Oncogenic Hits Research Articles

Abstract 4800: A next-gen animal model to Study PIK3CA-mTOR driven HPV-related oral malignancies

Abstract The rising incidence of HPV-associated malignancies, especially for head and neck squamous cell carcinoma (HNSCC), has highlighted the urgent need to understand the role of HPV-16 E6/E7 oncogenes in cancer initiation and progression. For that propose we decided to generate a doxycycline inducible E6/E7 transgenic mouse targeting E6/E7 onco-proteins to the basal epithelia layer, including the stem cell compartment, through a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA). This system allows to turn on the expression of HPV genes after birth, minimizing the negative effect that viral E6/E7 expression may have during embryonic development, and mimicking the natural infection process that usually happens in adults after initiation of sexual activities. Interestingly, we found that after doxycycline induction, both E6 and E7 were highly expressed, resulting in a clear increase in the number of proliferative cells even in the suprabasal layers, which results in rapid epidermal hyperplasia. However, in our animal mouse model HPV-16 E6/E7 expression alone was not sufficient to induce spontaneous squamous carcinomas (SCC). Due that we decided to challenge the oncogenic role of HPV using a classical chemical carcinogenesis protocol (DMBA-TPA). These mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, TPA. These data suggest that only few oncogenic hits may be sufficient to induce cancer in humans already infected with HPV-16 E6/E7. Then, we decided to elucidate whether or not a few genomic alterations in combination with HPV-16 expression may be sufficient to induce cancer. One of the most frequent mutations in HNSCC, including HPV-associated cancers, are those affecting the PI3K-mTOR pathway. Based on that, we engineered a genetically defined mouse model combining HPV-16 E6/E7 and PIk3CAH1047R expression under the control of a cytokeratin 14 promoter driven tamoxifen-regulated Cre recombinase (cK14-CreERTM). Surprisingly, we found that upon tamoxifen activation mice develop oral cancer in only few weeks. Furthermore, our HPV-related cancer mouse models exhibited a marker increase of mTOR activation which is a key oncogenic driver in malignant progression to SCC. Remarkably, rapamycin, an mTOR inhibitor, was able to block tumor development, supporting the potential clinical use of direct and indirect mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies. Citation Format: Juan Luis Callejas-Valera, Ramiro Iglesias-Bartolome, Panomwat Amornphimoltham, Julia Palacios-Garcia, Daniel Martin, Joseph A. Califano, Alfredo A. Molinolo, J.Silvio Gutkind. A next-gen animal model to Study PIK3CA-mTOR driven HPV-related oral malignancies [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 4800. doi:10.1158/1538-7445.AM2017-4800

Role of SOX11 and Genetic Events Cooperating with Cyclin D1 in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm, incurable with current therapies. The t(11;14)(q13;q32) involving cyclin D1 is considered the first oncogenic hit found in virtually all MCLs. However, additional secondary genomic alterations are essential for complete transformation. MCLs are genetically very unstable with several genetic alterations associated with its high proliferative behavior involving several oncogenic pathways. Furthermore, SOX11 is overexpressed in the majority of conventional MCLs (cMCL), including cyclin D1-negative cases, but absent in non-nodal leukemic MCL with indolent clinical behavior (nnMCL). Recent data have revealed the potential oncogenic role of SOX11 in MCL biology, highlighting its implication in tumor aggressiveness and progression. This review addresses the implication of SOX11 overexpression and frequent genetic lesions, cooperating with cyclin D1 underlying the pathogenesis of this aggressive disease.

2% of Healthy Newborns Reveal ETV6-RUNX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency.To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based retrospective screening of 300 cord blood samples from Danish newborns.The GIPFEL method is capable of detecting the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In contrast to previously used RNA-based methods, it relies on DNA as sample material, which is more stable than RNA. In the case of ETV6-RUNX1-positive leukemia GIPFEL exploits the unique presence of a genomic fragment joining material from chromosome 12 and 21. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. The ligation site is independent of the translocation point within the individual DNA circle. Digestion of the breakpoint regions of the ETV6 and RUNX1 gene with the restriction enzyme SacI generates fragments smaller than 50 kb. Primer pairs amplify the complete set of theoretically predicted circularized fragments requiring 37 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from mononuclear cells from cord blood samples of 300 newborns that were cryopreserved within 24 h (median 12 h) from birth. After B cell enrichment and column purification of DNA, the DNA was subjected to SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gelelectrophoresis and Sanger sequencing to validate the result and to identify the breakpoint region. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In previously published proof-of-principle blinded studies we tested 61 samples obtained from ETV6-RUNX1-positive ALL patients. Without any unspecific result, 64% for ETV6-RUNX1 fusion genes were detected in that sample set. The sensitivity of the technique was estimated to be 10-4, i.e. one translocation carrying cell within 10,000 normal cells can theoretically be detected.Within the analyzed cohort of 300 healthy newborns 6 screened positive for the ETV6-RUNX1 translocation (2%) (Table 1). Further 700 cord blood samples are currently screened.Table 1: 6 of 300 cord blood samples from healthy newborns screened positive for the ETV6-RUNX1 translocation using the GIPFEL technique (Fueller E*, Schaefer D* et al. PloS One 2014, 9(8): e104419). Number of the positively tested healthy newborn within the cohort, used primers, and introns of RUNX1 and ETV6 affected by the translocation are presented.Our results indicate that the actual incidence of ETV6-RUNX1-positive cells in healthy newborns might be even higher than previously assumed, potentially due to instability of the ETV6-RUNX1 RNA transcript in preserved cord blood samples. This would hint at a comparably low penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and further strengthen the importance of secondary environmentally caused or spontaneously occurring cooperating oncogenic lesions for ETV6-RUNX1-positive childhood leukemia to emerge. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair.

The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens.

Triggering the TCR Developmental Checkpoint Activates a Therapeutically Targetable Tumor Suppressive Pathway in T-cell Leukemia.

Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ε chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. T-ALLs are aggressive malignant lymphoid proliferations of T-cell precursors characterized by high relapse rates and poor prognosis, calling for the search for novel therapeutic options. Here, we report that the lineage-specific TCR/CD3 developmental checkpoint controlling cell death in normal T-cell progenitors remains switchable to induce massive tumor cell apoptosis in T-ALL and is amenable to preclinical therapeutic intervention. Cancer Discov; 6(9); 972-85. ©2016 AACR.See related commentary by Lemonnier and Mak, p. 946This article is highlighted in the In This Issue feature, p. 932.

MTOR inhibition prevents rapid-onset of carcinogen-induced malignancies in a novel inducible HPV-16 E6/E7 mouse model.

The rising incidence of human papillomavirus (HPV)-associated malignancies, especially for oropharyngeal cancers, has highlighted the urgent need to understand how the interplay between high-risk HPV oncogenes and carcinogenic exposure results in squamous cell carcinoma (SCC) development. Here, we describe an inducible mouse model expressing high risk HPV-16 E6/E7 oncoproteins in adults, bypassing the impact of these viral genes during development. HPV-16 E6/E7 genes were targeted to the basal squamous epithelia in transgenic mice using a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA) system. After doxycycline induction, both E6 and E7 were highly expressed, resulting in rapid epidermal hyperplasia with a remarkable expansion of the proliferative cell compartment to the suprabasal layers. Surprisingly, in spite of the massive growth of epithelial cells and their stem cell progenitors, HPV-E6/E7 expression was not sufficient to trigger mTOR activation, a key oncogenic driver in HPV-associated malignancies, and malignant progression to SCC. However, these mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Thus, only few oncogenic hits may be sufficient to induce cancer in E6/E7 expressing cells. All HPV-E6/E7 expressing SCC lesions exhibited increased mTOR activation. Remarkably, rapamycin, an mTOR inhibitor, abolished tumor development when administered to HPV-E6/E7 mice prior to DMBA exposure. Our findings revealed that mTOR inhibition protects HPV-E6/E7 expressing tissues form SCC development upon carcinogen exposure, thus supporting the potential clinical use of mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies.

Abstract 4385: Live-imaging the interface between homeostasis and cancer initiation

Abstract While cancer-associated mutations are most frequently found within tumors, it has been established that these same mutations can be present in even phenotypically normal organs, such as the skin. This raises the question as to how the switch from normal to malignant is initiated within the interface between homeostasis and cancer. One challenge to understanding the initiating events towards malignancy is the inability to follow the same cells over time in an intact mammal. Specifically, this roadblock hinders the ability to understand both the role of specific cells and how their location contributes to their growth, whether that growth be normal or cancerous. To overcome this, we have established a novel live imaging approach to track cellular behaviors in normal, healthy skin. This approach has previously enabled our lab to demonstrate that location dictates the fate and behaviors of hair follicle stem cells during skin regeneration (Rompolas et al. Nature 2013). We are now utilizing this approach to capture the emergence of the cellular behaviors that lead to Hras-driven skin tumors in live mice. To begin to characterize the initiating events of skin cancer, we utilized a model of oncogenic Hras (HrasG12V), which has been established to give rise to skin papillomas. Using targeted, inducible genetic drivers, we activated HrasG12V at low levels and restricted the activation to the cycling, stem-cell rich portion of the hair follicle. Strikingly, we have observed that the presence of HrasG12V cells within the cycling portion of the hair follicle are not only tolerated, but can also be eliminated, supporting the idea that the surrounding wild-type tissue has the capability to both integrate and overcome the presence of a single oncogenic hit. Furthermore, within this targeted genetic system, we found that the initial position of the mutant cell impacted the resultant phenotype of the tissue. To next understand these behaviors at the cellular level, we utilized a viral approach to directly activate and fluorescently tag HrasG12V at clonal levels throughout the skin epithelium allowing us to observe interactions between wild-type and mutant cells. Furthermore, within the upper portion of the hair follicle and epidermis, which (unlike the cycling portion) is immune permissive, we observed the release of exosomes by mutant cells and their subsequent clearing by both epithelial and immune cells. Currently, we are introducing a second mutation, loss of TGFβ signaling, to the oncogenic Hras system which is a mutational combination well established to produce cutaneous Squamous Cell Carcinoma (cSCC). This system will provide us with a spectrum of phenotypes from wild-type, to benign yet aberrant (papillomas), to malignant (cSCC) and allow us to interrogate how interactions among different cell types (wild-type, single mutants, and double mutants) within particular niches influence the overall fate of the tissue to initiate disease. Citation Format: Cristiana Pineda, Catherine Martone, Katie Suozzi, Samara Brown, Slobodan Beronja, Valentina Greco. Live-imaging the interface between homeostasis and cancer initiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4385.

65. Oncolytic Measles Virus Differentially Affects Mitochondrial Biogenesis in Transformed versus Non-Transformed BM-Derived MSCs

Vaccine strain measles virus (MV) is oncolytic in numerous models of malignancy. The mechanism behind the selectivity of MV for transformed cells is poorly understood. To investigate further, an established step-wise model of cellular transformation was used; in which progressive oncogenic hits were stably and additively expressed in human bone marrow derived mesenchymal stromal cells following retroviral transfer of human telomerase reverse transcriptase (hTERT), human papilloma virus16 E6 and E7 (3H), SV40 small T antigen (4+V), finally, H-RAS (5H) (Funes at al, 2007). The most highly transformed cells (5H) were more permissive to oncolytic MV infection than any of the less transformed counterparts, with significantly greater viral titres. MV-induced cell-death increased progressively with progressive transformation. This was not explained by any differences in MV receptors CD46, SLAM or Nectin-4 expression. Investigation of anti-viral type 1 IFN response in this model 24 and 48 hours post MV infection (hpi) by ELISA demonstrated a robust induction of IFNβ (to a lesser extent IFNα) in hTERT cells, which was significantly and progressively reduced in 3H, 4+V and 5H according to level of transformation, suggesting that defective IFN pathway is a potential mechanism for the enhanced MV permissiveness observed in transformed cells. Examination of integrity of the RLR signalling pathway, which triggers IFNα/β production, revealed that expression levels of RIG-I, MDA5 and MAVS, determined by RQ-PCR at 6, 12, 24 and 48 hpi, were lowest in 5H and highest in hTERT cells, proposing a role for the RLR pathway in MV-mediated oncolysis. To gain insight into the differential effects of MV infection in transformed versus non-transformed cells, metabolic effects post MV infection were investigated. A significant increase in oxygen consumption rate (OCR) was observed 24 hpi, proportional to the stage of transformation, followed by a steep decline at 48 hpi. This increase was abrogated in the presence of fusion inhibitory peptide (FIP), implicating the role of syncytia formation, the cytopathic effect of MV infection, in MV-mediated oncolysis in vitro. A similar pattern was observed in ATP levels post MV infection; highest at 24hpi in 4+V and 5H and dropping off dramatically at 48hpi when cell viability is compromised. To further characterize the consistent increase in OCR and ATP levels upon MV infection of the more transformed cell lines, we sought to assess mitochondrial biogenesis using the fluorescent probes TMRM, Calcein AM and Hoechst. Preliminary results indicated significantly increasing numbers of mitochondria per cell at 24 hpi principally in 4+V (16%) and 5H (~29%) cells compared to their uninfected counterparts potentially explaining the observed high levels of OCR and ATP. Altogether, our data suggests that altered mitochondrial numbers and functions correlate with the degree of MV permissiveness and thus MV-induced cell death. Mitochondrial mass was also seen to be increased with MV infection. However, this effect was not specific to transformed cells. Metabolomics and further evaluations will be crucial to comprehensively assess differences in metabolism between cells in this model and to understand the role of bioenergetics in the differential levels of MV-mediated oncolysis.

Clonal Mutations and Clonal Hierarchy in Pediatric Myeloid Neoplasms

Pediatric myeloid neoplasms are a clinically and genetically heterogeneous group of clonal diseases. Unlike in adults inherited predisposition plays a major role in leukemogenesis. A number of germline mutations are known to increase the risk of somatically occurring myeloid transformation. These include the genes associated with the classical inherited bone marrow failure syndromes, but also transcriptopathies resulting from RUNX1, CEBPA or GATA2 deficiency. Herein we review the most significant findings from recent years and our present experience on the clonal landscape of myeloid malignancies in children with a focus on pediatric MDS and JMML. In both disease entities, monosomy 7 is the most frequent chromosomal aberration. It is likely that haploinsufficiency of genes located on chromosome 7 contributes to clonal evolution. Several of the more than 1000 lost genes were postulated as mechanistic drivers. However, in most cases additional somatic mutations not related to chromosome 7 are found at diagnosis, making it difficult to pinpoint the initial oncogenic hit. Driver somatic hits frequently encountered in adults with MDS, affecting TET2, DNMT3A or the spliceosome genes, do not play a major role in the pathogenesis of childhood disease. Also, considering the young age, environmental effects and age-related pre-malignant clonal mutations can be disregarded. In fact, GATA2 germline mutations can be considered the most common "initial hit" in pediatric MDS. In our experience, GATA2 deficiency accounts for 37% of primary pediatric MDS with monosomy 7. The prevalence of GATA2 mutations in MDS with monosomy 7 increases dramatically in adolescence where they are found in up to 72%. In comparison, 16% of pediatric patients with MDS and trisomy 8 have an underlying GATA2deficiency. The somatic mutational landscape in childhood MDS is associated with cytogenetic subgroups, i.e. SETBP1 and ASXL1 mutations are overrepresented in cases with monosomy 7. Interestingly, longitudinal NGS analysis and single colony sequencing indicate that SETBP1 lesions precede the development of ASXL1 mutations. We identified the genes SETBP1, ASXL1, NRAS, KRAS, RUNX1, PTPN11 and BCOR/ BCORL as the most frequent targets of recurrent mutations in a large cohort of pediatric patients with primary MDS. The biological significance of these genomic changes is currently not understood and warrants further studies. In contrast to pediatric MDS, signaling pathways involved in JMML have been better defined during recent years. Somatic aberrations in PTPN11, NRAS, KRAS, CBL, and NF1 genes lead to a constitutively overactive RAS pathway in leukemic cells. Additional activating mutations were identified in some JMML patients, including the RAS pathway gene RRAS, and concomitant mutations in SETBP1 and JAK3. The latter were postulated to develop as subclonal events during disease progression and to confer poor prognosis. However most recently, very rare clones with SETBP1 mutation were reported to be present at diagnosis in as much as 30% of JMML patients. This might indicate that multiple clones with different clonal composition arise during initial stages of disease, and therapy-driven selection pressure results in the expansion of these subclones. It is not clear how the somatic mutations interact with the epigenetic landscape in JMML clones, especially with regard to promoter hypermethylation, and what are the definitive biological consequences. In summary, a number of germline and somatic mutations have been identified which can initiate the development of abnormal hematopoiesis in children. Premalignant stem cells undergo evolutionary selection and produce somatically altered malignant clones resulting in myeloid transformation. DisclosuresNo relevant conflicts of interest to declare.

Somatic Mutation and Isotype Switch Events Outside Igv Gene Loci Provide New Evidence for a Role of Antigenic Challenge in Mantle Cell Lymphoma Etiology

Mantle cell lymphoma (MCL) represents 5-10% of B-cell lymphomas and generally exhibits an aggressive clinical behavior. t(11;14) translocation, the genetic hallmark and early initiating event of MCL pathogenesis, lead to the constitutive CCND1 expression and consequently to cell-cycle deregulation. However, t(11;14) is also present in the blood from rare healthy individuals indicating that alone the t(11;14) is not sufficientfor malignant transformation. It has long been assumed in MCL that t(11;14)+ cells were actually carried by circulating naive and antigen-inexperienced B-cells. Yet, several reports, including long-term persistence of t(11;14)+ cells in lymphoma free-patients, biased immunoglobulin heavy variable chain gene (IgVH) repertoire and evidence of subset of cases with mutated IgVH genes led us to question this model and further investigate the role of antigenic challenge in MCL etiologic origin.As a first approach, we sought to determine the class-switch recombination (CSR) status of t(11;14)+ cells in MCL patients. CSR, as somatic hypermutation (SHM), are diversification mechanisms of the B cell receptor induced upon antigen encounter in the germinal center (GC) reaction, and absent in naive cells. Evidence for ongoing switch events in MCL has been previously reported with detection of mature switch transcripts, however in MCL cells, sIgM(D) expression remains clearly evident suggesting that effective deletional switch events occur infrequently on the functional allele. To characterize CSR in t(11;14)+ cells, we thus took advantage of their unique BCL1/IGH translocation signature assuming that BCL1/IGH fusions do not prevent CSR to occur on the downstream constant region of the IGH locus. Using 2 long-range PCR assays designed to amplify unswitched BCL1/Sµ and switched BCL1/Sγ regions, DNA from 30 MCL patients differing by their IgVH status were tested. Of the MCL cases, we confirmed that 7 of 30 (23%) were unmutated (UM, 100% germline homology) and 23 of 30 (77%) were mutated (M), considering as mutated all cases exhibiting any level of SHM (ranging from minimal: >0.3% to high rate: 7.0%). Although mutated BCRs were common in most MCL cases, we found that only 1 out of 30 MCL cases underwent CSR to IgG on the non-productive, translocated allele. Accordingly, this case carried a mutated IgVH gene with 97.5% germline identity in line with a GC/antigen-experienced signature of the tumor cells. Contrary to SHM events of the IgVH gene region, CSR deletional events appeared very infrequent in the bulk of MCL cells both on the functional and translocated allele, contrasting from findings in other GC-derived non-Hodgkin lymphomas where the same selective pressure to maintain a sIgM is accompanied by deletional switch events on the non-functional allele. To get more insights into a role of antigenic drive in MCL and AID targeting outside the VH gene loci, we next evaluated the clonal evolution of t(11;14)+ cells from 11 MCL cases (2 UM and 9 M) by analyzing SHM in the Sμ region on the translocated allele using sequencing of LR-PCR amplicons (with 2 subclones/patient). We found that 63% (7 out of 11) of BCL1/IGH clones display significant levels of SHM in the Sµ together with intra-Sµ indels (2/11) compatible with off-target AID-mediated events linked to chronic(?) antigen encounter. In addition, we found a good correlation between mutated BCR and the SHM levels in Sμ regions for a given patient. Importantly, in one instance subclones from a MCL case displayed intraclonal diversification (despite the fact that the expected rate of SHM is much lower in the Sµ region than in the IgVH region), indicative of an ongoing AID activity at least in a subset of MCL cases. In conclusion, we found that CSR events, although detectable in some MCL cases, do not appear to participate predominantly or to be selected during MCL lymphomagenesis. By contrast, analysis of SHM profiles both on the functional and the non-functional translocated allele provide evidence for an active AID activity in MCL cells inside and outside V-gene loci, highlighting the propensity of some t(11;14)+ clones to acquire a complementary oncogenic hit over time as a result of recurrent antigen challenge. DisclosuresNo relevant conflicts of interest to declare.

Abstract A15: A human mini brain model of pediatric GBM

Abstract Glioblastoma (GBM) is the most devastating brain cancer with dismal prognosis both in adults and children and accounts for 16% of primary brain tumors. Despite aggressive therapeutic approaches, the majority of patients die within one year after diagnosis. Giving the limitation of current GBM mouse models, which do not reflect the biological properties of human tumors, in particular, tumor heterogeneity and pathogenesis, there is a growing need for new models. Current in vitro 3D models involve the use of hydrogel scaffolds and cell lines, which do not represent a viable developmental context where the tumor mass interacts with and invades the surrounding brain tissues. Starting with pluripotent human embryonic stem cells we reproduced a 3D tissue structure (so-called cerebral organoid) representing a developing human brain in vitro. We engineered these mini-brains to express oncogenic hits responsible for the derivation of tumors in pediatric GBM patients. Pediatric GBM is driven by a combination of epigenetic and genetic hits including the expression of a mutant histone H3 variant H3F3A (mutated at K27M or G34R) or a gain of function mutant isocitrate dehydrogenase IDH1R132H in combination with the loss of the histone remodeling factor ATRX and/or p53 tumor suppressor. We used a combination of these oncogenic hits to create a model of tumor engineering representing the first 3D human tissue model of GBM. This model combines the accurate multi-lineage differentiation and physiology of human brain with the easiness of manipulation of in vitro systems. H&E staining of the modified organoids shows remarkable dysplasia reminiscent of human primary glioma. Different genetic hits result in different type of dysplasia with IDH1R132H showing distinct morphology, suggestive of a broad range of applicability to model oncogenic-dependence in pediatric GBM. Cerebral organoids may also be used as a model to study tumor invasion starting with glioma stem-like cells (GSCs, which are believed to be the tumor-initiating cells in GBM). Indeed 923 GSCs (which are not invasive in mice brain) quickly and spontaneously (within few days) invade a co-cultured cerebral organoid. This study is the first to engineer a human CNS tumor in vitro. The model carries a tremendous potential to uncover the pathogenesis and initiation of brain cancer in a pediatric setting. Note: This abstract was not presented at the conference. Citation Format: Amin Ismail. A human mini brain model of pediatric GBM. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A15.

Abstract LB-138: Use of cerebral organoids to model pediatric gliomas with H3.3K27M and H3.3G34R

Abstract Glioblastoma (GBM) is the most devastating brain cancer with dismal prognosis both in adults and children and accounts for 16% of primary brain tumors. Despite aggressive therapeutic approaches, the majority of patients die within one year after diagnosis. Giving the limitation of current GBM mouse models, which do not reflect the biological properties of human tumors, in particular, tumor heterogeneity and pathogenesis, there is a growing need for new models. Current in vitro 3D models involve the use of hydrogel scaffolds and cell lines, which do not represent a viable developmental context where the tumor mass interacts with and invades the surrounding brain tissues. Starting with pluripotent human embryonic stem cells we reproduced a 3D tissue structure (so-called cerebral organoid) representing a developing human brain in vitro. We engineered these mini-brains to express oncogenic hits responsible for the derivation of tumors in pediatric GBM patients. Pediatric GBM is driven by a combination of epigenetic and genetic hits including the expression of a mutant histone H3 variant H3F3A (mutated at K27M or G34R) or a gain of function mutant isocitrate dehydrogenase IDH1R132H in combination with the loss of the histone remodeling factor ATRX and/or p53 tumor suppressor. We used a combination of these oncogenic hits to create a model of tumor engineering representing the first 3D human tissue model of GBM. This model combines the accurate multi-lineage differentiation and physiology of human brain with the easiness of manipulation of in vitro systems. H&E staining of the modified organoids shows remarkable dysplasia reminiscent of human primary glioma. Different genetic hits result in different type of dysplasia with IDH1R132H showing distinct morphology, suggestive of a broad range of applicability to model oncogenic-dependence in pediatric GBM. Cerebral organoids may also be used as a model to study tumor invasion starting with glioma stem-like cells (GSCs, which are believed to be the tumor-initiating cells in GBM). Indeed 923 GSCs (which are not invasive in mice brain) quickly and spontaneously (within few days) invade a co-cultured cerebral organoid. This study is the first to engineer a human CNS tumor in vitro. The model carries a tremendous potential to uncover the pathogenesis and initiation of brain cancer in pediatric settings. Citation Format: Amin Ismail. Use of cerebral organoids to model pediatric gliomas with H3.3K27M and H3.3G34R. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-138. doi:10.1158/1538-7445.AM2015-LB-138

Abstract SY45-01: Defining the origin of tumor heterogeneity in epithelial cancers

Abstract Different theories have been proposed to explain tumor heterogeneity including the cancer cell of origin. Here, we developed genetically engineered mouse models allowing lineage tracing together with oncogenic activation in different cell lineages, and assessed whether the cancer cell of origin controls tumor heterogeneity. Mouse skin squamous cell carcinoma (SCC) arising from the interfollicular epidermis (IFE) are all well-differentiated SCCs. In contrast, tumors that arise from the same oncogenic hits in the hair follicle (HF) lineages displayed considerable heterogeneity ranging from well-differentiated SCCs to fibroblastic-like cancers exhibiting epithelial to mesenchymal transition (EMT) features. I will discuss further evidence that the cancer cell of origin controls EMT-related tumor heterogeneity and the underlying molecular mechanisms that promote multilineage differentiation, tumor propagation, EMT and metastasis in primary skin SCCs. Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumors that are classified into different histological and molecular subtypes. PIK3CA and p53 are the two most frequently mutated genes and are associated with different types of human breast cancers. I will present new results uncovering the cellular origin of mammary tumors. These results have important implications for our understanding of the mechanisms controlling tumor heterogeneity and the development of new strategies to block tumor initiation. This work is supported by the WELBIO, the FNRS, the Fondation Bettencourt-Schuler and the Fondation Baillet-Latour. Citation Format: Cedric Blanpain. Defining the origin of tumor heterogeneity in epithelial cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY45-01. doi:10.1158/1538-7445.AM2015-SY45-01

Developmental-stage-dependent transcriptional response to leukaemic oncogene expression

Acute myeloid leukaemia (AML) is characterized by a block in myeloid differentiation the stage of which is dependent on the nature of the transforming oncogene and the developmental stage of the oncogenic hit. This is also true for the t(8;21) translocation that gives rise to the RUNX1-ETO fusion protein and initiates the most common form of human AML. Here we study the differentiation of mouse embryonic stem cells expressing an inducible RUNX1-ETO gene into blood cells as a model, combined with genome-wide analyses of transcription factor binding and gene expression. RUNX1-ETO interferes with both the activating and repressive function of its normal counterpart, RUNX1, at early and late stages of blood cell development. However, the response of the transcriptional network to RUNX1-ETO expression is developmental stage specific, highlighting the molecular mechanisms determining specific target cell expansion after an oncogenic hit.

Gipfel – a Novel Method for Unbiased Molecular ETV6-RUNX1 Screening of Healthy Newborns

Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations that frequently occur in utero in preleukemic cells. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell ALL. A previous study suggested that ETV6-RUNX1 positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1 positive ALL in children by a factor of 100. This finding indicated that secondary, but relatively rare cooperating oncogenic hits are necessary for the development of overt leukemia. However, later studies could not confirm this high incidence of preleukemic cells in newborns.To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based screening of ≈1000 cord blood samples from healthy newborns.The GIPFEL method is capable to detect the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In the case of ETV6-RUNX1 positive leukemia, GIPFEL exploits the unique presence of a genomic fragment joining material from chromosomes 12 and 21 in the translocation-positive cells. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. Importantly, the ligation site is independent of the translocation point within the individual DNA circle. The published breakpoint regions of the ETV6 and RUNX1 genes involved in the translocation were analyzed in silico for restriction sites that allow digestion of all possible translocation events to yield fragments smaller than approximately 50 kb. This condition was met for ETV6-RUNX1 breakpoints by digestion with SacI. Primer pairs were designed amplifying the complete set of theoretically predicted circularized fragments requiring 36 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from cell lines, diagnostic specimens from ALL patients, peripheral blood from healthy donors and cord blood samples from newborns by column purification. The equivalent of approximately 4x105 cells (2.5 µg DNA) was subjected to the SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product.In a first proof-of-principle study employing the ETV6-RUNX1 translocation positive cell line REH, process optimization close to the theoretical limits was carried out. Cell dilution and mixing studies revealed that under optimal conditions approximately 40 translocation positive cells (=10-4) present in the input DNA are sufficient to produce a reliable output signal. The method was next tested in a blinded study with 60 samples obtained from ETV6-RUNX1 diagnostic ALL samples. ETV6-RUNX1 samples positive at 10-4, being diluted from these diagnostic samples, still gave a reliable output signal. There was no false positive result. Detection coverage (=sensitivity) was 64%. This method was then applied to a retrospective sample set of cryopreserved anonymized cord blood samples of ≈1000 healthy newborns to determine frequency and levels of translocation-positive cells. First results will be presented.In conclusion this population-based study will allow an estimate of the actual incidence of ETV6-RUNX1 positive preleukemic cells in healthy newborns. The results will enable us to evaluate the penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and will provide a basis for the assessment of potential secondary environmental or spontaneously occurring cooperating oncogenic lesions in ETV6-RUNX1 positive childhood leukemia. DisclosuresNo relevant conflicts of interest to declare.

Cytoskeletal regulatory gene expression and migratory properties of B-cell progenitors are affected by the ETV6-RUNX1 rearrangement.

Although the ETV6-RUNX1 fusion is a frequent initiating event in childhood leukemia, its role in leukemogenesis is only partly understood. The main impact of the fusion itself is to generate and sustain a clone of clinically silent preleukemic B-cell progenitors (BCP). Additional oncogenic hits, occurring even several years later, are required for overt disease. The understanding of the features and interactions of ETV6-RUNX1-positive cells during this "latency" period may explain how these silent cells can persist and whether they could be prone to additional genetic changes. In this study, two in vitro murine models were used to investigate whether ETV6-RUNX1 alters the cellular adhesion and migration properties of BCP. ETV6-RUNX1-expressing cells showed a significant defect in the chemotactic response to CXCL12, caused by a block in CXCR4 signaling, as demonstrated by inhibition of CXCL12-associated calcium flux and lack of ERK phosphorylation. Moreover, the induction of ETV6-RUNX1 caused changes in the expression of cell-surface adhesion molecules. The expression of genes regulating the cytoskeleton was also affected, resulting in a block of CDC42 signaling. The abnormalities described here could alter the interaction of ETV6-RUNX1 preleukemic BCP with the microenvironment and contribute to the pathogenesis of the disease. Alterations in the expression of cytoskeletal regulatory genes and migration properties of BCP represent early events in the evolution of the disease, from the preleukemic phase to the clinical onset, and suggest new strategies for effective eradication of leukemia.

IDH mutations in liver cell plasticity and biliary cancer

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer associated with the bile ducts within the liver. These tumors are characterized by frequent gain-of-function mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes—that are also common in subsets of neural, haematopoietic and bone tumors, but rare or absent in the other types of gastrointestinal malignancy. Mutant IDH acts through a novel mechanism of oncogenesis, producing high levels of the metabolite 2-hydroxyglutarate, which interferes with the function of α-ketoglutarate-dependent enzymes that regulate diverse cellular processes including histone demethylation and DNA modification. Recently, we used in vitro stem cell systems and genetically engineered mouse models (GEMMs) to demonstrate that mutant IDH promotes ICC formation by blocking hepatocyte differentiation and increasing pools of hepatic progenitors that are susceptible to additional oncogenic hits leading to ICC. We found that silencing of HNF4A—encoding a master transcriptional regulator of hepatocyte identity and quiescence—was critical to mutant IDH-mediated inhibition of liver differentiation. In line with these findings, human ICC with IDH mutations are characterized by a hepatic progenitor cell transcriptional signature suggesting that they are a distinct ICC subtype as compared to IDH wild type tumors. The role of mutant IDH in controlling hepatic differentiation state suggests the potential of newly developed inhibitors of the mutant enzyme as a form of differentiation therapy in a solid tumor.

Characterization of pancreatic ductal adenocarcinoma patients using whole-transcriptome sequencing and copy number analysis by SNPs array techniques.

e15192 Background: Our challenge it is to implemented a whole transcriptome massively parallel sequencing (RNASeq) study and copy number analysis to better understand the PDAC molecular biology. Methods: We collected 16 PDAC samples by ultrasound-guided biopsy or surgical specimen for DNA and RNA extraction. All the samples were analyzed by RNASeq, performed at 75x2 bp on a HiScanSQ Illumina platform. Single nucleotide variants (SNVs) were detected with SNVMix2 and filterend on dbSNP, 1,000 genomes, Cosmic. Non-synonimous SNVs were analyzed with SNPs&GO and PROVEAN. Thirteen samples were analyzed by high resolution copy number analysis (CNA) on Affymetrix SNP array 6.0. Results: RNAseq results in an average of 264 coding non-synonymous novel SNVs (ranging from 146 to 374) and 16 novel Indels (ranging from 6 to 24) for each sample, of which respectively a mean of 11.2% were declared as disease related and somatic events, and 34.7% were frameshift somatic insertions or deletions. We highlighted the major oncogenic hits of PDAC, confirming KRAS mutations (93.7%) and inactivation of CDKN2A (50%), SMAD4 (50%), and TP53 (56%). One case negative for KRAS showed a G13D NRAS mutation. We also detected gene fusion in 10 samples for a total of 23 different intra- or inter-chromosomal rearrangements even if no recurrent fusion transcript was found. Most rearrangements were inter-chromosomal (66%) and did not change the reading-frame (51%). Trough SNP-Array we identified macroscopic and cryptic cytogenetic alterations in 85% of patients. Gains were observed in chromosome arms 6p, 12p, 18q and 19q that harbor KRAS, GATA6, AKT2 and CCND3,while deletions were found on chromosome arms 1p, 9p, 6p, 18q, 10q, 15q, 17p, 21q, 19p, involving TP53, CDKN2A/B, SMAD4, RUNX2, ARID1A, PTEN and STK11. Conclusions: Genetic alterations in PDCA involve several pathways such as cell migration, TGFß signaling, apoptosis, cell proliferation and particularly DNA damage repair. However, not all tumors show alterations of these signaling, and key mutations appeared to differ from one cancer to another. PDAC still represents a challenge and innovative approaches are needed to eradicate the disease.

Nucleotide metabolism, oncogene-induced senescence and cancer

Senescence is defined as a stable cell growth arrest. Oncogene-induced senescence (OIS) occurs when an activated oncogene is expressed in a normal cell. OIS acts as a bona fide tumor suppressor mechanism by driving stable growth arrest of cancer progenitor cells harboring the initial oncogenic hit. OIS is often characterized by aberrant DNA replication and the associated DNA damage response. Nucleotides, in particular deoxyribonucleotide triphosphates (dNTPs), are necessary for both DNA replication and repair. Imbalanced dNTP pools play a role in a number of human diseases, including during the early stages of cancer development. This review will highlight what is currently known about the role of decreased nucleotide metabolism in OIS, how nucleotide metabolism leads to transformation and tumor progression, and how this pathway can be targeted as a cancer therapeutic by inducing senescence of cancer cells.

Retroviral insertional mutagenesis in telomerase-immortalized hepatocytes identifies RIPK4 as novel tumor suppressor in human hepatocarcinogenesis

Carcinogenesis is a multistep process involving alterations in various cellular pathways. The critical genetic events driving the evolution of primary liver cancer, specifically hepatoblastoma and hepatocellular carcinoma (HCC), are still poorly understood. However, telomere stabilization is acknowledged as prerequisite for cancer progression in humans. In this project, human fetal hepatocytes were utilized as a cell culture model for untransformed, proliferating human liver cells, with telomerase activation as first oncogenic hit. To elucidate critical downstream genetic events driving further transformation of immortalized liver cells, we used retroviral insertional mutagenesis as an unbiased approach to induce genetic alterations. Following isolation of hyperproliferating, provirus-bearing cell clones, we monitored cancer-associated growth properties and characterized changes toward a malignant phenotype. Three transformed clones with the ability to form colonies in soft agar were expanded. As proof-of-principle for our experimental setup, we identified a transforming insertion on chromosome 8 within the pleiomorphic adenoma gene 1 (PLAG1), resulting in a 20-fold increase in PLAG1 expression. Upregulation of PLAG1 has already been described to promote human hepatoblastoma development. In a separate clone, a transforming insertion was detected in close proximity to the receptor-interacting serine-threonine kinase 4 (RIPK4) with an approximately eightfold suppression in RIPK4 expression. As validation for this currently unknown driver in hepatocarcinogenesis, we examined RIPK4 expression in human HCC samples and confirmed a significant suppression of RIPK4 in 80% of the samples. Furthermore, overexpression of RIPK4 in transformed human fetal hepatocytes resulted in an almost complete elimination of anchorage-independent growth. On the basis of these data, we propose RIPK4 as a novel putative tumor suppressor in human hepatocarcinogenesis.