Dominant-negative P53 Research Articles

Mitochondrially targeted p53 or DBD subdomain is superior to wild type p53 in ovarian cancer cells even with strong dominant negative mutant p53

BackgroundWhile tumor suppressor p53 functions primarily as a transcription factor in the nucleus, cellular stress can cause p53 to translocate to the mitochondria and directly trigger a rapid apoptotic response. We have previously shown that fusing p53 (or its DNA binding domain, DBD, alone) to the mitochondrial targeting signal (MTS) from Bak or Bax can target p53 to the mitochondria and induce apoptosis in gynecological cancer cell lines including cervical cancer cells (HeLa; wt p53), ovarian cancer cells (SKOV-3; p53 267del non-expressing), and breast cancer cells (T47D; L194F p53 mutation). However, p53 with Bak or Bax MTSs have not been previously tested in cancers with strong dominant negative (DN) mutant p53 which are capable of inactivating wt p53 by homo-oligomerization. Since p53-Bak or Bax MTS constructs act as monomers, they are not subject to DN inhibition. For this study, the utility of p53-Bak or p53-Bax MTS constructs was tested for ovarian cancers which are known to have varying p53 statuses, including a strong DN contact mutant p53 (Ovcar-3 cells), a p53 DN structural mutant (Kuramochi cells), and a p53 wild type, low expressing cells (ID8).ResultsOur mitochondrial p53 constructs were tested for their ability to localize to the mitochondria in both mutant non-expressing p53 (Skov-3) and p53 structural mutant (Kuramochi) cell lines using fluorescence microscopy and a nuclear transcriptional activity assay. The apoptotic activity of these mitochondrial constructs was determined using a mitochondrial outer membrane depolarization assay (TMRE), caspase assay, and a late stage cell death assay (7-AAD). We also tested the possibility of using our constructs with paclitaxel, the current standard of care in ovarian cancer treatment. Our data indicates that our mitochondrial p53 constructs are able to effectively localize to the mitochondria in cancer cells with structural mutant p53 and induce apoptosis in many ovarian cancer cell lines with different p53 statuses. These constructs can also be used in combination with paclitaxel for an increased apoptotic effect.ConclusionsThe results suggest that targeting p53 to mitochondria can be a new strategy for ovarian cancer treatment.

Dominant-negative p53 mutant R248Q increases the motile and invasive activities of oral squamous cell carcinoma cells.

The tumor suppressor gene TP53 (gene) codes for a transcription factor which transactivates its target genes responsible for cell cycle arrest, DNA repair, apoptosis, and senescence. TP53 is well known to be the most frequent target of genetic mutations in nearly half of human cancers including oral squamous cell carcinoma (OSCC). Many p53 mutants including R248Q and R248W not only lose its tumor-suppressor activities, but also interfere with the functions of wild-type p53; this is so-called dominant-negative (DN) mutation. The DN p53 mutation is a predictor of poor outcome in patients with various cancers, and also a risk factor for metastatic recurrence in patients with OSCC. Recently it has been reported that DN p53 mutants acquire new oncogenic activities, which is named gain-of-function (GOF). This study aimed at determining whether R248Q and R248W were involved in OSCC cells' acquiring aggressive phenotypes, using SAS, HSC4 and Ca9-22 cell lines. First, two mutants p53, R248Q and R248W, were respectively transfected into SAS cells harboring recessive-type p53 (E336X). As a result, SAS cells expressing R248Q showed highly spreading, motile and invasive activities compared to parent or mock-transfected cells whereas those expressing R248W did not increase those activities. Secondly, in HSC4 cells harboring R248Q and Ca9-22 cells harboring R248W, expressions of the mutants p53 were inhibited by the transfection with siRNAs targeting p53. The inhibition of the mutants p53 decreased spreading, motile and invasive activities of HSC4 cells whereas it did not affect those activities of Ca9-22 cells. These findings suggest that R248Q p53 mutation, but not R248W p53 mutation, induces more motile and invasive potentials in human OSCC cells.

Analysis across multiple tumor types provides no evidence that mutant p53 exerts dominant negative activity

Missense mutations in the TP53-binding domain predominate, and >30% of these occur in just eight codons. Dominant negative properties of mutant p53, taken together with the mutation susceptibility of the nucleotides in the codon, are believed to explain the prevalence of specific mutations, including hot spots. We analyzed multiple tumor types and found no difference in clinical characteristics or survival between patients with dominant negative p53 mutant tumors and those with TP53 mutations that are predicted to be non-dominant negative. The rate tumors underwent loss of heterozygosity in these respective mutation classes was nearly identical, suggesting that presence of stable, mutant protein with predicted dominant negative activity does not reduce selective pressure to inactivate the wild-type allele. Our data suggest all inactivating mutations of TP53 are equal, and the frequency of dominant negative, hot spot mutations is likely driven more by the relative mutability of the DNA at specific codons.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

MYC-driven malignant transformation of mature murine B cells requires inhibition of both intrinsic apoptosis and p53 activity.

Increased expression of the oncogene MYC is a common feature of many B-cell malignancies, however MYC overexpression by itself is not sufficient for transformation, and additional genetic events are required, although the exact nature of these remains unknown. In patients and in transgenic mouse models, oncogenic transformation may occur in B cells at various differentiation stages interacting with complex microenvironments. B-cell oncogenesis often occurs after prolonged periods of time, making it difficult to accurately identify the genetic events required for transformation. An in vitro system, where malignant transformation of primary B cells could be analyzed, would facilitate the identification of genetic events required for transformation. Here, we describe such a system and show that primary murine B cells rapidly become transformed upon forced expression of MYC, in conjunction with simultaneous inhibition of the ARF/p53 axis via overexpression of BMI1, as well as through downregulation of p19ARF or expression of a dominant-negative p53 and suppression of intrinsic apoptosis through overexpression of BCLXL or MCL1. Established tumor cells remained addicted to expression of the lymphoma-inducing genes. In mice, transformed cells rapidly established fatal B-cell lymphomas. Our results suggest that transformation of normal mature B cells into lymphomas can occur as a consequence of three defined events.

Assessing Therapeutic Efficacy of MEK Inhibition in a KRASG12C-Driven Mouse Model of Lung Cancer.

Purpose: Despite the challenge to directly target mutant KRAS due to its high GTP affinity, some agents are under development against downstream signaling pathways, such as MEK inhibitors. However, it remains controversial whether MEK inhibitors can boost current chemotherapy in KRAS-mutant lung tumors in clinic. Considering the genomic heterogeneity among patients with lung cancer, it is valuable to test potential therapeutics in KRAS mutation-driven mouse models.Experimental Design: We first compared the pERK1/2 level in lung cancer samples with different KRAS substitutions and generated a new genetically engineered mouse model whose tumor was driven by KRAS G12C, the most common KRAS mutation in lung cancer. Next, we evaluated the efficacy of selumetinib or its combination with chemotherapy, in KRASG12C tumors compared with KRASG12D tumors. Moreover, we generated KRASG12C/p53R270H model to explore the role of a dominant negative p53 mutation detected in patients in responsiveness to MEK inhibition.Results: We determined higher pERK1/2 in KRASG12C lung tumors compared with KRASG12D Using mouse models, we further identified that KRASG12C tumors are significantly more sensitive to selumetinib compared with KrasG12D tumors. MEK inhibition significantly increased chemotherapeutic efficacy and progression-free survival of KRASG12C mice. Interestingly, p53 co-mutation rendered KRASG12C lung tumors less sensitive to combination treatment with selumetinib and chemotherapy.Conclusions: Our data demonstrate that unique KRAS mutations and concurrent mutations in tumor-suppressor genes are important factors for lung tumor responses to MEK inhibitor. Our preclinical study supports further clinical evaluation of combined MEK inhibition and chemotherapy for lung cancer patients harboring KRAS G12C and wild-type p53 status. Clin Cancer Res; 24(19); 4854-64. ©2018 AACR.

Roles of p53, NF-κB and the androgen receptor in controlling NGAL expression in prostate cancer cell lines.

Neutrophil gelatinase-associated lipocalin (NGAL a.k.a lipocalin 2, lnc2) is a secreted protein which can form a complex with matrix metalloproteinase-9 (MMP9). This MMP9/NGAL complex has been associated with metastasis. MMP9 and NGAL are detected in the urine of patients afflicted with many different types of cancer, including prostate cancer. The effects of p53, NF-κB and the androgen receptor (AR) on the expression of NGAL was examined in four prostate cancer cell lines. Prostate cancer cell lines that are AR negative and expressed either mutant or no p53 (DU145 and PC3) displayed higher levels of NGAL expression compared to the prostate cancer cell lines (LNCaP and 22Rv-1) which are AR positive and express wild type (WT) p53. Introduction of WT-p53 into the PC3 prostate cancer cell line, resulted in reduction of the levels of NGAL expression. Conversely, introduction of dominant negative (DN) p53 or a retroviral construct expressing NF-κB into LNCaP cells increased NGAL expression. NGAL expression had functional effects on the ability of the cells to form colonies in soft agar. Whereas suppression of WT-53 in LNCaP cells increased NGAL expression, the introduction of WT-p53 suppressed NGAL transcription activity in PC3 prostate cells which normally express high level of NGAL. NF-κB and p53 were determined to regulate NGAL expression by positive and negative mechanisms, respectively. Our data indicate that prostate cancer growth, progression and sensitivity to chemotherapeutic drugs are regulated in part by NGAL and may involve complex interactions between NGAL, MMP9, NF-κB and p53.

O6-methylguanine–induced transcriptional mutagenesis reduces p53 tumor-suppressor function

Altered protein function due to mutagenesis plays an important role in disease development. This is perhaps most evident in tumorigenesis and the associated loss or gain of function of tumor-suppressor genes and oncogenes. The extent to which lesion-induced transcriptional mutagenesis (TM) influences protein function and its contribution to the development of disease is not well understood. In this study, the impact of O6-methylguanine on the transcription fidelity of p53 and the subsequent effects on the protein's function as a regulator of cell death and cell-cycle arrest were examined in human cells. Levels of TM were determined by RNA-sequencing. In cells with active DNA repair, misincorporation of uridine opposite the lesion occurred in 0.14% of the transcripts and increased to 14.7% when repair by alkylguanine-DNA alkyltransferase was compromised. Expression of the dominant-negative p53 R248W mutant due to TM significantly reduced the transactivation of several established p53 target genes that mediate the tumor-suppressor function, including CDKN1A (p21) and BBC3 (PUMA). This resulted in deregulated signaling through the retinoblastoma protein and loss of G1/S cell-cycle checkpoint function. In addition, we observed impaired activation of apoptosis coupled to the reduction of the tumor-suppressor functions of p53. Taking these findings together, this work provides evidence that TM can induce phenotypic changes in mammalian cells that have important implications for the role of TM in tumorigenesis.

MTA1 promotes viability and motility in nasopharyngeal carcinoma by modulating IQGAP1 expression.

Nasopharyngeal carcinoma (NPC) is frequently seen in Chinese, especially the population that resides in southeast China. Metastasis-associated protein 1 (MTA1) is a chromatin modifier and plays a role in tumor cell metastasis. IQGAP1 is a ubiquitously expressed protein that contributes to cytoskeleton remodeling. This study aimed to investigate the role of MTA1 and IQGAP1 in NPC malignant transformation. MTA1 and IQGAP1 expression in NPC (n = 43) and control tissues (n = 31) were detected using qRT-PCR, immunoblot, and immunohistochemistry. MTA1 was overexpressed in CNE-1 and CNE-2 cell line by pcDNA3.1/MTA1 transfection. Dominant-negative p53 was transfected to inhibit p53 activity. si-IQGAP1 or dominant-negative IQGAP1 (IQGAP1ΔGRD) was used to suppress IQGAP1 activity. Cell proliferation was measured by CKK-8 assay. Cell migration was evaluated by Transwell assay. The results showed that MTA1 and IQGAP1 were highly expressed in NPC tissues compared with the controls. Forced expression of MTA1 accelerated cell proliferation and migration and upregulated IQGAP1 expression in a p53-independent way. Knockdown of IQGAP1 or transfection of dominant-negative IQGAP1 impeded tumor cell proliferation and migration as well as PI3K/Akt signaling induced by MTA1. In conclusion, MTA1 participates in NPC malignant transformation via regulating IQGAP1 expression and PI3K/Akt signaling pathway.

Reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells.

Fallopian tube secretory epithelial cells (FTSECs) have been suggested to be the source of high-grade serous ovarian carcinoma (HGSOC). Although several genetic alterations are known to be involved in HGSOC development, the minimal requirements remain unclear. We aimed to identify oncogenic mutations indispensable for HGSOC development in a stepwise model, using immortalized FTSECs. FTSECs were isolated from clinical samples and immortalized by overexpression of cyclin D1, CDK4R24C, and hTERT. Oncogenic mutations in the p53, c-Myc, and RAS/PI3K pathways were mimicked by lentiviral transduction. We found two distinct patterns of gene alteration essential for HGSOC development: p53/KRAS/AKT and p53/KRAS/c-Myc. Dominant-negative p53, alone or combined with oncogenic KRAS (KRASV12), constitutively active AKT (CA-AKT), and c-Myc, did not induce tumorigenesis in immortalized cells; however, overexpression of CA-AKT or c-Myc, along with dominant-negative p53 and KRASV12, conferred tumorigenic potential. Transformed FTSECs formed tumors in nude mice that were grossly, histologically, and immunohistochemically similar to human HGSOCs. Interestingly, mice harboring tumors with c-Myc amplifications displayed extensive metastases, consistent with the increased dissemination in their human counterparts. Thus, aberrant p53/KRASV12/c-Myc or p53/KRASV12/PI3K-AKT signaling was the minimum requirement for FTSEC carcinogenesis. The model based on this evidence could shed light on the early stages of HGSOC development.

Abstract 5760: Identifying cell of origin for Group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Although surgery, radiation and high-dose chemotherapy have led to increased survival rates, many patients still die from their disease and those who survive often suffer severe long-term side effects. Group 3 MB is the most aggressive subtype of MB, which is often resistant to therapy, and patients with this tumor have an extremely poor prognosis. Thus, developing novel and more effective therapeutic approaches is desperately needed for treating this disease. Improved approaches are likely to come from a deeper understanding of the molecular and cellular basis of the disease. Group 3 MB is characterized by large cell/anaplastic histology with a high proliferative index and is associated with MYC amplification or overexpression. Our previous studies suggest that MYC-driven MB can be generated by infecting granule neuron progenitors (GNPs, Math1+) or neonatal cerebellar stem cells/progenitors (CD133+) with viruses encoding MYC and dominant negative p53 (DNp53) and transplanting these cells into the cerebella of immunocompromised mice (NSG). Although this model resembles Group 3 MB in terms of histology and gene expression, it most likely does not faithfully recapitulate human MB because mutation and deletion of p53 are rarely detected in human Group 3 MB at diagnosis. This observation prompted us to ask whether there might be a cell type in which the MYC oncogene alone is sufficient to induce MB. Sox2 expression has been found in neural stem cell/progenitor cells in cerebellum. Moreover, it has been reported that ~70% human Group 3 MB express Sox2, and that its expression is correlated with poor prognosis. Thus we hypothesize that Sox2-positive cells with MYC overexpression can develop tumors. We FACS-sorted Sox2GFP-positive or Sox2GFP-negative cells from Sox2-GFP mice at P5, infected these cells with MYC virus and transplanted them into the cerebella of NSG mice. Animals (n=10) receiving Sox2-positive cells developed highly aggressive tumors and had to be sacrificed within 3-6 weeks. In contrast, only one of the animals (n=10) that received Sox2-negative cells developed a tumor at 8 weeks. These results suggest that the MYC oncogene alone is sufficient to induce tumorigenesis in Sox2-positive cells, without requiring loss of p53. Further studies demonstrate that the tumors that were generated from Sox2-positive cells resemble Group 3 MB at a histological, immunohistochemical and molecular level, indicating that Group 3 MB may originate from Sox2-positive cells during cerebellar development. We will use this model to investigate the mechanisms that make these cells sensitive to transformation. These studies will pave the way for the development of novel approaches to treating this devastating tumor. Citation Format: Ran Tao, Najiba Murad, Zhenhua Xu, Yanxin Pei. Identifying cell of origin for Group 3 medulloblastoma [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 5760. doi:10.1158/1538-7445.AM2017-5760

Abstract 5755: In vitro reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells

Abstract Purpose: Recent studies suggest that fallopian tube secretory epithelial cells (FTSECs) are potential cells-of-origin for high-grade serous ovarian carcinoma (HGSOC). Several genetic alterations are involved in HGSOC carcinogenesis, but the minimal requirement for tumor initiation remains unclear. Therefore, we sought to identify oncogenic mutations indispensable for HGSOC carcinogenesis in a stepwise model using immortalized FTSECs. Experimental Design: We established an in vitro stepwise carcinogenesis model using immortalized FTSECs overexpressing cyclin D1, CDK4R24C, and hTERT, and mimicked select genetic abnormalities identified as gene alterations essential for carcinogenesis, including p53, c-Myc, or RAS/PI3K pathway mutations. Results: Our analyses revealed two distinct patterns of gene alterations essential for HGSOC carcinogenesis: p53/KRAS/AKT and p53/KRAS/c-Myc. Dominant-negative p53 expression alone or in combination with oncogenic KRAS (KRASV12), constitutively active AKT (CA-AKT), or c-Myc in immortalized cells failed to induce a tumorigenic phenotype; however, overexpression of either CA-AKT or c-Myc in combination with dominant-negative p53 and KRASV12 was sufficient to confer tumorigenic potential. Importantly, all transformed FTSECs formed tumors in xenograft mice, which were grossly, histologically, and immunohistochemically similar to human HGSOC. Interestingly, mice harboring tumors with c-Myc amplifications displayed extensive metastases, consistent with the increased dissemination observed in their human counterparts. C-Myc is associated with cell proliferation in vitro; therefore, this genetic abnormality may promote HGSOC progression. Conclusions: Collectively, our data showed that aberrant p53/KRASV12/c-Myc or p53/KRASV12/PI3K-AKT signaling is the minimal requirement for FTSEC carcinogenesis. Moreover, the model generated with this evidence will likely facilitate analysis of early events in HGSOC carcinogenesis. Citation Format: Kohei Nakamura, Kentaro Nakayama, Tohru Kiyono, Noriyoshi Ishikawa, Toshiko Minamoto, Tomoka Ishibashi, Kaori Sanuki, Hitomi Yamashita, Ruriko Ono, Kouji Iida, Hiroki Sasamori, Razia Sultana, Mohammad Mahmud Hossain, Masako Ishikawa, Satoru Kyo. In vitro reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5755. doi:10.1158/1538-7445.AM2017-5755

Abstract 313: Tbata induces G2/M cell cycle arrest and sensitizes osteosarcoma cells to etoposide in a p53-independent manner

Abstract Thymus, brain, and testes-associated (Tbata) is a negative control cell-cycle gene highly expressed in murine thymic epithelial cells (TECs). Tbata protein binds to Uba3, inhibiting formation of Nedd8 E1 and subsequent target degradation via neddylation of several cell cycle control proteins needed for G2/M transition, which may be a major mechanism of TEC growth arrest during thymic involution. Etoposide is a cytotoxic drug which targets the enzyme toposimerase II — increased 2-3 fold during the G2 phase. To further characterize effect of Tbata on the cell cycle, we modified p53 wild-type U2OS cells derived from human osteosarcoma to express a Tbata/mCherry fusion protein when mifepristone is added to culture media. Tbata-expressing cells identified on flow cytometry did exhibit growth arrest, with quantitative assessment of DNA content in U2OS cells by flow cytometry establishing that 32% of Tbata/mCherry-expressing cells were in the G2 phase when exposed to mifepristone for 24 hours, compared to 11% of mCherry-expressing cells. Gene expression studies were consistent with these results. Cells expressing Tbata/mCherry were also more sensitive to etoposide at 0.5, 1, and 5 times the IC50 dose. To test whether p53 function was required, we further modified the cells to overexpress a dominant negative p53 mutant along with Tbata/mCherry. Similar G2 arrest and increased sensitivity to etoposide were observed, indicating that the effects of Tbata did not require normal p53 function. Potential Tbata analogues or mimetics may therefore be used as an adjunct to G2-targeted chemotherapy. Citation Format: Milos D. Miljkovic, Francis A. Flomerfelt, Ronald E. Gress. Tbata induces G2/M cell cycle arrest and sensitizes osteosarcoma cells to etoposide in a p53-independent manner [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 313. doi:10.1158/1538-7445.AM2017-313

IMMU-09. P53 REGULATES IMMUNOGENICITY OF MEDULLOBLASTOMA

Immunotherapy has recently emerged as an important strategy for treating cancer, but has not been tested extensively in pediatric brain tumors. To determine whether immunotherapy would be effective in this context, we need robust animal models of brain tumors that have a fully functional immune system. We recently created genetically engineered mouse models of Group 3 (Myc-driven) medulloblastoma, the most aggressive and lethal form of the disease. These models are generated by transducing cerebellar stem cells with viruses encoding Myc and dominant negative p53 (MP model), or Myc and GFI1 (MG model), and transplanting them into the cerebellum of an adult immunodeficient (ID) mouse. To evaluate whether these models can be used to study immunotherapy, we transplanted tumor cells into syngeneic, immunocompetent (IC) hosts. While MP tumors grew robustly in both ID and IC hosts, MG tumors failed to grow when transplanted into IC mice. Depletion of T cells rescued this effect, supporting the notion that MG tumors are being recognized and rejected by the immune system. Notably, knockdown or inactivation of p53 in MG tumors also allowed them to grow in IC hosts, suggesting that p53 mutations may render tumor cells invisible to the immune system. Ongoing studies are focused on understanding the basis of p53-mediated immune regulation, and on developing strategies to overcome it. These studies will provide insight into the interactions between medulloblastoma cells and the immune system, and identify novel approaches to immunotherapy of this tumor. Since p53 is mutated in a large proportion of human cancers, these studies may also have important implications for immunotherapy of other cancers.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.

Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use.

DiSCoVERing Innovative Therapies for Rare Tumors: Combining Genetically Accurate Disease Models with In Silico Analysis to Identify Novel Therapeutic Targets.

We used human stem and progenitor cells to develop a genetically accurate novel model of MYC-driven Group 3 medulloblastoma. We also developed a new informatics method, Disease-model Signature versus Compound-Variety Enriched Response ("DiSCoVER"), to identify novel therapeutics that target this specific disease subtype. Human neural stem and progenitor cells derived from the cerebellar anlage were transduced with oncogenic elements associated with aggressive medulloblastoma. An in silico analysis method for screening drug sensitivity databases (DiSCoVER) was used in multiple drug sensitivity datasets. We validated the top hits from this analysis in vitro and in vivo Human neural stem and progenitor cells transformed with c-MYC, dominant-negative p53, constitutively active AKT and hTERT formed tumors in mice that recapitulated Group 3 medulloblastoma in terms of pathology and expression profile. DiSCoVER analysis predicted that aggressive MYC-driven Group 3 medulloblastoma would be sensitive to cyclin-dependent kinase (CDK) inhibitors. The CDK 4/6 inhibitor palbociclib decreased proliferation, increased apoptosis, and significantly extended the survival of mice with orthotopic medulloblastoma xenografts. We present a new method to generate genetically accurate models of rare tumors, and a companion computational methodology to find therapeutic interventions that target them. We validated our human neural stem cell model of MYC-driven Group 3 medulloblastoma and showed that CDK 4/6 inhibitors are active against this subgroup. Our results suggest that palbociclib is a potential effective treatment for poor prognosis MYC-driven Group 3 medulloblastoma tumors in carefully selected patients. Clin Cancer Res; 22(15); 3903-14. ©2016 AACR.

Abstract 2476: DiSCoVERing innovative therapies for rare tumors: Combining genetically accurate disease models with advanced in silico analysis to identify novel therapeutic targets

Abstract The lack of genetically accurate models that recapitulate the genetics of human tumors hampers the development of new treatments for rare tumor types. One strategy to create an accurate model would be to use stem cells from a cancer's tissue of origin and add key oncogenic driver elements in combinations that are seen in tumors. These elements should also drive oncogenic transformation relevant to that particular tumor type. To validate this strategy, we used human neural stem and progenitor cells derived from the cerebellar anlage to develop a model of Group 3 MYC-driven medulloblastoma, a rare and frequently fatal pediatric brain tumor. The cells were transduced with c-MYC, dominant-negative p53, constitutively active AKT and hTERT, key oncogenic driver elements associated with aggressive medulloblastoma and injected into moice as orthotopic xerographs. These cells formed tumors in the brains of mice and resulted in decreased survival to a median of 117 days. These tumors histologically, pathologically, and genetically resembled the MYC-driven Group 3 medulloblastoma tumors. In contrast, human neural stem cells immortalized with SV40 did not form tumors in mice. To facilitate rapid screening of a large number of available drugs, we developed an in silico analysis technique, DiSCoVER (Disease-model Signature vs. Compound-Variety Enriched Response) that uses the expression profile of the human neural stem cell models and existing drug sensitivity databases to identify novel therapeutic targets. The DiSCoVER analysis predicted aggressive medulloblastoma would be sensitive to CDK inhibitors. Among the top hits were the CDK4/6 inhibitor palbociclib. Indeed, treatment of Palbociclib in human neural stem cell medulloblastoma model and high-serum medulloblastoma cell lines led to decreased proliferation by an average of 62% as measured by MTS assay compared to vehicle control (p<0.01). Palbociclib treatment also increased the percentage of apoptotic cells by an average of 150% as measured by cleaved caspase-3 immunofluorescence and cleaved-PARP western blot compared to vehicle control (p< 0.04). Treatment with palbociclib significantly extended the survival of mice with orthotopic medulloblastoma xenografts (p = 0.003). We validated our human neural stem cell model of Group 3 medulloblastoma and show that this model, as well as patient-derived high MYC medulloblastoma models, are sensitive to CDK4/6 inhibition in vitro and in vivo. Our results strongly suggest that Palbociclib can be an effective treatment for poor-prognosis MYC-driven medulloblastomas in carefully selected patients (Group 3/C1). In summary, we have presented here a new method of generating genetically accurate models for rare tumors using region-specific human stem and progenitor cells and a novel analysis technique to find therapeutics to target them. Citation Format: Allison Rose Hanaford, Tenley C. Archer, Antoinette Price, Ulf Kahlert, Jarek Maciaczyk, Nikkhah Guido, Wlliam Kim, Tobias Ehrenberger, Paul Clemons, Vlado Dancik, Brinton Seashore-Ludlow, Vasanthi Viswanathan,, Michelle Stewart, Matthew Rees, Alykahn Shamji, William Hahn, Stuart Schreiber, Ernest Fraenkel, Scott Pomeroy, Jill Mesirov, Pablo Tamayo, Charles G. Eberhart, Eric H. Raabe. DiSCoVERing innovative therapies for rare tumors: Combining genetically accurate disease models with advanced in silico analysis to identify novel therapeutic targets. [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 2476.

MiRNA-126 Orchestrates an Oncogenic Program in B Cell Precursor Acute Lymphoblastic Leukemia

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.

Transcriptional and translational regulation of CCAAT/Enhancer Binding Protein β expression by P53

The transcription factor CCAAT/enhancer‐binding protein β (C/EBPβ) is highly expressed in various tissues during development, including liver, adipose tissue, blood cells, lung and the endocrine pancreas. Two isoforms of C/EBPβ namely liver‐enriched activator protein (LAP) and liver‐enriched inhibitor protein (LIP) translated from the same mRNA have been described. The LAP and LIP isoforms share the same C‐terminal DNA binding domain except LAP has an extra N‐terminal activation domain. Probably due to its higher affinity for its DNA cognate sequences, the LIP can counteract the effects of LAP isoforms even at substoichiometric levels. In vitro and in vivo experiments with cultured cells and knockout mice demonstrated that C/EBPβ play a critical role in the regulation of myofibroblast differentiation and bleomycin induced pulmonary fibrosis. However, the regulatory mechanism of C/EBPβ gene expression is unclear. Analysis of the C/EBPβ promoter sequence identified a conserved P53 response element (P53RE) in the C/EBPβ gene promoter. Gel shift mobility and chromatin immunoprecipitation assays confirmed that P53 binds to the P53RE. Promoter reporter assays and real time PCR analysis of samples from fibroblasts transiently transfected with P53 or dominant negative P53 expression plasmids indicated that P53 was a repressor of C/EBPβ gene transcription. Western blot analysis with protein extracts from similarly transfected fibroblasts or P53 deficient fibroblasts showed that P53 differentially regulated translation of the C/EBPβ LAP and LIP isoforms through the regulation of eIF4E and eIF4E‐BP1. Further studies with constructs containing the LAP 5′ untranslated region or the LIP 5′ untranslated region showed that the 5′ untranslated region is important in differential control of C/EBPβ LAP and LIP translation. Thus P53 could regulate C/EBPβ gene expression at both transcriptional and translational levels.Support or Funding InformationThis work was supported by grants HL052285 and HL112880 from the National Institute of Health.

Abstract P3-14-07: Intra-cellular dsRNA receptor RIG-I: A ubiquitous novel target for treatment of chemotherapy drug resistant breast cancer

Abstract INTRODUCTION: Toll-like receptor 3 (TLR3), the cell surface receptor for double stranded RNA (dsRNA), expressed in ∼40% of advanced primary breast cancers (T2/T3/N+) has been shown to be an effective therapeutic target for synthetic dsRNA poly A:U in combination with radiation and adjuvant chemotherapy, producing a significant decrease in risk of metastatic relapse (HR 1.85-2.0; 1.03-3.89; Cancer Res;71:1607). AIM: The present pre-clinical study aimed to explore the therapeutic potential of a ubiquitously expressed intracellular dsRNA receptor, retinoic-acid-inducible gene-I (RIG-I) - a cytoplasmic pathogen recognition receptor directed at pathogen-associated molecular pattern (PAMP) motifs to differentiate viral from cellular dsRNAs. We have shown Ampligen, poly I:C12U (a synthetic dsRNA polymer designed to rapidly degrade in vivo to prevent the toxicity of long dsRNA polymers such as poly I:C) is capable of entering cells in its fragmented dsRNA oligomeric form (<1–2 kb) optimal for activation of intra-cellular RIG-I (J Exp Med 2008;205:1601–1610). METHODOLOGY & RESULTS: Preliminary experiments with Ampligen on several human breast cancer cell lines (MCF-7 & MDA-MB 453) and normal human mammary epithelial & fibroblast cell lines (HMEC & HFC) unexpectedly showed it to consistently cause a significant loss of cell viability (CellTiter-Glo Luminescent Cell Viability Assay) in p53-deficient drug resistant (5-FU/doxorubicin) MDA-MB 453 cell line in contrast to cell growth arrest (Guava cell Cycle Assay) in p53 wild type MCF-7 cancer and the two non-neoplastic cells lines. This selective effect was confirmed using syngeneic clones of MCF-7 breast cancer cells stably transfected with a dominant negative p53 construct or vector alone: p53-function blocked (DD1) vs p53-function active (EV1) MCF-7 cell lines, respectively (J Nucl Med 2006; 47:1525–1530), and shown to be associated with RIG-I specific mRNA induction (RT-PCR) and Type I interferon pathway activation both inhibited by BX795 (selective inhibitor of IRF3 activation and IFN-b production). Decitabine (DNA demethylating drug capable of intra-cellular generation of dsRNA through transcriptional activation of Alu retrotransposons - PNAS 2012; December 10: E89–E98 ) was next tested as an alternative source of intra-cellular dsRNA, and found to produce results similar to Ampligen on DD1 and EV1. Work is in progress to examine dose response and time-course relationships of the effects Ampligen or decitabine added singly or in combination with chemotherapeutic drug (e.g. doxorubicin) on DD1 and triple negative breast cancer cell lines such as MDA-MB 453 to explore potential therapeutically most effective protocols. PROVISIONAL CONCLUSIONS: Intra-cellular dsRNA receptor RIG-I constitutively expressed in all cells offers a more ubiquitous target compared to TLR3 for the treatment of breast cancer. Ampligen and decitabine with their selective growth inhibitory effect on drug resistant p53-deficient breast cancer cell lines, merit testing as novel drugs for treatment of p53-deficient drug resistant breast cancer e.g. triple negative breast cancer frequently (∼70%) associated with drug resistance and altered p53 status. Citation Format: Jasani B, Navabi N, Barrett-Lee P, Thompson A, Chester J, Mason M. Intra-cellular dsRNA receptor RIG-I: A ubiquitous novel target for treatment of chemotherapy drug resistant breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-07.