancer cells, which may help to guide the use of alternating therapeutic strategies for enhanced clinical responses in drug-resistant cancers.

BackgroundIn non-small cell lung cancer (NSCLC), PD-L1 expression on either tumor cells (TC) or both TC and tumor-infiltrating immune cells (IC) is currently the most used biomarker in cancer immunotherapy. However, the mechanisms involved in PD-L1 regulation are not fully understood. To provide better insight in these mechanisms, a multiangular analysis approach was used to combine protein and mRNA expression with several clinicopathological characteristics.Patients and methodsArchival tissues from 640 early stage, resected NSCLC patients were analyzed with immunohistochemistry for expression of PD-L1 and CD8 infiltration. In addition, mutational status and expression of a selection of immune genes involved in the PD-L1/PD-1 axis and T-cell response was determined.ResultsTumors with high PD-L1 expression on TC or on IC represent two subsets of NSCLC with minimal overlap. We observed that PD-L1 expression on IC irrespective of expression on TC is a good marker for inflammation within tumors. In the tumors with the highest IC expression and absent TC expression an association with reduced IFNγ downstream signaling in tumor cells was observed.ConclusionsThese results show that PD-L1 expression on TC and IC are both independent hallmarks of the inflamed phenotype in NSCLC, and TC-negative/IC-high tumors can also be categorized as inflamed. The lack of correlation between PD-L1 TC and IC expression in this subgroup may be caused by impaired IFNγ signaling in tumor cells. These findings may bring a better understanding of the tumor-immune system interaction and the clinical relevance of PD-L1 expression on IC irrespective of PD-L1 expression on TC.

Background Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary. Results We developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations. Conclusions XenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR. Electronic supplementary material The online version of this article (10.1186/s12859-018-2353-5) contains supplementary material, which is available to authorized users.

Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.

Tumor organoids are 3D cultures of cancer cells. They can be derived from the tumor of each individual patient, thereby providing an attractive ex vivo assay to tailor treatment. Using patient-derived tumor organoids for this purpose requires that organoids derived from biopsies maintain the genetic diversity of the in vivo tumor. In this study tumor biopsies were obtained from 14 patients with metastatic colorectal cancer (i) to test the feasibility of organoid culture from metastatic biopsy specimens and (ii) to compare the genetic diversity of patient-derived tumor organoids and the original tumor biopsy. Genetic analysis was performed using SOLiD sequencing for 1,977 cancer-relevant genes. Copy number profiles were generated from sequencing data using CopywriteR. Here we demonstrate that organoid cultures can be established from tumor biopsies of patients with metastatic colorectal cancer with a success rate of 71%. Genetic analysis showed that organoids reflect the metastasis from which they were derived. Ninety percent of somatic mutations were shared between organoids and biopsies from the same patient, and the DNA copy number profiles of organoids and the corresponding original tumor show a correlation of 0.89. Most importantly, none of the mutations that were found exclusively in either the tumor or organoid culture are in driver genes or genes amenable for drug targeting. These findings support further exploration of patient-derived organoids as an ex vivo platform to personalize anticancer treatment.

The development of targeted inhibitors, like vemurafenib, has greatly improved the clinical outcome of BRAF(V600E) metastatic melanoma. However, resistance to such compounds represents a formidable problem. Using whole-exome sequencing and functional analyses, we have investigated the nature and pleiotropy of vemurafenib resistance in a melanoma patient carrying multiple drug-resistant metastases. Resistance was caused by a plethora of mechanisms, all of which reactivated the MAPK pathway. In addition to three independent amplifications and an aberrant form of BRAF(V600E), we identified a new activating insertion in MEK1. This MEK1(T55delins) (RT) mutation could be traced back to a fraction of the pre-treatment lesion and not only provided protection against vemurafenib but also promoted local invasion of transplanted melanomas. Analysis of patient-derived xenografts (PDX) from therapy-refractory metastases revealed that multiple resistance mechanisms were present within one metastasis. This heterogeneity, both inter- and intra-tumorally, caused an incomplete capture in the PDX of the resistance mechanisms observed in the patient. In conclusion, vemurafenib resistance in a single patient can be established through distinct events, which may be preexisting. Furthermore, our results indicate that PDX may not harbor the full genetic heterogeneity seen in the patient's melanoma.

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three-dimensional imaging. In all patients, only a subset of lesions progressed. Next-generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib-resistant patient-derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.

Background: The majority of breast cancer patients are diagnosed with ERα-positive breast cancer. Most ERα-positive patients are treated with adjuvant endocrine therapy — typically tamoxifen or aromatase inhibitors — to block cellular proliferation. Although these treatments are considered successful, resistance is common. Notably, cross-resistance between the two types of therapies is not always observed suggesting molecular heterogeneity and underlining the need for development of personalized treatments. The Anastrozole, Fulvestrant or Tamoxifen Exposure — Response in molecular profile study (AFTER study, NCT00738777) aims to investigate prospectively whether short-term treatment can induce molecular changes indicative of pre-operative therapy response. Study Design: ERα-positive breast cancer patients are included in this open-label multicenter study. Post-menopausal patients are randomized between tamoxifen, anastrozole and fulvestrant and pre-menopausal and male patients receive tamoxifen. Treatment occurs during the pre-operative window between diagnosis and surgery (4±2 weeks). Clinical characteristics collected are ERα/PR and HER2 status as well as lymph-node status. The primary endpoint is the decrease in tumor cell proliferation, as assessed by Ki67 gene expression and published cell proliferation gene expression signatures. All data are collected from both pre- and post-treatment samples. Additionally, we will compare the changes induced by treatment in gene expression, ERα/DNA binding interactions, DNA copy number, endoxifen and estradiol levels. Results: Among 67 patients currently enrolled, we examined the data from the subset of 28 tamoxifen treated patients. ERα and PR levels did not differ significantly between pre- and post-treatment. All tumors were HER2-negative. Proliferation examined by Ki67 (IHC and gene expression, MKI67) was significantly lower in post-treatment samples (P < 0.01). A significant association was identified with the change in gene expression proliferation signature score and change in MKI67 (rho = 0.7, P < 0.001). We identified two samples, which changed from MammaPrint (MP) low-risk to high-risk among 17 pairs with data. One sample's score was on the cutoff for high-risk definition. Interestingly, the second sample also had an increase in Ki67 gene expression and proliferation gene signature score in the post-treatment sample. Overall, ERα/DNA binding interaction regions overlapped significantly more among post-treatment samples as compared to pre-treatment samples (P Citation Format: Linn SC, Severson TM, Nevedomskaya E, Peeters J, van Rossum A, Kuilman T, Krijgsman O, Goossens I, Glas A, Koornstra R, Peeper D, Wesseling J, Simon I, Wessels L, Zwart W. Neoadjuvant tamoxifen therapy synchronizes ERα binding and gene expression profiles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-06.

New clinical opportunities are needed to increase immunotherapy (IT) benefit. Whereas IFNγ-pathway mutations cause IT resistance, we find that IFNγ receptor-deficient tumors remain remarkably susceptible to T-cell elimination. By integrating transcriptomics, proteomics and genome-wide CRISPR-Cas9 screening with clinical data, we defined the IFNγ-independent tumor landscape and uncovered an immunotherapeutic opportunity. Ablation of TRAF2 enhanced T-cell elimination of both melanoma and lung cancer cells by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP. Corroborating these observations clinically, we identified recurring TRAF2 mutations that reduced T-cell sensitivity of tumor cells. Furthermore, both IT response duration and overall survival were lower for tumors that had acquired TNF pathway mutations on IT, suggesting immune-editing by TNF pressure. These results functionally annotate the tumor genome landscape of IFNγ-independent T-cell cytotoxicity, and merit clinical exploration of TRAF2 for cancer immunotherapy. Citation Format: Daniel S. Peeper, David Vredevoogd, Thomas Kuilman. T-cell-induced tumor vulnerability discovery in an IFNγ-independent genomic landscape [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B033.

Estrogen receptor alpha (ERα)-positive breast cancers are frequently treated with tamoxifen, but resistance is common. It remains elusive how tamoxifen resistance occurs and predictive biomarkers for treatment outcome are needed. Because most biomarker discovery studies are performed using pre-treatment surgical resections, the effects of tamoxifen therapy directly on the tumor cell in vivo remain unexamined. In this study, we assessed DNA copy number, gene expression profiles and ERα/chromatin binding landscapes on breast tumor specimens, both before and after neoadjuvant tamoxifen treatment. We observed neoadjuvant tamoxifen treatment synchronized ERα/chromatin interactions and downstream gene expression, indicating that hormonal therapy reduces inter-tumor molecular variability. ERα-synchronized sites are associated with dynamic FOXA1 action at these sites, which is under control of growth factor signaling. Genes associated with tamoxifen-synchronized sites are capable of differentiating patients for tamoxifen benefit. Due to the direct effects of therapeutics on ERα behavior and transcriptional output, our study highlights the added value of biomarker discovery studies after neoadjuvant drug exposure.

Almost half a century after the first reports describing the limited replicative potential of primary cells in culture, there is now overwhelming evidence for the existence of “cellular senescence” in vivo. It is being recognized as a critical feature of mammalian cells to suppress tumorigenesis, acting alongside cell death programs. Here, we review the various features of cellular senescence and discuss their contribution to tumor suppression. Additionally, we highlight the power and limitations of the biomarkers currently used to identify senescent cells in vitro and in vivo.

Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analysis of their RNA and DNA profiles is challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin can result both in the generation of false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. Therefore, we developed the open-source R-package XenofilteR, which separates mouse from human sequence reads based on the number of discordant base pairs between each read and the reference genomes. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human whole genome and whole exome DNA sequence data. This analysis revealed that XenofilteR removes >99.9% of sequence reads of mouse origin while retaining sequence reads of human origin. The filtering allowed for mutation analysis of PDX samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic mutations present in the original tumor. These findings were further validated in breast cancer and melanoma PDX samples, confirming the retrieval of accurate variant allele frequencies and somatic mutations. In conclusion, XenofilteR accurately dissects sequence reads from mouse and human origin in PDX sequence data, thereby outperforming currently available tools. Citation Format: Oscar Krijgsman, Roelof JC Kluin, Kristel Kemper, Thomas Kuilman, Julian R. de Ruiter, Vivek Iyer, Josep V. Forment, Paulien Cornelissen-Steijger, Iris de Rink, Petra ter Brugge, Ji-Ying Song, Sjoerd Klarenbeek, Ultan McDermott, Jos Jonkers, Arno Velds, David J. Adams, Daniel S. Peeper. XenofilteR: Computational dissection of mouse and human reads in PDX and xenograft sequence data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1041.

SummaryOncogene-induced cellular senescence (OIS) is emerging as a potent cancer-protective response to oncogenic events, serving to eliminate early neoplastic cells from the proliferative pool. Using combined genetic and bioinformatic analysis, we find that OIS is linked specifically to the activation of an inflammatory transcriptome. Induced genes included the pleiotropic cytokine interleukin-6 (IL-6), which upon secretion by senescent cells acted mitogenically in a paracrine fashion. Unexpectedly, IL-6 was also required for the execution of OIS, but in a cell-autonomous mode. Its depletion caused the inflammatory network to collapse and abolished senescence entry and maintenance. Furthermore, we demonstrate that the transcription factor C/EBPβ cooperates with IL-6 to amplify the activation of the inflammatory network, including IL-8. In human colon adenomas, IL-8 specifically colocalized with arrested, p16INK4A-positive epithelium. We propose a model in which the context-dependent cytostatic and promitogenic functions of specific interleukins contribute to connect senescence with an inflammatory phenotype and cancer.

Breast cancers with BRCA1 germline mutation have a characteristic DNA copy number (CN) pattern. We developed a test that assigns CN profiles to be ‘BRCA1‐like’ or ‘non‐BRCA1‐like’, which refers to resembling a BRCA1‐mutated tumor or resembling a tumor without a BRCA1 mutation, respectively. Approximately one third of the BRCA1‐like breast cancers have a BRCA1 mutation, one third has hypermethylation of the BRCA1 promoter and one third has an unknown reason for being BRCA1‐like. This classification is indicative of patients' response to high dose alkylating and platinum containing chemotherapy regimens, which targets the inability of BRCA1 deficient cells to repair DNA double strand breaks. We investigated whether this classification can be reliably obtained with next generation sequencing and copy number platforms other than the bacterial artificial chromosome (BAC) array Comparative Genomic Hybridization (aCGH) on which it was originally developed. We investigated samples from 230 breast cancer patients for which a CN profile had been generated on two to five platforms, comprising low coverage CN sequencing, CN extraction from targeted sequencing panels (CopywriteR), Affymetrix SNP6.0, 135K/720K oligonucleotide aCGH, Affymetrix Oncoscan FFPE (MIP) technology, 3K BAC and 32K BAC aCGH. Pairwise comparison of genomic position‐mapped profiles from the original aCGH platform and other platforms revealed concordance. For most cases, biological differences between samples exceeded the differences between platforms within one sample. We observed the same classification across different platforms in over 80% of the patients and kappa values of at least 0.36. Differential classification could be attributed to CN profiles that were not strongly associated to one class. In conclusion, we have shown that the genomic regions that define our BRCA1‐like classifier are robustly measured by different CN profiling technologies, providing the possibility to retro‐ and prospectively investigate BRCA1‐like classification across a wide range of CN platforms., Highlights We investigated concordance of BRCA1‐like classification of copy number profiles across technologies.We included array‐based and (targeted) next generation sequencing profiling techniques.BRCA1‐like classification was highly concordant across techniques and datasets.10% misclassification can be attributed to poorer quality, weak class association and experimental variation.We concluded that BRCA1‐like classification of breast cancer is robust across techniques and datasets.

Current methods for detection of copy number variants (CNV) and aberrations (CNA) from targeted sequencing data are based on the depth of coverage of captured exons. Accurate CNA determination is complicated by uneven genomic distribution and non-uniform capture efficiency of targeted exons. Here we present CopywriteR, which eludes these problems by exploiting ‘off-target’ sequence reads. CopywriteR allows for extracting uniformly distributed copy number information, can be used without reference, and can be applied to sequencing data obtained from various techniques including chromatin immunoprecipitation and target enrichment on small gene panels. CopywriteR outperforms existing methods and constitutes a widely applicable alternative to available tools. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0617-1) contains supplementary material, which is available to authorized users.

Cellular senescence, a growth-arrest program that limits the lifespan of mammalian cells and prevents unlimited cell proliferation, is attracting considerable interest because of its links to tumour suppression. Using a mouse model in which the oncogene Ras is activated in the haematopoietic compartment of bone marrow, Braig et al. show that cellular senescence can block lymphoma development. Genetic inactivation of the histone methyltransferase Suv39h1 that controls senescence by ‘epigenetic’ modification of DNA-associated proteins, or a pharmacological approach that mimics loss of this enzyme, allow the formation of malignant lymphomas in response to oncogenic Ras. This work has important implications for both tumour development and tumour therapy. Michaloglou et al. report that oncogene-induced senescence may be a physiologically important process in humans, keeping moles in a benign state for many years: unchecked they develop into malignant melanomas. Chen et al. also find that cellular senescence blocks tumorigenesis in vivo: they show that acting together, the p53 tumour suppressor and the cellular senescence system can prevent prostate cancer induction in mice by the PTEN mutation. Collado et al. show that cellular senescence is a defining feature of Ras-initiated premalignant tumours; this could prove valuable in the diagnosis and prognosis of cancer. See the web focus . Most normal mammalian cells have a finite lifespan1, thought to constitute a protective mechanism against unlimited proliferation2,3,4. This phenomenon, called senescence, is driven by telomere attrition, which triggers the induction of tumour suppressors including p16INK4a (ref. 5). In cultured cells, senescence can be elicited prematurely by oncogenes6; however, whether such oncogene-induced senescence represents a physiological process has long been debated. Human naevi (moles) are benign tumours of melanocytes that frequently harbour oncogenic mutations (predominantly V600E, where valine is substituted for glutamic acid) in BRAF7, a protein kinase and downstream effector of Ras. Nonetheless, naevi typically remain in a growth-arrested state for decades and only rarely progress into malignancy (melanoma)8,9,10. This raises the question of whether naevi undergo BRAFV600E-induced senescence. Here we show that sustained BRAFV600E expression in human melanocytes induces cell cycle arrest, which is accompanied by the induction of both p16INK4a and senescence-associated acidic β-galactosidase (SA-β-Gal) activity, a commonly used senescence marker. Validating these results in vivo, congenital naevi are invariably positive for SA-β-Gal, demonstrating the presence of this classical senescence-associated marker in a largely growth-arrested, neoplastic human lesion. In growth-arrested melanocytes, both in vitro and in situ, we observed a marked mosaic induction of p16INK4a, suggesting that factors other than p16INK4a contribute to protection against BRAFV600E-driven proliferation. Naevi do not appear to suffer from telomere attrition, arguing in favour of an active oncogene-driven senescence process, rather than a loss of replicative potential. Thus, both in vitro and in vivo, BRAFV600E-expressing melanocytes display classical hallmarks of senescence, suggesting that oncogene-induced senescence represents a genuine protective physiological process.

Recent advances in the understanding of tumor immunology have led to the development of various immunotherapeutic strategies, some of which show unprecedented clinical efficacies. Two of the most effective treatments, ipilimumab and pembrolizumab / nivolumab target the immune checkpoints factors CTLA-4 and PD-1, respectively. While not all patients respond to immunotherapy, those that do frequently show long-lasting clinical responses. These accomplishments have led to a surge in research focusing on the question how the immune system can be stimulated for more common durable therapy responses. A current focus of tumor immunological research is the identification of mechanisms allowing cancer cells to evade recognition and/or killing by the immune system while facing immunotherapy. For example, abrogation of interferon-γ signaling pathway has been shown to result in escape of immune surveillance after pembrolizumab treatment. However, it is likely that other tumor cell-intrinsic mechanisms play a role, too. To uncover novel pathways that are involved in resistance to immune checkpoint inhibitors, we aim to identify transcriptional changes in tumor cells that result from effector T-cell exposure. To this end, we established a co-culture platform of a panel of Mart1-expressing melanoma cell lines with primary CD8+ T cells expressing a matching TCR. The cell lines display varying degrees of sensitivity to T cells, allowing us to bioinformatically uncover differential expression depending on this range of sensitivities. Rather than concentrating on single genes, we have focused on pathways, gene sets and ontologies. These analyses have been integrated with various publically available transcriptomic data sets to identify a set of mechanisms that may contribute to T-cell mediated killing. We will systematically abrogate key factors using CRISPR technology in our co-culture platform and assess whether this affects T-cell mediated killing. Our ultimate goal is to test whether these factors, or their signaling pathways, are involved in resistance to immunotherapy in patients, and to develop new strategies to prevent immunotherapy resistance. Note: This abstract was not presented at the meeting. Citation Format: Thomas Kuilman, David Vredevoogd, Daniel S. Peeper. Uncovering novel signaling pathways that mediate tumor cell killing by T effector cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5627. doi:10.1158/1538-7445.AM2017-5627

The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast. We use phosphoproteome analysis of mitotic exit to identify Cdk targets that are dephosphorylated at the time of cytokinesis. We then apply a new and widely applicable tool to generate conditionally phosphorylated proteins to identify those whose dephosphorylation is required for cytokinesis. This approach identifies Aip1, Ede1 and Inn1 as cytokinetic regulators. Our results suggest that cytokinesis is coordinately controlled by the master cell cycle regulator Cdk together with its counteracting phosphatase and that it is executed by concerted dephosphorylation of Cdk targets involved in several cell biological processes.

Substrate dephosphorylation by the cyclin-dependent kinase (Cdk)-opposing phosphatase, Cdc14, is vital for many events during budding yeast mitotic exit. Cdc14 is sequestered in the nucleolus through inhibitory binding to Net1, from which it is released in anaphase following Net1 phosphorylation. Initial Net1 phosphorylation depends on Cdk itself, in conjunction with proteins of the Cdc14 Early Anaphase Release (FEAR) network. Later on, the Mitotic Exit Network (MEN) signaling cascade maintains Cdc14 release. An important unresolved question is how Cdc14 activity can increase in early anaphase, while Cdk activity, that is required for Net1 phosphorylation, decreases and the MEN is not yet active. Here we show that the nuclear rim protein Nur1 interacts with Net1 and, in its Cdk phosphorylated form, inhibits Cdc14 release. Nur1 is dephosphorylated by Cdc14 in early anaphase, relieving the inhibition and promoting further Cdc14 release. Nur1 dephosphorylation thus describes a positive feedback loop in Cdc14 phosphatase activation during mitotic exit, required for faithful chromosome segregation and completion of the cell division cycle., Author Summary During the cell cycle, a specific sequence of events leads to the formation of two daughter cells from one mother cell. Progression through the cell cycle is tightly controlled, with events occurring in the right place at the right time. Exactly how this is achieved is still being elucidated. In budding yeast, the events occurring during the final cell cycle phase – “mitotic exit” – are controlled by the phosphatase Cdc14. It is kept sequestered and inactive until it is needed for mitotic exit, at which time it is rapidly released. In this study, we have identified a new regulator of Cdc14 activity, the protein Nur1. In a series of experiments, we saw that Nur1 acts both upstream and downstream of Cdc14 activation, thereby creating a positive feedback loop. On the one hand, Nur1 contributes to inhibiting Cdc14 until the start of mitotic exit. On the other hand, through the actions of Cdc14 itself, Nur1 is disabled as an opponent of the phosphatase. This creates a robust system, rapidly switching between two opposing states and thus driving forward the mitotic exit transition.

International audience; Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.

Background: The Anastrozole, Fulvestrant or Tamoxifen Exposure—Response in molecular profile (AFTER study, NCT00738777) aims to investigate the molecular basis of therapy response to neoadjuvant treatment in Estrogen Receptor alpha (ERα) breast cancer patients. ERα breast cancer makes up the majority of breast cancer diagnosed in the world each year. In these patients, tumor cell proliferation is driven by transcriptional activation through ERα binding DNA at specific genomic locations. To block this proliferation, endocrine therapies such as tamoxifen have been developed. While endocrine therapy is considered a successful targeted treatment, resistance is common. Patients who recur are typically given a different type of endocrine therapy and frequently respond well, suggesting ERα breast cancer is a heterogeneous disease made up of subtypes responsive to different treatments. We hypothesize drug exposure induces molecular changes in the tumor and the basis for these alterations may provide insight into the phenomenon of therapy resistance. Study Design and Methods: In the AFTER study ERα breast cancer patients are randomized for treatment arm in a multi-institutional study. Male and pre-menopausal patients receive tamoxifen. The primary endpoint is decreased tumor cell proliferation assessed by Ki67 gene expression. Tumor samples are taken at the time of diagnosis (biopsy) and surgery (primary tumor) for molecular and clinical assays. Treatment occurs during the pre-operative window after diagnosis. All data are collected at both timepoints. Molecular data collected include gene expression and ERα/DNA binding patterns and DNA copy number profiles. Clinical data comprise ERα/PR/HER2 and Ki67 immunohistochemical staining (IHC) and lymphnode status. Results: 67 patients are currently enrolled in the study. We examined the subset of tamoxifen treated patients (n=28). All tumors were HER2-negative. ERα/PR showed no significant difference between pre- and post-treatment levels. Tumor cell proliferation was significantly lower in post-treatment samples (Ki67 gene expression P < 0.01). ERα/DNA interaction regions were more frequently overlapping in the post-treatment samples compared with the pre-treatment samples. Change in published gene expression proliferation signatures was significantly correlated with change in Ki67 gene expression (ρ = 0.7, P Conclusions: Substantial synchronization in ERα/DNA binding and gene expression pattern was observed after treatment, while DNA copy number patterns remained stable. Our results indicate neoadjuvant tamoxifen induces molecular changes in a heterogeneous group of breast cancers associated with change in proliferation and ERα/DNA binding landscape. Importantly, while most tumors show these changes, some do not. Follow-up data will demonstrate whether taking samples after treatment exposure may be a powerful tool to help understand response to treatment. Citation Format: Tesa M. Severson, Ekaterina Nevedomskaya, Justine Peeters, Annelot van Rossum, Thomas Kuilman, Oscar Krijgsman, Rutger Koornstra, Daniel Peeper, Jelle Wesseling, Iris M. Simon, Lodewyk Wessels, Wilbert Zwart, Sabine C. Linn. Effects on epigenomic, transcriptomic, and genomic landscape of neoadjuvant tamoxifen in estrogen receptor alpha breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A38.

Oncogene-induced cellular senescence constitutes a strong anti-proliferative response, which can be set in motion following either oncogene activation or loss of tumour suppressor signalling. It serves to limit the expansion of early neoplastic cells and as such is a potent cancer-protective response to oncogenic events. Recently emerging evidence points to a crucial role in oncogene-induced cellular senescence for the 'senescence-messaging secretome' or SMS, setting the stage for cross-talk between senescent cells and their environment. How are such signals integrated into a coordinated response and what are the implications of this unexpected finding?

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

International audience; In Saccharomyces cerevisiae, dicentric chromosomes stemming from telomere fusions preferentially break at the fusion. This process restores a normal karyotype and protects chromosomes from the detrimental consequences of accidental fusions. Here, we address the molecular basis of this rescue pathway. We observe that tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. We also show that condensins generate forces sufficient to rapidly refold dicentrics prior to breakage by cytokinesis and are essential to the preferential breakage at telomere fusions. Thus, the rescue of fused telomeres results from a condensin- and Rap1-driven chromosome folding that favors fusion entrapment where abscission takes place. Because a close spacing between the DNA-bound Rap1 molecules is essential to this process, Rap1 may act by stalling condensins.