Pten Allele Research Articles

Abstract 1257: CHD5 suppresses glioblastoma by inhibiting MYC

Abstract Fundamentally, cancer is a disease caused by genetic and epigenetic alterations that converge to reprogram gene expression networks, leading to unrestrained proliferation. Glioblastoma (GBM) is the most prevalent and aggressive primary brain cancer, median survival being approximately one year and the 5-year survival rate being only 5%. Despite decades of effort, this devastating picture has not appreciably improved and effective therapies have been elusive for GBM patients. A better understanding of GBM biology is essential for developing more effective therapies. Although several genetic alterations have been implicated in gliomagenesis, driving genetic factors responsible for GBM are still obscure, and the interplay between genetic alterations and epigenetic dysregulation is largely undocumented during the gliomagenic process. Here, we show that CHD5 — a gene mapping to the 1p36 chromosomal region that is notoriously deleted in many cancers, is recurrently deleted in over 20% of GBM cases and is further downregulated through epigenetic means. To delineate how CHD5 loss promotes gliomagenesis, we established a glioma-prone mouse model harboring conditional alleles of Pten, Trp53, and Chd5. We developed an engineered mouse neural stem cell (NSCs)-based strategy to elicit glioma formation, as NSCs are the major cell-of-origin for GBM. We show that Chd5 loss significantly impairs NSCs differentiation while promoting proliferation, transformation, and gliomagenesis in vivo. Mechanistically, Chd5 forms a Chd3-containing but Chd4-independent NuRD complex that directly binds the Myc promoter and super enhancers to govern Myc expression in normal NSCs, with the Snf2 domain of Chd5 playing the most critical chromatin regulating role in the context of GBM. Chd5 loss triggers the remodeling of chromatin, enhances accessibility, and augments Myc expression, leading to the formation of highly aggressive glioma. Furthermore, reactivating CHD5 in human GBM xenograft models significantly extends survival. This work demonstrates the tumor suppressive role of CHD5 in gliomagenesis, providing therapeutic insights for cancers with 1p36 deletions. In parallel, this work presents a powerful strategy for rapidly interrogating gene function during gliomagenesis using engineering NSCs, and the generation of novel mouse models with classic clinical features of GBM, offering new approaches for elucidating GBM biology that can inform on therapeutic opportunities for treating patients with GBM. Citation Format: Xueqin Sherine Sun, Alea Mills. CHD5 suppresses glioblastoma by inhibiting MYC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1257.

Abstract B014: CHD5 suppresses glioblastoma by inhibiting MYC

Abstract Fundamentally, cancer is a disease caused by genetic and epigenetic alterations that converge to reprogram gene expression networks, leading to unrestrained proliferation. Glioblastoma (GBM) is the most prevalent and aggressive primary brain cancer, median survival being approximately one year and the 5-year survival rate being only 5%. Despite decades of effort, this devastating picture has not appreciably improved and effective therapies have been elusive for GBM patients. A better understanding of GBM biology is essential for developing more effective therapies. Although several genetic alterations have been implicated in gliomagenesis, driving genetic factors responsible for GBM are still obscure, and the interplay between genetic alterations and epigenetic dysregulation is largely undocumented during the gliomagenic process. Here, we show that CHD5 — a gene mapping to the 1p36 chromosomal region that is notoriously deleted in many cancers, is recurrently deleted in over 20% of GBM cases and is further downregulated through epigenetic means. To delineate how CHD5 loss promotes gliomagenesis, we established a glioma-prone mouse model harboring conditional alleles of Pten, Trp53, and Chd5. We developed an engineered mouse neural stem cell (NSCs)-based strategy to elicit glioma formation, as NSCs are the major cell-of-origin for GBM. We show that Chd5 loss significantly impairs NSCs differentiation while promoting proliferation, transformation, and gliomagenesis in vivo. Mechanistically, Chd5 forms a Chd3-containing but Chd4-independent NuRD complex that directly binds the Myc promoter and super enhancers to govern Myc expression in normal NSCs, with the Snf2 domain of Chd5 playing the most critical chromatin regulating role in the context of GBM. Chd5 loss triggers the remodeling of chromatin, enhances accessibility, and augments Myc expression, leading to the formation of highly aggressive glioma. Furthermore, reactivating CHD5 in human GBM xenograft models significantly extends survival. This work demonstrates the tumor suppressive role of CHD5 in gliomagenesis, providing therapeutic insights for cancers with 1p36 deletions. In parallel, this work presents a powerful strategy for rapidly interrogating gene function during gliomagenesis using engineering NSCs and highlights the generation of novel mouse models with classic clinical features of GBM, offering new approaches for elucidating GBM biology that can inform on therapeutic opportunities for treating patients with GBM. Citation Format: Xueqin Sherine Sun, Alea Mills. CHD5 suppresses glioblastoma by inhibiting MYC [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B014.

Pharmacogenetic study of phosphatase and tensin homolog polymorphism (rs701848) in childhood epilepsy: relation to circulating Wnt signaling

ABSTRACT Objective The present study aimed at evaluating the potential contribution of Phosphatase and Tensin Homolog (PTEN) and its gene polymorphism (PTEN rs701848 T/C) in relation to Wingless/integrase-1 (Wnt) signaling in childhood epilepsy and the impact of antiepileptic medications on their serum levels. Methods This study included 100 children with epilepsy (50 pharmacoresistant and 50 pharmacoresponsive) and 50 matched controls. All subjects had their genotypes for the PTEN rs701848T/C polymorphism assessed using TaqManTM assays and real-time PCR. By using the sandwich ELISA technique, the blood concentrations of PTEN and Wnt3a were measured. Results Serum Wnt3a levels in epileptic patients were significantly higher than in the control group, p < 0.001. Children with epilepsy who received oxcarbazepine had considerably lower serum Wnt3a levels than those who didn’t, p < 0.001.With an AUC of 0.71, the cutoff value for diagnosing epilepsy as serum Wnt3a > 6.2 ng/mL has a sensitivity of 55% and a specificity of 80%. When compared to controls, epileptic children had considerably more (TT) genotype and less (TC and CC) genotypes, p < 0.05 for all. Epileptic children had significantly higher (T) allele frequency than controls, p = 0.006 with OR (95%CI) = 1.962(1.206–3.192). Pharmacoresistant epileptic children had significantly higher (TT) genotype compared to pharmacoresponsive type (p = 0.020). Conclusion We originally found a strong association between PTEN rs701848 T/C and childhood epilepsy, in particular pharmacoresistant type. Serum Wnt3a levels increased in epilepsy, but were not significantly different between different alleles of PTEN. In pharmaco-responsive children Wnt3a levels differed significantly between the different PTEN genotypes. Antiepileptics may affect Wnt3a levels.

PTEN Protein Phosphatase Activity Is Not Required for Tumour Suppression in the Mouse Prostate.

Loss PTEN function is one of the most common events driving aggressive prostate cancers and biochemically, PTEN is a lipid phosphatase which opposes the activation of the oncogenic PI3K-AKT signalling network. However, PTEN also has additional potential mechanisms of action, including protein phosphatase activity. Using a mutant enzyme, PTEN Y138L, which selectively lacks protein phosphatase activity, we characterised genetically modified mice lacking either the full function of PTEN in the prostate gland or only lacking protein phosphatase activity. The phenotypes of mice carrying a single allele of either wild-type Pten or PtenY138L in the prostate were similar, with common prostatic intraepithelial neoplasia (PIN) and similar gene expression profiles. However, the latter group, lacking PTEN protein phosphatase activity additionally showed lymphocyte infiltration around PIN and an increased immune cell gene expression signature. Prostate adenocarcinoma, elevated proliferation and AKT activation were only frequently observed when PTEN was fully deleted. We also identify a common gene expression signature of PTEN loss conserved in other studies (including Nkx3.1, Tnf and Cd44). We provide further insight into tumour development in the prostate driven by loss of PTEN function and show that PTEN protein phosphatase activity is not required for tumour suppression.

Integrating thousands of PTEN variant activity and abundance measurements reveals variant subgroups and new dominant negatives in cancers

BackgroundPTEN is a multi-functional tumor suppressor protein regulating cell growth, immune signaling, neuronal function, and genome stability. Experimental characterization can help guide the clinical interpretation of the thousands of germline or somatic PTEN variants observed in patients. Two large-scale mutational datasets, one for PTEN variant intracellular abundance encompassing 4112 missense variants and one for lipid phosphatase activity encompassing 7244 variants, were recently published. The combined information from these datasets can reveal variant-specific phenotypes that may underlie various clinical presentations, but this has not been comprehensively examined, particularly for somatic PTEN variants observed in cancers.MethodsHere, we add to these efforts by measuring the intracellular abundance of 764 new PTEN variants and refining abundance measurements for 3351 previously studied variants. We use this expanded and refined PTEN abundance dataset to explore the mutational patterns governing PTEN intracellular abundance, and then incorporate the phosphatase activity data to subdivide PTEN variants into four functionally distinct groups.ResultsThis analysis revealed a set of highly abundant but lipid phosphatase defective variants that could act in a dominant-negative fashion to suppress PTEN activity. Two of these variants were, indeed, capable of dysregulating Akt signaling in cells harboring a WT PTEN allele. Both variants were observed in multiple breast or uterine tumors, demonstrating the disease relevance of these high abundance, inactive variants.ConclusionsWe show that multidimensional, large-scale variant functional data, when paired with public cancer genomics datasets and follow-up assays, can improve understanding of uncharacterized cancer-associated variants, and provide better insights into how they contribute to oncogenesis.

PTEN Hamartoma Tumor Syndrome: Skin Manifestations and Insights Into Their Molecular Pathogenesis.

Germline PTEN pathogenic variants cause a spectrum of disorders collectively labeled PTEN Hamartoma Tumor Syndrome (PHTS) and featured by hamartomas, developmental anomalies and increased cancer risk. Studies on experimental models provided evidence that PTEN is a “haploinsufficient” tumor-suppressor gene, however, mechanisms involved in the pathogenesis of clinical manifestations in PHTS patients remain elusive. Beyond analyzing clinical and molecular features of a series of 20 Italian PHTS patients, we performed molecular investigations to explore the mechanisms involved in the pathogenesis of PTEN-associated manifestations, with special focus on mucocutaneous manifestations. Typical mucocutaneous features were present in all patients assessed, confirming that these are the most important clue to the diagnosis. The most frequent were papules located in the trunk or extremities (73.7%), oral mucosa papules (68.4%), acral/palmoplantar keratosis and facial papules (both 57.9%), according with literature data. Molecular analyses on one trichilemmoma suggested that the wild-type PTEN allele was retained and expressed, reinforcing the evidence that PTEN does not require a second somatic hit to initiate pathogenic processes. Unexpectedly, one patient also displayed a cutaneous phenotype consistent with atypical mole/melanoma syndrome; no variants were detected in known melanoma genes, but Whole Exome Sequencing showed the rare truncating variant c.495G>A in the CDH13 gene that might have cooperated with PTEN-haploinsufficiency to generate such phenotype. Our findings confirm the reproducibility of known PHTS manifestations in real-world practice, highlighting the role of mucocutaneous manifestations in facilitating prompt diagnosis of the syndrome, and provide some insights into the pathogenic process induced by PTEN alterations, which may contribute to its understanding.

Abstract IA003: Radiotherapy effects on tumor microenvironment

Abstract Radiation therapy (RT) exerts multiple effects on the tumor and its microenvironment, affecting immunity, metabolism and the microbiome (Heather M. McGee et al. Clin Cancer Res 2019;25:2969-2974). Radiation has profound and sustained effects on non-malignant tissue, including epithelial to mesenchymal transition (EMT) and carcinogenesis. Seminal experiments from Barcellos-Hoff' s lab originally demonstrated the capacity of RT to induce EMT of non-malignant epithelial cells (Andarawewa, KL et al: Cancer Res 2007; 67: (18). September 15, 2007). Burma et al demonstrated in an in vivo model of gliomas engineered after loss of one allele of Trp53 and Pten, that mice were highly susceptible to radiation-induced gliomagenesis in a linear energy transfer (LET)-dependent manner, possibly dependent on the increasing degree of irreparable DNA breaks with increasing LET (Todorova PK et al Cancer Res 2007; 67: (18). September 15, 2007). Interestingly, the histopathology of RT-induced high grade gliomas in this model mimics that of human glioblastoma, with similar patterns of necrosis, pseudo-palisading and nuclear atypia. This effect has translational implications, in view of the fact that the adjacent irradiated brain after standard post-operative RT to GBM is the most common site of recurrence (Gupta K and Burns TC: Frontiers in Oncology, 2018). Radiation is a powerful inducer of immunogenic cell death (ICD) of cancer cells, by releasing of DAMPs such as high mobility group box protein 1 (HMGB1), ATP and translocation of calreticulin to cell surface. In response to DNA damage, cytosolic DNA released from the nucleus mitochondria activates sensors like cGAS/STING, leading to an increase in IFNβ, that recruits and activates BAFT3+ dendritic cells, crucial agents for cross-presentation and cross-priming of CD8+ T cells. Radiation also increases the trafficking of activated CD8+ T cells by releasing CXCL16, a chemokine that binds to CXCR6 (Matsumura et al., J Immunol 181:3099-3107 2008). Recent evidence has demonstrated how as part of DNA damage response to RT, tumor mutations are expressed and can generate neoantigens that contribute to sustained immune responses in patients (Nature Medicine, 2018; 24;12:1845). Consequently, RT can recruit both the innate and adaptive immune response and, in combination with immune checkpoint blockade (ICB) can convert the irradiated tumor into an in situ vaccine. The choice of technical parameters like radiation dose rate, dose and fractionation and the type of field chosen, all impact on the success of RT and immunotherapy combinations, as demonstrated by impairment of immune response when nodal regions are included in the field treated by RT and ICB (Marciscano A et al: Clin Cancer Res: 4(20) October 15, 2018). The widespread access to radiotherapy centers and the ease to include a standard modality like RT in clinical trials have have rendered it one of the tools of modern immunotherapy of cancer, by exploiting RT effects on tumor and TME, beyond its classical cytocidal potential. Citation Format: Silvia Formenti. Radiotherapy effects on tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA003.

Inactivation of TRP53, PTEN, RB1, and/or CDH1 in the ovarian surface epithelium induces ovarian cancer transformation and metastasis.

Ovarian cancer (OvCa) remains the most common cause of death from gynecological malignancies. Genetically engineered mouse models have been used to study initiation, origin, progression, and/or mechanisms of OvCa. Based on the clinical features of OvCa, we examined a quadruple combination of pathway perturbations including PTEN, TRP53, RB1, and/or CDH1. To characterize the cancer-promoting events in the ovarian surface epithelium (OSE), Amhr2cre/+ mice were used to ablate floxed alleles of Pten, Trp53, and Cdh1, which were crossed with TgK19GT121 mice to inactivate RB1 in KRT19-expressing cells. Inactivation of PTEN, TRP53, and RB1 with or without CDH1 led to the development of type I low-grade OvCa with enlarged serous papillary carcinomas and some high-grade serous carcinomas (HGSCs) in older mice. Initiation of epithelial hyperplasia and micropapillary carcinoma started earlier at 1 month in the triple mutations of Trp53, Pten, and Rb1 mice as compared to 2 months in quadruple mutations of Trp53, Pten, Rb1, and Cdh1 mice, whereas both genotypes eventually developed enlarged proliferating tumors that invaded into the ovary at 3-4 months. Mice with triple and quadruple mutations developed HGSC and/or metastatic tumors, which disseminated into the peritoneal cavity at 4-6 months. In summary, inactivation of PTEN, TRP53, and RB1 initiates OvCa from the OSE. Additional ablation of CDH1 further increased persistence of tumor dissemination and ascites fluid accumulation enhancing peritoneal metastasis.

Radiation-Induced DNA Damage Cooperates with Heterozygosity of TP53 and PTEN to Generate High-Grade Gliomas.

Glioblastomas are lethal brain tumors that are treated with conventional radiation (X-rays and gamma rays) or particle radiation (protons and carbon ions). Paradoxically, radiation is also a risk factor for GBM development, raising the possibility that radiotherapy of brain tumors could promote tumor recurrence or trigger secondary gliomas. In this study, we determined whether tumor suppressor losses commonly displayed by patients with GBM confer susceptibility to radiation-induced glioma. Mice with Nestin-Cre-driven deletions of Trp53 and Pten alleles were intracranially irradiated with X-rays or charged particles of increasing atomic number and linear energy transfer (LET). Mice with loss of one allele each of Trp53 and Pten did not develop spontaneous gliomas, but were highly susceptible to radiation-induced gliomagenesis. Tumor development frequency after exposure to high-LET particle radiation was significantly higher compared with X-rays, in accordance with the irreparability of DNA double-strand breaks (DSB) induced by high-LET radiation. All resultant gliomas, regardless of radiation quality, presented histopathologic features of grade IV lesions and harbored populations of cancer stem-like cells with tumor-propagating properties. Furthermore, all tumors displayed concomitant loss of heterozygosity of Trp53 and Pten along with frequent amplification of the Met receptor tyrosine kinase, which conferred a stem cell phenotype to tumor cells. Our results demonstrate that radiation-induced DSBs cooperate with preexisting tumor suppressor losses to generate high-grade gliomas. Moreover, our mouse model can be used for studies on radiation-induced development of GBM and therapeutic strategies. SIGNIFICANCE: This study uncovers mechanisms by which ionizing radiation, especially particle radiation, promote GBM development or recurrence.

Abstract 4621: Developing genetically modified homograft models of mouse prostate cancer for efficacy evaluation of combinatory immunotherapies

Abstract Despite the early approval of Sipuleucel-T for metastatic castration-resistant prostate cancer, which often perceived as a milestone achievement in cancer immunotherapy, subsequent progress in prostate cancer immunotherapy development has been limited by disappointing results with tumor vaccines and its resistance to immune checkpoint inhibitors, such as PD1 &amp; PD-L1. It is now generally accepted that we need to tackle prostate cancer by combinatorial approaches of chemo-, targeted- and immuno-therapies. Highly relevant preclinical models are very much needed for proof of principle efficacy evaluation. Genetically engineered mouse models (GEMM) recapitulate key aspects of human prostate cancer in both histo- &amp; molecular- pathology. Among those, PTEN loss of function (LOF) in prostate epithelium is one of the central events in human prostate cancer. PTEN haploinsufficiency in mice is sufficient to drive mouse prostatic intraepithelial neoplasia (PIN) formation, while loss of both alleles of PTEN in mouse prostate leads to hyperplasia at 4 weeks, PIN at 6 weeks, and fully invasive adenocarcinoma from 12 weeks of age. PTEN null tumors are also resistant to androgen depletion. In the meantime, although KRAS mutation are not often seen in human prostate cancer, activation of MAPK pathway often happens in advanced tumor. Mutant KRAS or BRAF can robustly promote mouse prostate cancer progression. Prostate specific Pten null and Kras G12D; Pten null mouse prostate cancer models have been well characterized by a number of labs. However, parental GEMM models are difficult to be used for pharmacological studies due to the spontaneous nature of tumor onset and progression. The compound mutant mice are also costly to breed. Here we report generation of transplantable mouse prostate cancer homograft models by passaging the primary tumor subcutaneously in the C57BL/6 mice. These mouse tumors featuring Pbsn-Cre;LSL-KrasG12D/+;Ptenflox/flox or Pbsn-Cre;Ptenflox/flox retain morphological similarity to moderate to poorly differentiated human prostate cancer. These homograft tumors show high levels of M2 macrophage infiltration. Growth of these mouse prostate tumors are resistant to androgen depletion. They are also resistant to the treatment of AR inhibitor enzalutamide, only marginally responsive to docetaxel, but sensitive to treatments with mTOR inhibitor everolimus. When treated with immune checkpoint antibodies, i.e. PD1 and CTLA4, these tumors showed increased T-cell infiltration, but very modest growth inhibition. We are now testing a variety of combinatory therapies with chemotherapies, immune modulators and immune checkpoint antibodies. The results will be presented at the meeting. Citation Format: Jessie Jingjing Wang, Yanrui Song, Annie Xiaoyu An, Likun Zhang, Henry Qixiang Li, Davy Xuesong Ouyang. Developing genetically modified homograft models of mouse prostate cancer for efficacy evaluation of combinatory immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4621.

PTEN loss in prostatic adenocarcinoma correlates with specific adverse histologic features (intraductal carcinoma, cribriform Gleason pattern 4 and stromogenic carcinoma)

The loss of PTEN tumor suppressor gene is one of the most common somatic genetic aberrations in prostate cancer (PCa) and is frequently associated with high-risk disease. Deletion or mutation of at least one PTEN allele has been reported to occur in 20% to 40% of localized PCa and up to 60% of metastases. The goal of this study was to determine if somatic alteration detected by PTEN immunohistochemical loss of expression is associated with specific histologic features. Two hundred sixty prostate core needle biopsies with PCa were assessed for PTEN loss using an analytically validated immunohistochemical assay. Blinded to PTEN status, each tumor was assessed for the Grade Group (GG) and the presence or absence of nine epithelial features. Presence of stromogenic PCa was also assessed and defined as grade 3 reactive tumor stroma as previously described: the presence of carcinoma associated stromal response with epithelial to stroma ratio of greater than 50% reactive stroma. Eight-eight (34%) cases exhibited PTEN loss while 172 (66%) had intact PTEN. PTEN loss was significantly (P < 0.05) associated with increasing GG, poorly formed glands (74% of total cases with loss vs 49% of intact), and three well-validated unfavorable pathological features: intraductal carcinoma of the prostate (IDC-P) (69% of total cases with loss vs 12% of intact), cribriform Gleason pattern 4 (38% of total cases with loss vs 10% of intact) and stromogenic PCa (23% of total cases with loss vs 6% of intact). IDC-P had the highest relative risk (4.993, 95% confidence interval, 3.451-7.223, P < 0.001) for PTEN loss. At least one of these three unfavorable pathological features were present in 67% of PCa exhibiting PTEN loss, while only 11% of PCa exhibited PTEN loss when none of these three unfavorable pathological features were present. PCa with PTEN loss demonstrates a strong correlation with known unfavorable histologic features, particularly IDC-P. This is the first study showing the association of PTEN loss with stromogenic PCa.

Exploration of the Role of Tumor Suppressor Genes Foxo1 and PTEN in the Tumorigenesis of Mouse Natural Killer-Cell Lymphoma

To explore whether tumor suppressor gene Foxo1 and PTEN play a critical role in the tumorigenesis of mouse natural killer-cell lymphoma. NKp46-iCre mice were crossed with mice carrying floxed Foxo1 alleles (Foxo1fl/fl) as well as floxed PTEN alleles (PTENfl/fl) to generate mice in which Foxo1 and PTEN in NK cells were knock-out, referred as Foxo1△NKPTEN△NK. The growth and development of the mice and tumor formation were observed. The flow cytometry was used to detect the percentages of NK cells in main lymphatic organs. B16F10 metanoma model of tumor metastasis was utilized to investigate NK cell-mediated tumor surveillance in vivo after NK cells special deletion of Foxol and PTEN. The mouse model with NK cell-special Foxo1 and PTEN double knockout was established. Compared with control group (Foxo1fl/flPTENfl/fl mice), Foxo1△NKPTEN△NK mice were born alive and appeared to be healthy over a period of 46 weeks. No spontaneous tumor formation was observed at this stage. There were no significant differences in NK cell percentages of gated lymphocytes from various organs including blood, bone marrow, peripheral lymph node and spleen between Foxo1△NKPTEN△NK mice and Foxo1fl/flPTENfl/fl mice [PB: 4.76%±0.46% vs 4.17%±0.64% (P＞0.05, n=8); BM: 1.13%±0.23% vs 1.31%±0.10% (P＞0.05, n=8) ; LN: 0.50%±0.10% vs 0.85%±0.20% (P＞0.05, n=8); SP: 4.41%±0.65% vs 3.50%±0.24% (P＞0.05, n=8)]. B16F10 melanoma metastasis model of tumor was established, No differences in median survival time were observed in the 2 types of mice (P＞0.05, n=13). The simultaneous deletion of the Foxo1 and PTEN genes may not plays significant role in the tumorigenesis of mouse natural killer-cell lymphoma and NK cell-mediated tumor surveillance in vivo.

Targeting MYC-Driven B-Cell Lymphoma By Inhibition of the Histone Methyltransferase DOT1L

Aberrant activation of the MYC proto-oncogene is a recurrent feature in human B-cell lymphomas of diverse sub-types, correlating with adverse prognosis and therapy resistance. Direct pharmacological MYC-targeting has proved difficult, but recent studies have shown that targeting chromatin regulators critical for MYC-driven oncogenesis may provide alternative avenues for therapeutic intervention. Recently, it has been demonstrated that MYC-driven oncogenesis in certain solid tumors is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L. We hypothesized that B-cell lymphomas with hyperactive MYC-signaling might be responsive to DOT1L inhibition. In order to test this hypothesis, we tested the effect of the DOT1L inhibitor Pinometostat (EPZ-5676) on a panel of human B-cell lymphoma cell lines featuring elevated MYC. Pinometostat treatment reduced global H3K79 methylation levels, accompanied by a time and dose-dependent decrease in proliferation of several Burkitt's lymphoma cell lines including P493-6, Daudi and Raji. We observed that key MYC-target genes including CDK4, PPAT and NPM1 were significantly downregulated upon Pinometostat treatment, suggesting that DOT1L is required for the transcriptional activation of MYC-target genes in these cells. Pinometostat-treated B-lymphoma cells showed a significant decrease of cells in S-phase compared to controls as assessed by BrdU-labeling assays. Similar results were also obtained in a panel of B-cell lymphoma cell lines with MYC-rearrangements including mantle cell lymphoma (MCL) cell lines Jeko-1, JVM2, Mino-1 and Maver-1 and the diffused large B-cell lymphoma (DLBCL) cell line Karpas 422.Next, we sought to investigate whether the DOT1L-dependence of MYC-driven B-cell lymphoma could be reproduced in a well-defined model of MYC-driven B-cell lymphoma. Towards this end, we utilized a mouse model in which expression of the Cre recombinase from a B cell specific promoter leads to ectopic expression of a transgenic human MYC allele and concomitant deletion of the tumor suppressor Pten in B cells. Similar to our in vitro studies, Pinometostat treatment led to a significant reduction in proliferation of B-cell lymphoma cells from these mice with an IC50 of 0.5 µM.Furthermore, we sought to ascertain whether these findings reflected on-target effects related to DOT1L inhibition. Therefore, we deleted DOT1L using CRISPR/Cas9 in B-cell lymphoma cell lines and assessed the effect on proliferation using competitive-proliferation assays. We observed that DOT1L-deletion progressively diminished the relative growth of anti-DOT1L sgRNA-expressing P493-6 and Jeko1 cells compared to non-targeted cells invitro. In order to test the requirement for DOT1L in lymphoma propagation in vivo, we performed intravenous injections of equal number of Jeko-1 cells with either anti-DOT1L or anti-Renilla control sgRNAs into sub-lethally irradiated non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID) mice. Mice injected with control anti-Renilla sgRNAs succumbed to disease with a median latency of 34 days while the latency of disease in the anti-DOT1L sgRNA cohort was 45 days. In summary, DOT1L depletion significantly delayed disease latency in this invivo disseminated model of B-cell lymphoma (P=0.02).We then performed transcriptomic analyses of Pinometostat-treated B-cell lymphoma cell lines compared to DMSO-treated counterparts using RNA-seq. Gene-set enrichment analysis (GSEA) of RNA-seq data from three different B-cell lymphoma cell lines demonstrated that Pinometostat treatment significantly decreased the expression of MYC-target genes. In order to investigate the intriguing role of DOT1L in regulating MYC-target gene expression, we used ChIP-seq to assess the genome-wide occupancy of MYC following DOT1L inhibitor treatment. Strikingly, our studies demonstrated that DOT1L inhibition significantly reduced the chromatin occupancy of MYC.Taken together, our experiments demonstrate the role of DOT1L in MYC-driven B-cell lymphoma pathogenesis invitro and invivo. Furthermore, our genome-wide studies demonstrate the importance of DOT1L for genomic MYC occupancy. Based on these findings, we propose that therapeutic DOT1L targeting may be a viable strategy in MYC-driven B-cell lymphoma. DisclosuresWeigert:Roche: Research Funding; Novartis: Research Funding. Rickert:Pfizer: Employment. Ren:Elli Lilly: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

Establishment of a transgenic mouse to model ETV7 expressing human tumors

The ETS transcription factor ETV7 has been characterized as a hematopoietic oncoprotein, which requires cooperating mutations for its leukemogenic activity. Although the ETV7 gene is highly conserved among vertebrates, part of the rodents, including Mus musculus, deleted the Etv7 gene locus. Many human hematopoietic malignancies upregulate ETV7 expression but contrary to ETV7’s role in oncogenesis, its physiological role in normal tissues is unknown. To determine the physiological function of ETV7 in vivo and determine its role in tumorigenesis in a mouse model, we have generated an ETV7 transgenic mouse that carries a single copy of human BAC DNA containing the ETV7 gene locus and its regulatory sequences. ETV7 heterozygous (ETV7Tg+/WT) mice were fertile, normal in size and born at a normal Mendelian frequency. They had a normal blood count, did not display any gross physical or behavioral abnormalities, and were not tumor-prone. The ETV7 expression pattern in hematopoietic cells of ETV7Tg+/WT mice is very similar to that in human hematopoietic cells. To examine the oncogenic potential of ETV7 in vivo, we crossed ETV7Tg+/WT mice with tumor-prone mouse models. ETV7 greatly accelerated loss of Pten (phosphatase and tensin homolog)-evoked leukemogenesis in PtenΔ/ΔETV7Tg+/WT mice after deletion of the conditional Pten allele. Consistent with this observation, ETV7 expression enhanced the colony-forming and self-renewal activities of primary myeloid Pten−/− cells. In this study we established a transgenic mouse in which we can more accurately model ETV7-associated human tumorigenesis in vivo.

PO-207 Ferritin-engineered nanoparticles as targeted drug delivery system for cancer treatment

IntroductionT cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy that originates from malignant transformation of T cell progenitors. It accounts for 15% of paediatric and 25% of adult ALLs. Most frequent genetic alterations associated with this neoplasia are activating mutations in NOTCH1 and loss of function mutation for FBXW7 and PTEN. Current chemotherapeutic approaches demonstrate good cure and survival rates in patients. However prognosis of relapsed patients is still dismal. Therefore there is an urgent need to identify new targets for the development of more effective therapeutic compounds.Material and methodsCRISPR/Cas9 editing technology was used to generate mosaic mutant animals in Xenopus tropicalis, a true diploid amphibian. sgRNAs were designed to induce activating (i.e. truncating) mutations in notch1* and loss-of-function mutations in fbxw7 and pten. Cas9 recombinant protein and different sgRNA combinations were injected in 4 cell stage embryos. Animals were raised for 6 weeks. Liver, spleen, kidneys and thymus were dissected and further processed.Results and discussionsCombined injections of sgRNAs that targeted notch1 and pten resulted in mosaic mutant animals that exhibited pale appearance, enlarged spleen and haemorrhagic hindlimbs. Furthermore, sudden unexplained full penetrant mortality was observed around the time of metamorphosis. From diseased animals, genomic DNA was extracted from dissected thymi and the targeted gene loci were PCR-amplified and deep sequenced. Interestingly, several thymi showed clonal enrichment of a single cell population with a unique INDEL mutation pattern in one notch1 allele and two pten alleles, indicative of a leukemic cell population in the mosaic mutant animals. Exploiting a concept of clonal enrichment and negative selection for mutations in genes essential for T-ALL formation, we are using multiplexed gene inactivation to identify T-ALL dependency factors and hence explore novel routes for targeted therapy. We are currently verifying the feasibility of this experimental concept by multiplexed injections of pten and notch1 together with myc. Furthermore, we are validating our model by conducting flow cytometric and immunohistochemical analysis of the blood.ConclusionWe generated a genetic T–ALL model in X. tropicalis by co-targeting of notch1* and pten. We expect this model to provide new opportunities for identification and validation of novel driver genes and dependency factors for T-ALL.

Abstract 4074: Pten haploinsufficiency promotes tumor invasion and carcinogenesis in mouse colon epithelium with Apc deficiency

Abstract Background and Aim: The loss of PTEN (phosphatase and tensin homologue) expression in human colorectal cancer (CRC) is found in approximately 40% of cases, and its functional contribution is not fully understood. To address this question, the colon tumors from the Pten-deficient mice with a disruption of the Adenomatous polyposis coli (Apc) were compared with those from the mice with Apc deficiency alone. Method: To recapitulate human CRC, mouse models carrying transgenes regulated by a 9.5-kb fragment containing sequences from the human CDX2 promoter (CDX2P9.5), which were previously shown to have tightly restricted transgene expression in the colon epithelium, were prepared. CDX2P9.5-NLSCre;Apcflox/+;Ptenflox/+ (referred to as CPC;Apc+Pten mouse) and CDX2P9.5-NLSCre;Apcflox/+(;Pten+/+) mice (referred to as CPC;Apc mouse) were generated, and the overall survival and tumor phenotypes (number, size and severity) at 4, 6, 9, 12 and 15 weeks of age were compared between the two models. In addition, the loss of heterozygosity status of the Apc and Pten wild-type alleles in tumor tissue and normal mucosa in the two mouse models were compared with multiplex polymerase chain reaction (PCR), and Pten transcripts and protein expression were evaluated by quantitative reverse transcription PCR and immunohistochemistry, respectively. Results: CPC;Apc+Pten mice had a significantly shorter life span than CPC;Apc mice (median survival time of CPC;Apc+Pten mice and CPC;Apc mice: 14.4 weeks [n=20] vs. 21.6 weeks [n=20], respectively). The CPC;Apc+Pten mice had more tumors than CPC;Apc mice at all time points measured, but the tumor size in CPC;Apc+Pten mice was significantly larger than that in CPC;Apc mice only at 9 weeks of age. Invasion into the submucosa was more frequently observed in CPC;Apc+Pten mice than in CPC;Apc mice (CPC;Apc+Pten mice and CPC;Apc mice: 36.3%/58.3% and 4.9%/10% at 12/15 weeks of age, respectively). Regarding the findings on multiplex PCR, while the tumors from both models showed biallelic Apc inactivation, the tumors from CPC;Apc+Pten mice showed no loss of the wild-type allele of Pten. Regarding the Pten transcript levels, the levels in the tumors and mucosa from CPC;Apc+Pten mice were almost half of those in CPC;Apc mice. The Pten protein expression level in tumors from CPC;Apc+Pten mice was lower than that in tumors from CPC;Apc mice but not completely suppressed. Conclusion: Pten promotes tumor invasion and carcinogenesis without two hits on the gene. Consistent with the previous finding that two hits on the PTEN gene was a rare event in human CRC with PTEN loss, our data strongly suggest the haploinsufficient tumor-suppressive activity of Pten. These findings show that this CPC;Apc+Pten mouse model recapitulates human CRC with PTEN loss. Citation Format: Haruki Sada, Takao Hinoi, Hiroaki Niitsu, Masatoshi Kochi, Naoya Sakamoto, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, Hideki Ohdan. Pten haploinsufficiency promotes tumor invasion and carcinogenesis in mouse colon epithelium with Apc deficiency [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 4074.

Abstract SY22-02: Metabolic vulnerability of PTEN mutant cancer

Abstract Signaling pathways that act as drivers of cancer cell growth and proliferation activate metabolic pathways required to generate the building blocks required for cell doubling. Loss of PTEN activates PI3K, AKT and mTOR to stimulate glucose and glutamine flux to allow DNA replication. One of the metabolic pathways that is activated after deletion of both alleles of PTEN is the de novo pyrimidine synthesis pathway. PTEN null cells show evidence of increased DNA replication and low levels of damage at replication forks that can be rescued by providing supplementary uridine. Interruption of de novo pyrimidine synthesis with an inhibitor of dihydroorotate dehydrogenase (DHODH) increased DNA damage at replication forks of PTEN null cells that ultimately led to chromosome breaks detected in mitosis and cell death. This cellular vulnerability to inhibition of DHODH was rescued with uridine, demonstrating the specificity of the effect. Exploration of the mechanism that led to vulnerability of PTEN null cells demonstrated that it was due to defective ATR signaling at replication forks that was a consequence of activated AKT, which phosphorylate and attenuate TOPBP1 and CHK1. Complementation of PTEN null cells with mutants of TOPBP1 and CHK1 that cannot be phosphorylated by AKT rescued the cells from DNA damage and cell death due to inhibition of DHODH. Tumor cell lines lacking PTEN tended to be more sensitive to DHODH inhibition than PTEN wild type cells were. In conclusion, the attenuated ATR signaling and increased flux of nucleotides to newly synthesized DNA at replication forks makes PTEN mutant cells vulnerable to disruption of de novo pyrimidine synthesis. Citation Format: Ramon E. Parsons. Metabolic vulnerability of PTEN mutant cancer [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 SY22-02.

Characterization of novel cell lines derived from a MYC-driven murine model of lethal metastatic adenocarcinoma of the prostate.

Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer. We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively. Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection. These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.

Abstract PR11: Investigating lung cancer cells-of-origin using three-dimensional organoid cultures

Abstract There are many distinct epithelial cell types in the adult lung that could give rise to non-small cell lung cancer. These cell types include tracheal basal stem cells, tracheal club cells, distal lung bronchioalveolar stem cells (BASCs), and distal lung alveolar type 2 cells (AT2). My laboratory is using mouse models to test directly which lung stem and progenitor cells are most fit to serve as cells-of-origin of lung adenocarcinoma and lung squamous cell carcinoma. Previously, we demonstrated that tracheal club cells and BASCs are the likely cells-of-origin for KRASG12D; Lkb1-null adenosquamous tumors. The same strategy is now being used to test which cells can give rise to adenocarcinomas using the LSL:EGFRT90M/L858R (LSL=lox-stop-lox) mouse strain, or squamous cell carcinoma using the Lkb1flox/flox; Ptenflox/flox mouse strain. Briefly, tracheas and lungs from Cre-naïve mice are enzymatically dissociated, and fluorescence activated cell sorting (FACS) is used to enrich for four distinct populations: NGFR+/Sca1+ tracheal basal cells, NGFR-/Sca1+ tracheal club cells, Sca1+ distal lung BASCs, and Sca1- distal lung AT2 cells. Each population is then incubated with either adeno-GFP control virus or adeno-Cre virus to induce EGFR T790M/L858R expression or delete both alleles of Lkb1 and Pten. Three dimensional air-liquid-interface cultures are then used to expand the populations as organoids. For EGFR organoids, both basal and club cells give rise to larger, faster-growing bronchiolar organoids after EGFR is induced. AT2 cells similarly give rise to much larger alveolar organoids after EGFR induction, and BASCs change from giving rise to both bronchiolar and alveolar organoids to nearly exclusively alveolar. For Lkb1/Pten organoids, all four populations can tolerate deletion of the two genes, but it is only the basal cell and club cell organoids that progress to a squamous morphology in vitro. While both BASC and AT2 organoids continue to grow, they appear phenotypically normal. Transplant assays to assess malignancy and RNA-seq and ChIP-seq studies to explore gene transcription and epigenetic changes are under way. These experiments will shed light on the cells of the adult lung that give rise to genotype-specific tumors, and will aid us to better classify lung cancer heterogeneity from a cell-of-origin perspective. Citation Format: Kwok-Kin Wong, Carla Kim, Christine Fillmore Brainson. Investigating lung cancer cells-of-origin using three-dimensional organoid cultures [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR11.

Novel transgenic knockout model of basal-squamous bladder cancer.

459 Background: The recent description of the highly aggressive Basal-Squamous molecular subtype of bladder cancer (BLCa) requires the development of new in vivo models for the study of this clinical entity. Although loss of the transcription factor Forkhead box A1 ( FOXA1) is significantly associated with the Basal-Squamous subtype, cooperating genetic alterations are unknown. Herein, we identify genetic alterations that potentially cooperate with FOXA1 loss to drive tumorigenesis and/or the Basal-Squamous phenotype and develop a novel transgenic model for the study of Basal-Squamous BLCa. Methods: We interrogated The Cancer Genome Atlas (TCGA) BLCa study to identify common genetic alterations associated with FOXA1 copy number (CN) loss. Based on our results, we utilized a bladder-specific Cre-LoxP ( Uroplakin II-Cre) to constitutively knock-out (KO) Foxa1 and/or Pten in mouse urothelium. Two cohorts of mice were aged for 6 and 12 months, and a third cohort was exposed to the bladder-specific carcinogen N-butyl-(4-hydroxybutyl)-nitrosamine (BBN; 0.05%) for 12 weeks. Following all experiments, the bladder tissue was isolated and characterized. Results: The TCGA data revealed that combined CN loss of both FOXA1 and PTEN occurs in 17% of MI BLCa patients. At 6 months of age, our mice developed urothelial hyperplasia, whereas at 12 months of age, double KO ( Foxa1-/-/Pten-/-) mice developed carcinoma in situ (CIS) with SqD, enriched for basal (Krt5/6 and 14) and reduced in luminal (Gata3 and Pparγ) markers. Increased tumor stage was significantly associated with combined KO of at least one allele of Foxa1 and Pten following BBN exposure. Moreover, upregulation of apoptosis related genes was observed by RNA-sequencing data. Conclusions: In advanced BLCa, CN loss of FOXA1 and PTEN is common. Genetic ablation of Foxa1 and Pten results in CIS with SqD and a pathologic profile consistent with the Basal-Squamous subtype and accelerated tumor onset following carcinogen exposure. Therefore, we describe the development of a novel model for the study of Basal-Squamous BLCa potentially useful for preclinical studies.