Wild-type Cancers Research Articles

Clinicopathologic characteristics of NRG1 fusion-positive cancers: A single-institution study.

3129 Background: NRG1 rearrangements are oncogenic drivers across several tumor types. Chimeric proteins encoded by NRG1 fusions activate HER3, resulting in heterodimerization with HER2 and activation of downstream signaling. Preclinical and preliminary clinical data suggest that targeting HER3 may be an effective treatment strategy for patients with NRG1 fusion-positive tumors. We aimed to describe the clinical and genomic characteristics of patients identified at our institution with NRG1 fusions. Methods: We analyzed results from prospective targeted exome and/or RNA sequencing performed at Memorial Sloan Kettering between 2014-2018 involving > 30,000 samples. NRG1 fusion-positive tumors were identified and these cases were manually reviewed. Results: NRG1 fusions were detected in 24 patients. Cancer types included lung (N = 9), pancreas (N = 7), breast (N = 5), prostate (N = 1), gallbladder (N = 1), diffuse large B-cell lymphoma (N = 1), and cancer of unknown primary (N = 1). 6/9 lung cancers had mucinous differentiation. The majority of patients were Caucasian (N = 17), half were female (N = 12) and ages ranged from 24-82 years-old. Targeted exome sequencing identified the fusion in 10/23 cases tested, including 2 not confirmed by RNA. The remaining 14 were detected using RNA. Fusion partners included CD74 (N = 6), SLC3A2 (N = 2), SDC4 (N = 2), ATP1B1 (N = 2), FOXA1 (N = 1), SLCA4 (N = 1), ROCK1 (N = 1), TNKS (N = 1), CCND1 (N = 1), PAK1 (N = 1), STAU3 (N = 1), RAD51 (N = 1), CD44 (N = 1), NCOR1 (N = 1), RBPMS (N = 1), and WHSC1L1 (N = 1). Pancreas cancers were KRAS wild-type and lung cancers had no co-occurring alterations in ALK, ROS1, EGFR, RET, MET, RAS, or RAF. All tumors were microsatellite stable. A durable response was achieved with anti-HER3 antibody therapy (GSK2849330) in a patient with a CD74- NRG1-rearranged invasive mucinous adenocarcinoma (previously reported). Four patients treated with an irreversible small molecule HER2 inhibitor (afatinib) did not respond to treatment, suggesting direct targeting of HER3 may be superior to HER2 inhibition in patients with NRG1 fusion-positive tumors. Conclusions: NRG1 fusions occur in several tumor types and may be amenable to targeting with HER3-directed therapy.

P53-Dependent Apoptotic Effect of Puromycin via Binding of Ribosomal Protein L5 and L11 to MDM2 and its Combination Effect with RITA or Doxorubicin.

Among ribosomal proteins essential for protein synthesis, the functions of ribosomal protein L5 (RPL5) and RPL11 still remain unclear to date. Here, the roles of RPL5 and RPL11 were investigated in association with p53/p21 signaling in the antitumor effect of puromycin mainly in HCT116 and H1299 cancer cells. Cell proliferation assays using 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays and colony formation assays, cell cycle analysis, Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed in cancer cells. Puromycin exerted cytotoxic and anti-proliferative effects in p53 wild-type HCT116 more than in p53 null H1299 cells. Consistently, puromycin increased sub-G1, cleaved Poly (ADP-ribose) polymerase (PARP), activated p53, p21, and Mouse double minute 2 homolog (MDM2), and attenuated expression of c-Myc in HCT116 cells. Notably, puromycin upregulated the expression of RPL5 and RPL11 to directly bind to MDM2 in HCT116 cells. Conversely, deletion of RPL5 and RPL11 blocked the activation of p53, p21, and MDM2 in HCT116 cells. Also, puromycin enhanced the antitumor effect with reactivating p53 and inducing tumor apoptosis (RITA) or doxorubicin in HCT116 cells. These findings suggest that puromycin induces p53-dependent apoptosis via upregulation of RPL5 or RPL11 for binding with MDM2, and so can be used more effectively in p53 wild-type cancers by combination with RITA or doxorubicin.

Wild-Type IDH Enzymes as Actionable Targets for Cancer Therapy.

Isocitrate dehydrogenases (IDHs) are enzymes that catalyze the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (αKG) and CO2. The discovery of IDH1 and IDH2 mutations in several malignancies has brought to the approval of drugs targeting IDH1/2 mutants in cancers. Here, we summarized findings addressing the impact of IDH mutants in rare pathologies and focused on the relevance of non-mutated IDH enzymes in tumors. Several pieces of evidence suggest that the enzymatic inhibition of IDHs may have therapeutic potentials also in wild-type IDH cancers. Moreover, IDHs inhibition could enhance the efficacy of canonical cancer therapies, such as chemotherapy, target therapy, and radiotherapy. However, further studies are required to elucidate whether IDH proteins are diagnostic/prognostic markers, instrumental for tumor initiation and maintenance, and could be exploited as targets for anticancer therapy. The development of wild-type IDH inhibitors is expected to improve our understanding of a potential non-oncogenic addition to IDH1/2 activities and to fully address their applicability in combination with other therapies.

The MDM2/MDMX-p53 Antagonist PM2 Radiosensitizes Wild-Type p53 Tumors

Radiotherapy amplifies p53 expression in cancer cells with wild-type (wt) p53. Blocking the negative regulators MDM2 and MDMX stabilizes p53 and may therefore potentiate radiotherapy outcomes. In this study, we investigate the efficacy of the novel anti-MDM2/X stapled peptide PM2 alone and in combination with external gamma radiation in vitro and in vivo PM2 therapy combined with radiotherapy elicited synergistic therapeutic effects compared with monotherapy in cells with wt p53 in both in vitro and in vivo assays, whereas these effects did not manifest in p53 -/- cells. Biodistribution and autoradiography of 125I-PM2 revealed high and retained uptake homogenously distributed throughout the tumor. In mice carrying wt p53 tumors, PM2 combined with radiotherapy significantly prolonged the median survival by 50%, whereas effects of PM2 therapy on mutant and p53 -/- tumors were negligible. PM2-dependent stabilization of p53 was confirmed with ex vivo immunohistochemistry. These data demonstrate the potential of the stapled peptide PM2 as a radiotherapy potentiator in vivo and suggest that clinical application of PM2 with radiotherapy in wt p53 cancers might improve tumor control.Significance: These findings contribute advances to cancer radiotherapy by using novel p53-reactivating stapled peptides as radiosensitizers in wild-type p53 cancers. Cancer Res; 78(17); 5084-93. ©2018 AACR.

Abstract 5560: PD-1/PD-L1 blockade enhances MDM2 inhibitor activity in p53 wild-type cancers

Abstract p53 is a transcription factor that plays a central role in guarding genomic stability of the cell through cell cycle arrest or induction of apoptosis. It has also been reported that p53 participates in the regulation of tumor immunity and in homeostatic regulation of immune responses. However, the immunomodulatory effect of p53 in tumor and the tumor microenvironment is not well understood. From gene expression and immunohistochemical analysis of pre- and post-treatment biopsies from patients treated with the MDM2 inhibitor NVP-CGM097, we observed upregulation of immune checkpoint transcripts (PD1 and PDL1), and an increase in the number of CD8+ tumor infiltrating lymphocytes. To investigate the immunomodulatory effect of p53 in more detail, we studied murine syngeneic models treated with NVP-HDM201, a potent and selective second generation MDM2 inhibitor. We performed multi-color FACS analysis on tumors and tumor draining lymph nodes. With NVP-HDM201 treatment, we observed increase in numbers of CD103+ DC cells, capable of antigen cross-presentation. Furthermore, NVP-HDM201 increased the percentage of Tbet+EOMES-T cells in tumors as well as tumor draining lymph nodes and also resulted in increased ratios of CD8+ Tcells/Treg in tumor. In addition, levels of both PD-L1 on CD45- cells and PD-1 on CD4+ T cells were increased. Importantly, PD-1 and PD-L1 blockade enhanced HDM201 activity in p53 WT syngeneic mouse models but not in p53 mutated models. The rate of complete tumor regressions (CR) was significantly increased with combination treatment as compared to either treatment alone. The animals that achieved complete regressions also developed long lasting anti-tumor memory against the specific tumor cells as evidenced by re-challenge experiments. Taken together, these results demonstrate that MDM2 inhibition triggered adaptive immunity which was further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in cancer patients with wildtype p53. Citation Format: Hui Qin Wang, Jinsheng Liang, Iain Mulford, Fiona Sharp, Swann Gaulis, Yan Chen, Gina Trabucco, David Quinn, Joseph D. Growney, Matthew J. Meyer, Juliet Williams, Peter Hammerman, Francesco Hofmann, Glenn Dranoff, Jeffrey Engelman, Jennifer Mataraza, Ensar Halilovic. PD-1/PD-L1 blockade enhances MDM2 inhibitor activity in p53 wild-type cancers [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 5560.

Abstract 4385: p53 activation induces a targetable dependence on FLIPL

Abstract Background The tumor suppressive functions of the p53 transcription factor are inactivated via mutations or suppressed through non-mutational mechanisms in almost all cancer cells. A better understanding of the mechanisms through which p53 differentially regulates cell cycle arrest and cell death is important to maximize benefits from wild-type p53-dependent therapeutic strategies Methods A panel of matched p53 wild-type and deficient colorectal cancer cell line models were studied, using Nutlin-3A and Oxaliplatin as direct and indirect p53 activating agents respectively. A number of molecular (Western blot, RT-PCR), phenotypic (cell death) and genomic analyses were used to investigate the importance of p53 and its downstream transcriptional programs. Results Here, we report that activation of pro-apoptotic p53 targets in colorectal cancer cells imposes a critical targetable dependence on the long splice form of the caspase-8 regulator FLIP (FLIPL) for survival. p53 binds the promoter of the FLIP gene (CFLAR) and upregulates FLIPL expression in response to the p53 agonist Nutlin-3A in a manner dependent on HDAC1/2/3 activity. As such, preventing FLIPL upregulation with the clinically relevant HDAC1/2/3-selective inhibitor Entinostat promotes apoptosis induction in response to Nutlin-3A (or p53-activating chemotherapy), which otherwise predominantly induces growth arrest despite upregulating a range of pro-apoptotic target genes. Cell death in response to Nutlin-3A in FLIPL-depleted cells is primarily mediated via caspase-8. However, in the absence of caspase-8, apoptosis is delayed, but not prevented and is mediated via caspase-10. Of note, the cell death induced in both caspase-8-proficient and -deficient cells is mediated via TRAIL-R2 in a ligand-independent manner. Conclusion In summary, this work has uncovered novel, clinically-relevant biology, in which p53-mediated upregulation of FLIPL primes cells for TRAIL-R2-mediated apoptosis and identifies FLIPL as a key target for overcoming resistance to p53-stabilising agents in p53 wild-type cancers. Moreover, we show the potential of combining Nutlin-3A (or other p53 activating chemotherapies) with the clinically relevant Class I HDAC inhibitor Entinostat for the treatment of p53 wild-type CRC, and identify FLIPL as a critical p53-induced signaling node, the inhibition of which is necessary to promote Nutlin-3A-induced apoptosis. Citation Format: Alexander McIntyre, Andrea Lees, Fiammetta Falcone, Gemma Gregg, Sessler Tamas, Gerard Quinn, Nyree Crawford, Darragh McArt, Phillip Dunne, Mark Lawler, Longley B. Daniel, Simon S. McDade. p53 activation induces a targetable dependence on FLIPL [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 4385.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Regulation of Monoallelic TERT Expression in Cancer Cells with Wildtype Promoters

During cell division, chromosomal telomere shortening that occurs due to the “end‐replication problem” can be compensated for by telomerase, a ribonucleoprotein enzyme that lengthens telomeres. Telomerase is active in stem cells, enabling maintenance of the telomeres, while it is normally inactive in somatic cells, leading to telomeres progressively shortening until a critical length triggers cell senescence or apoptosis. Importantly, telomerase is pathogenically reactivated in approximately 80–90% of human cancers. The expression of telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, specifically appears to be crucial to telomerase's tightly regulated activities; the Cech lab and others have shown that an absence of TERT expression is what prevents telomerase activity in human cells. While TERT expression levels are tightly regulated, with only 10–20% decrease in telomerase activity thought to be possibly deleterious for normal stem cell function, it is unclear how TERT expression becomes reactivated in many cancer types. Of particular interest is monoallelic expression (MAE) of TERT, which has been found in the majority of cancerous tissues. While some MAE cancer lines contain activating promoter mutations, other MAE cancer lines have no detectable mutations, raising the intriguing question of why TERT is reactivated in a monoallelic fashion in these wildtype (WT) cancers. To investigate the cause(s) of MAE expression in these WT cancer lines, we are investigating the allelic DNA CpG methylation pattern using bisulfite conversion cloning, histone modification markers using chromatin immunoprecipitation (ChIP), and transcription factor (TF) binding via ChIP, all within the TERT promoter region. Lines being investigated include multiple myeloma, pancreatic, lung, and glioblastoma cancer lines that have a single‐nucleotide polymorphism (SNP) in TERT exon 2 to enable identification of the active allele via reverse transcription PCR. While CpG hypermethylation is typically associated with silent gene expression, the TERT promoter paradoxically contains an apparently hypermethylated promoter in cancers actively expressing TERT. Our group recently found that in MAE cancers with TERT promoter mutations, the active allele is in fact hypomethylated (Stern et al., in press*), raising the question of whether this is similar in the WT TERT MAE lines. We have found that, in at least some of the WT MAE lines investigated, DNA CpG methylation patterns appear to be allelic, with one allele possessing hypermethylation and the other hypomethylation. We are currently investigating how this correlates with allelic expression, histone modification, and TF binding. Improved understanding of how TERT is reactivated in WT MAE cancers could help guide future drug discovery and cancer therapeutic efforts.Support or Funding InformationThis work was funded by National Institutes of Health grant R01 GM099705 to T.R.C.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rucaparib: a novel PARP inhibitor for BRCA advanced ovarian cancer.

Rucaparib is a potent small-molecule inhibitor of poly (ADP-ribose) polymerase (PARP) proteins (PARP-1, PARP-2 and PARP-3) that play an important role in repairing DNA damage and maintaining genomic stability. Tumors with mutations in BRCA1/2 or other homologous recombination deficiency (HRD) genes are particularly sensitive to PARP inhibitors because of “synthetic lethality”, whereby a therapeutic agent can take advantage of an intrinsic weakness in DNA repair. Rucaparib has been investigated in several preclinical and clinical studies showing promising activity in BRCA-mutant and BRCA–wild-type epithelial ovarian cancers (EOCs). Dose-escalation Phase I studies have established the recommended Phase II dose to be 600 mg twice a day for oral rucaparib. Phase II and III studies have defined its role as treatment for BRCA-mutant recurrent high-grade EOC and as maintenance treatment for platinum-sensitive relapsed EOC following response to platinum-based chemotherapy. Genomic loss of heterozygosity has also been investigated as a potential signature of HRD and as a potential predictive biomarker of response. Treatment-induced adverse events (AEs) have been observed in almost all patients treated with rucaparib, but mainly lower grade; with the most common being nausea, vomiting, asthenia/fatigue, anemia and transient transaminitis. The majority of AEs occurred early in treatment, were transient and have been easily managed with supportive treatment, dose interruption or discontinuation. This review will analyze the results of clinical trials investigating efficacy and safety of rucaparib in patients with ovarian cancer.

The role of APC in WNT pathway activation in serrated neoplasia

Conventional adenomas are initiated by APC gene mutation that activates the WNT signal. Serrated neoplasia is commonly initiated by BRAF or KRAS mutation. WNT pathway activation may also occur, however, to what extent this is owing to APC mutation is unknown. We examined aberrant nuclear β-catenin immunolocalization as a surrogate for WNT pathway activation and analyzed the entire APC gene coding sequence in serrated and conventional pathway polyps and cancers. WNT pathway activation was a common event in conventional pathway lesions with aberrant nuclear immunolocalization of β-catenin and truncating APC mutations in 90% and 89% of conventional adenomas and 82% and 70% of BRAF wild-type cancers, respectively. WNT pathway activation was seen to a lesser extent in serrated pathway lesions. It occurred at the transition to dysplasia in serrated polyps with a significant increase in nuclear β-catenin labeling from sessile serrated adenomas (10%) to sessile serrated adenomas with dysplasia (55%) and traditional serrated adenomas (9%) to traditional serrated adenomas with dysplasia (39%) (P=0.0001). However, unlike the conventional pathway, truncating APC mutations were rare in the serrated pathway lesions especially sessile serrated adenomas even when dysplastic (15%) and in the BRAF mutant cancers with microsatellite instability that arise from them (8%). In contrast, APC missense mutations that were rare in conventional pathway adenomas and cancers (3% in BRAF wild-type cancers) were more frequent in BRAF mutant cancers with microsatellite instability (32%). We conclude that increased WNT signaling is important in the transition to malignancy in the serrated pathway but that APC mutation is less common and the spectrum of mutations is different than in conventional colorectal carcinogenesis. Moderate impact APC mutations and non-APC-related causes of increased WNT signaling may have a more important role in serrated neoplasia than the truncating APC mutations common in conventional adenomas.

S100B expression in breast cancer as a predictive marker for cancer metastasis

In the tumor microenvironment, soluble molecules play important role in the establishment of a pre-metastatic niche. The S100 calcium-binding protein family are inflammatory molecules that contribute to the development of a pro-inflammatory tumor microenvironment. S100B belongs to the S100 family and serum S100B (also known as S100beta) serves as a marker for metastasis in lung cancer, ovarian cancer and melanoma. However, the association between S100B and the metastasis of breast cancer is not yet well understood. In the present study, a relatively low S100B expression was observed in the tumor samples compared to normal breast tissue among online microarray datasets. When the estrogen receptor (ER)-negative breast cancer cell lines, MDA-MB-231 and Hs578T, were treated with recombinant human S100B, cell migration was significantly inhibited and epithelial cadherin expression was increased. Our results revealed that a high S100B expression predicted a good overall survival in patients with ER-negative breast cancer, and good distant metastases-free survival in all patients with breast cancer via the analysis of the KM plotter and SurvExpress databases. Although previous studies have indicated that the interaction of S100B with wild-type p53 inhibits p53 function, a high S100B expression is associated with a good prognosis in patients with p53 mutant and p53 wild-type breast cancers. On the whole, our findings demonstrate that S100B treatment suppresses the migratory capacity of ER-negative breast cancer and that S100B expression may serve a predictive marker for metastasis in breast cancer.

Dual Inhibition of Mdmx and Mdm2 Using an Alpha-Helical P53 Stapled Peptide (ALRN-6924) As a Novel Therapeutic Strategy in Acute Myeloid Leukemia

While the TP53 tumor suppressor gene is mutated in more than 50% of human tumors, in Acute Myeloid Leukemia (AML) TP53 mutations are rare, occurring in less than 10% of cases. Yet, functional inactivation of wild-type p53 due to non-mutational abnormalities occurs frequently in AML and other hematological malignancies. A major mechanism of p53 inactivation results from the overexpression of its endogenous inhibitors MDMX (also known as MDM4, HDMX, and HDM4) and MDM2 which are frequently overexpressed in various p53 wild-type human cancers, including AML. Strikingly, MDMX has been reported to be overexpressed in over 92% of AML cases, while MDM2 overexpression is less frequent. Pharmacological disruption of both these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in AML and other cancers with wild-type p53. Nonetheless, selective targeting of this pathway has thus far been limited to MDM2-only small molecule inhibitors which lack affinity for MDMX. ALRN-6924 is an optimized alpha helical p53 stapled peptide and first-in-class dual MDMX/MDM2 inhibitor which has recently entered phase I/II clinical testing (NCT02264613, NCT02909972) in solid tumors and lymphomas with, thus far, excellent tolerability and objective responses as single agent1. The goal of our study was to evaluate the molecular, cellular, and biochemical mechanisms of action of ALRN-6924 in AML.We used biochemical affinity studies as well as single molecule FISH and live single cell imaging to assess MDMX/MDM2 binding as well as p21 transactivation by p53 in response to ALRN-6924. Effects on cellular proliferation, apoptosis, DNA repair, cell cycle, clonogenic capacity, and serial replating were determined using AML cell lines and primary human AML patients' cells, including in leukemic stem (CD34+ CD38-) and progenitor (CD34+CD38+) cells. Genome-wide molecular effects were determined by RNA seq. P53 activity in a patient undergoing treatment with ALRN-6924 was measured by intracellular staining of p53 and its target gene p21 in CD34+ cells by flow cytometry. Furthermore, we evaluated ALRN-6924 activity in a xeno-transplantation model of human AML in NSG mice.We found that MDMX is significantly overexpressed in highly fractionated leukemic stem (Lin-CD34+CD38-CD90-) and progenitor (Lin-CD34+CD38+CD123+CD45+) cells in AML patients compared to identically sorted, age-matched healthy controls (p<0.0001). Dual MDMX/MDM2 inhibition using ALRN-6924 led to striking anti-leukemic effects in suspension culture of AML cell lines and primary cells, with negligible effects on healthy controls. ALRN-6924 robustly activated p53-dependent transcription at the single cell and single molecule level, and exhibited strong biochemical and molecular biological on-target activity in AML cell lines and primary cells in vitro, as well as in a patient who received ALRN-6924 treatment. Co-immunoprecipitation experiments demonstrated that ALRN-6924 disrupts both MDMX/P53 as well as MDM2/P53 interactions at relevant therapeutic dosages in AML cells. RNA seq data showed that dual MDMX/MDM2 inhibition led to global transcriptional activation of p53-dependent pathways in AML cells. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibited cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patients' cells, including in leukemic stem cell-enriched populations. Furthermore, ALRN-6924 disrupted functional clonogenic and serial replating capacity of AML cells, and showed superiority over MDM2-only inhibition. Most strikingly, ALRN-6924 led to highly significant improved survival in an AML xenograft model in vivo (p<0.0001).Our study provides insight into the molecular, cell biological, and in vivo effects of pharmacological dual MDMX/MDM2 inhibition in AML, which may have important implications for other MDMX/MDM2-related cancers. Our findings provide proof-of-concept for ALRN-6924 as a novel therapeutic in p53-wildtype AML, and provide a rationale for its further preclinical and clinical development in AML and other cancers.Furthermore, the success of targeting p53 raises the intriguing prospect that the same development path is possible for other helix-in-groove targets, and may thus pave the way for a new class of targeted therapeutics.1 ASCO 2017 annual meeting: J Clin Oncol 35, 2017 (suppl; abstr 2505) DisclosuresGuerlavais:Aileron: Employment. Annis:Aileron Therapeutics: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Aivado:Aileron: Employment. Steidl:Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding.

Abstract A25: Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer

Abstract Mutations in tumour suppressors and un-druggable oncogenes dominate the landscape of cancer driver genes. Only a minority of colon cancers have mutations in druggable cancer drivers, such as PIK3CA. Conversely, mutations in tumour suppressors such as APC and TP53 are frequent, as are mutations in the notoriously difficult to drug KRAS target. There is an urgent need for new therapeutics to target tumours driven by these mutations: immune checkpoint approaches are likely to only prove effective in the fraction of patients whose tumours bear high mutation loads, which is colon cancer may be restricted to the minority of mismatch repair deficient cancers. The concepts of non-oncogene addiction and synthetic lethality provide a conceptual framework for finding therapeutic targets in these cancers. We have used arrayed siRNA and pooled CRISPR-Cas9 libraries to screen a panel of isogenic and non-isogenic colon cancer cell lines under conditions designed to increase the cells dependency on oncogenic pathways. This panel contains cell lines with mutations in TP53, APC, KRAS, PIK3CA and/or FBXW7 for which we are aiming to identify co-dependencies that consistently segregate with genotype. Our screens have identified a number of novel targets for which reduced expression imposes specific fitness defects on cells with mutant PIK3CA, mutant TP53 or mutant FBXW7. Few of these targets were identified by both siRNA and CRISPR-Cas9 approaches. Furthermore, isogenic pairs of cell lines have not proved helpful for identifying synthetic lethal targets in the KRAS or PIK3CA genotypes. However, we believe the increased penetrance of the CRIPSR-Cas9 approach has uncovered novel candidate synthetic lethal genes that were not found by RNA interference. Although our current screens are not saturating, we have confirmed several previously reported genetic interactions including the requirements for expression of MDM2 in TP53 wild-type cancers and HK2 in colon cancer lines with KRAS mutations. The screens in the FBXW7 genotype were particularly interesting: in addition to identifying a potential synthetic lethal interaction with a target for which a drug has recently entered the clinic, we also found that FBXW7 itself seemed to be required in many of the colon cancers with FBXW7 mutations. This suggests that FBXW7 may sometimes act as an oncogene, rather than as a tumour suppressor in cancer initiation or progression. Finally, we have attempted to validate some of the candidate synthetic lethal genes identified by RNA interference via ultra-deep CRISPR-CAS9 pooled screening, extending the approach of the Vakoc lab (Nature Biotech. 33, 661-667, 2015). We can confirm this method can highlight functional domains of particular significance to a protein's function, but note that many-fold drop outs of sgRNAs can also occur where they target multiple sites in the genome, presumably via a DNA damage response. Citation Format: Jonathan D. Moore, Chantelle Hudson, Paul Russell, Gaganpreet Tiwana, David Walter, Ceri M. Wiggins, Joanne Yarker. Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A25.

Abstract 2807: Developing a variety of primary mouse tumor allograft models for evaluating efficacy of PARP inhibitor

Abstract Recent success of multiple poly (adenosine diphosphate [ADP]) ribose polymerase inhibitors (PARPi) in treating BRCA mutant ovarian cancers has fueled the development of PARPi in several areas, including expanding its use in other BRCA mutant or wildtype cancers; exploring resistant mechanisms and overcoming resistance; seeking for its combination with chemotherapy, radiotherapy and emerging immunotherapy to achieve persistent efficacy. Especially considering the mechanism of action of PARPi, its use can increase the mutational loads in tumors with defects in homologous recombination and DNA double strand break repair, which potentially would enhance the immunogenicity of the tumors. Therefore it would be very interesting to test the effectiveness of combining PARPi with anti-PD1/PD-L1 or anti-CTLA4. However, relevant animal models are lacking for such studies.Syngeneic mouse tumors offer about 30 viable in vivo models for proof of concept studies. However, these models suffer from several limitations: 1) only a few available and responsive to the current checkpoint inhibitors – for given strains of mouse, there are only very few choices of cells/disease types; 2) not mimicking patient tumors since they are in vitro immortalized cell lines; 3) not reflective of patient disease pathways thus unfit for common target agents, either in mono- or combination therapy. We have established allografts of spontaneous mouse tumors (MuPrime®) derived from genetically engineered mouse models (GEMMs) as a new type of immuno-oncology models with following advantages: 1) its primary nature of “stem cell diseases” and relevant tumor microenvironment; 2) its diverse cancer types/strains of mice; 3) engineered oncogenic drivers as seen in human diseases, deriving from a wide range of available GEMMs, suitable for targeted agents. Thus far, we have built a small library of allografts and are testing them to facilitate pharmacological investigation, particularly those of immuno-oncology.We have found a few models, including an APCmin/+ skin squamous cell carcinoma and a P53 null sarcoma responded well to PARPi Niraparib. Continuous treatment with Niraparib led to development of partial or complete resistance. We are now profiling the resistant models to find out their general mutation loads, as well as specific biomarkers render resistant mechanisms. We are also treating the tumors with mouse PD1 antibody to test whether presumably enhanced mutation loads would sensitize these tumors to immunotherapy. The data of these studies will be presented at the meeting. Citation Format: Gavin Jiagui Qu, Davy Xuesong Ouyang, Li Chen, Likun Zhang, Annie Xiaoyu An, Jie Cai, Henry Q. X. Li. Developing a variety of primary mouse tumor allograft models for evaluating efficacy of PARP inhibitor [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 2807. doi:10.1158/1538-7445.AM2017-2807

Ultrasonographic prediction of highly aggressive telomerase reverse transcriptase (TERT) promoter-mutated papillary thyroid cancer.

Telomerase reverse transcriptase promoter mutations are found in highly aggressive thyroid malignancies. Our aim was to define the ultrasonographic features of telomerase reverse transcriptase promoter-mutated papillary thyroid cancer and to evaluate their predictive performances. Ultrasonographic findings were reviewed for 185 patients with surgically confirmed papillary thyroid cancer between 1994 and 2004. Genomic DNA to identify telomerase reverse transcriptase promoter mutations was extracted from archived surgical specimens. Logistic regression analysis was performed to compare clinical factors and ultrasonographic findings between telomerase reverse transcriptase promoter-mutated and wild-type papillary thyroid cancers. A telomerase reverse transcriptase promoter mutation was detected in 8.1% (15 of 185) of specimens from papillary thyroid cancer patients with a strong trend toward increasing age. Nonparallel orientation and microlobulated margin were independent ultrasonographic findings for predicting telomerase reverse transcriptase promoter-mutated papillary thyroid cancer in patients over 50 years (odds ratio 5.898, 95% confidence interval 1.092-31.851, P = 0.039 for orientation; odds ratio 5.813, 95% confidence interval 1.320-25.602, P = 0.020 for margin). Prevalence of telomerase reverse transcriptase promoter mutations increased to 50.0% in papillary thyroid cancer patients older than 50 years with both ultrasonographic findings and was 0% in patients without either finding. For identifying telomerase reverse transcriptase promoter-mutated papillary thyroid cancer, ultrasonographic had 64.3% sensitivity, 80.8% specificity, 50.0% positive predictive value and 88.4% negative predictive value. Telomerase reverse transcriptase promoter-mutated papillary thyroid cancer could be suggested by the ultrasonographic features of nonparallel orientation and microlobulated margin in patients older than 50 years. Prebiopsy recognition of this unique ultrasonographic pattern would mandate the nodule fall into a "biopsy suggested" category.

Exon 7 splicing variant of estrogen receptor α is associated with pathological invasiveness in smoking-independent lung adenocarcinoma.

Patients with smoking-independent lung cancer mainly consist of females, yet the molecular background of this epidemiological feature, other than epidermal growth factor receptor (EGFR) mutation, remains unclear. Several studies have revealed the association between female hormone-associated factors and the prognosis of lung cancer, however the data remain inconsistent. The present study focused on the expression of estrogen receptor (ER)α in order to elucidate this association in smoking-independent lung cancer. Immunohistochemistry staining (IHC) of aromatase, ERα and ERβ was performed against formalin-treated tissues from 38 patients who had never-smoked who underwent complete surgical resection between 2012 and 2013. Among them, adequate RNA of the tumor and adjacent normal lung cancer was extracted from 31 matching deep frozen samples. Considering the IHC results, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to measure the expression level of 2 different exons of ERα, exon 6 and exon 7, which are part of the ligand binding domain of ERα, using the Taqman gene expression assay. Extra-nuclear expression of ERα using IHC demonstrated a statistically significant association with pathological invasiveness. RT-qPCR results exhibited a decreased expression of ERα exon 7 in invasive tumor tissues, compared with their adjacent normal tissues. This is consistent with the findings of previous in vitro studies indicating that extra-nuclear ERα were exon 7 splicing variants. No difference was observed in ERα exon 7 expression between normal and tumor tissues in non-invasive lung cancer tissues. When considering the EGFR mutation status, EGFR wild-type lung cancers exhibited decreased ERα exon 7 expression levels compared with EGFR mutated lung cancers. Extra-nuclear expression of ERα, which may represent exon 7 splicing variants of ERα, showed statistical association with pathological invasiveness in smoking-independent lung cancer. The post-translational splicing mechanism of ERα may be involved in the acquired invasiveness of smoking independent lung cancer.

Altered Treg and IL-1A Expression in the Immune Microenvironment of Lung Squamous-cell Cancer after EGFR Blockade

背景与目的精确靶向表皮生长因子受体（epidermal growth factor receptor, EGFR）的治疗在肺鳞癌、口腔和肠胃癌中取得了一定疗效，但会引发系统性炎症。本实验旨在探究EGFR抑制剂治疗癌引发的肿瘤内部免疫变化。方法我们通过含H-ras基因逆转录病毒转染EGFR基因缺失或野生型小鼠角质细胞，将改造的细胞同源移植至小鼠以成瘤，吉非替尼治疗荷瘤小鼠，流式细胞仪检测T细胞比例与程序性死亡受体1（programmed death 1, PD-1）表达，RT-PCR检测细胞因子与趋化因子的表达。结果敲除EGFR基因形成的肿瘤较野生型小，并且肿瘤微环境中浸润FoxP3+调节性T细胞（regulatory cells, Treg）细胞较少，FoxP3 RNA较少，程序性死亡受体1（programmed death 1, PD-1）阳性CD4+细胞比例降低。表明肿瘤细胞可以自主调节肿瘤微环境。野生型成瘤模型使用吉非替尼治疗1周显示，相对于对照组肿瘤较小；在这短期的药理模型中，同样观察到FoxP3+细胞、FoxP3 RNA减少的趋势，同时IL-1A/IL-1RA比例明显升高，表明相对短暂的系统性抑制EGFR信号通路可改变靶向肿瘤的免疫微环境。结论肿瘤细胞自发（基因）或系统性（药理作用）的抑制EGFR信号通路可减少肿瘤的生长和肿瘤微环境中Treg的渗透。EGFR依赖性Treg细胞增强肺鳞癌的生长，是EGFR抑制剂治疗的靶标。

PARP inhibitors in ovarian cancer: evidence, experience and clinical potential.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are considered one of the most active and exciting new therapies for the treatment of ovarian cancer. The anticancer activity of PARP inhibitors is based on the DNA repair vulnerability of many ovarian cancer cells, and multiple mechanisms of action of PARP inhibitors have been identified. As single agents, PARP inhibitors have demonstrated their greatest activity in ovarian cancer cells that harbor mutations in BRCA genes. Additionally, recent phase III studies have shown that single-agent PARP inhibitor activity extends beyond BRCA-related cancers and can benefit patients with ovarian cancers that do not have known BRCA mutations, especially when clinical characteristics such as platinum sensitivity and high-grade serous histology are present. PARP inhibitors have also been combined with chemotherapy, however, overlapping myelosuppression observed with PARP inhibitor and chemotherapy combinations has hampered development of these combinations. Contrariwise, PARP inhibitor and biologic agent combinations, specifically antiangiogenic agents, appear well tolerated and show promising activity in both BRCA mutated (BRCAm) and BRCA wild-type (BRCAwt) cancers. Currently, multiple clinical trials are underway examining the antitumor activity of PARP inhibitor combination therapy.

Abstract PR04: Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia

Abstract The WNT and MAPK signaling pathways are central to colorectal homeostasis. Mutation of the APC tumor suppressor gene activates the WNT pathway to initiate most conventional adenomas. By contrast, oncogenic BRAF mutation initiates development of sessile serrated adenomas (SSA) via activation of the MAPK pathway. The purpose of this study was to investigate the frequency, timing and mechanism of WNT pathway activation in serrated colorectal neoplasia. Methods: The WNT pathway was examined in a range of colorectal polyp and cancer subtypes. Aberrant nuclear immuno-localization of β-catenin was used as a surrogate for WNT pathway activation, compared to the normal membranous staining pattern. The entire coding region of the APC gene was sequenced using a custom enrichment panel that included 242 primer pairs (GeneRead DNAseq Targeted Panel, Qiagen) and the constructed library was sequenced using Illumina's HT TruSeq Kit. As another mechanism of activating WNT, mutations were also assessed in the E3 ubiquitin ligase genes RNF43 and ZNRF3 using Sanger sequencing. To test the interaction of the WNT and MAPK pathways in vivo, a conditional BRAF mutant mouse model was crossed with an inducible Cre driven by the murine Villin gene promoter, to restrict Cre expression and induction of the BRAF mutation to the intestine at 2 weeks. The chemical carcinogen azoxymethane was administered weekly for 6 weeks to activate the WNT pathway from 3 weeks of age. The potential therapeutic implication of targeting these pathways was tested in vitro using the porcupine inhibitor LGK974 and MEK inhibitor PD0325901. Results: The majority of conventional pathway polyps and cancers had nuclear localization of β-catenin (22/28, 79% adenomas and 36/42, 86% BRAF wildtype cancers) and mutations in APC (17/20, 85% adenomas and 19/30, 63% BRAF wild type cancers). Sessile serrated adenomas infrequently showed nuclear staining of β-catenin and 0/20 SSA had mutations. By contrast, the WNT signal was increased in the dysplastic component of SSA with a focus of dysplasia (76/137, 55%), however APC mutations were uncommon (3/20, 15%). Similarly, BRAF mutant cancers had nuclear β-catenin in 36/92 (39%) cancers compared to APC mutation in only 9/80 (11%) samples. This did not significantly differ when samples were stratified by microsatellite instability status. RNF43 and ZNRF3 were commonly mutated in BRAF mutant / microsatellite unstable cancers (47/54, 87% and 16/54, 30%, respectively) as well as BRAF mutant / microsatellite stable cancers (8/33, 24% and 5/33, 15%) compared to only 3/79 (4%) RNF43 mutations and 0/27 ZNRF3 mutations in BRAF wildtype cancers. This was accompanied by lower transcript expression for both RNF43 and ZNRF3 in BRAF mutant compared to BRAF wildtype cancers (P&amp;lt;0.0001). We further demonstrated the importance of the WNT pathway in progression of serrated neoplasia by administering azoxymethane to mice with BRAF mutant-induced intestinal hyperplasia. This accelerated the serrated phenotype from 0.6 polyps per mouse to 5.9 per mouse (P&amp;lt;0.001). These all showed typical murine serrated adenoma morphology. To investigate potential therapeutic implications we treated colorectal cancer cell lines with the porcupine inhibitor LGK974 and showed a 50% reduction in growth only in RNF43 and/or ZNRF3 mutant cell lines. In the BRAF / RNF43 / ZNRF3 mutant cell line RKO, LGK974 synergized with MEK inhibition to dramatically reduce growth by approximately 95%. Conclusions: The WNT pathway is commonly activated in serrated neoplasia at the transition to dysplasia. This is not due APC mutation but rather mutation of the upstream RNF43 and/or ZNRF43 genes or other WNT pathway targets. The combination of WNT and MAPK pathway inhibition offers potential for improving therapy for patients with serrated pathway cancers. This abstract is also being presented as Poster A21. Citation Format: Vicki Whitehall, Jennifer Borowsky, Catherine Bond, Mark Bettington, Sally-Ann Pearson, Murugan Kalimutho, John Liu, Troy Dumenil, Diane McKeone, Saara Jamieson, Winnie Fernando, Lochlan Fennell, Andrew Clouston, Christophe Rosty, Ian Brown, Neal Walker, Barbara Leggett. Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR04.

Mutations in TP53, PIK3CA, PTEN and other genes in EGFR mutated lung cancers: Correlation with clinical outcomes

IntroductionThe degree and duration of response to epidermal growth factor receptor (EGFR) inhibitors in EGFR mutated lung cancer are heterogeneous. We hypothesized that the concurrent genomic landscape of these tumors, which is currently unknown in view of the prevailing single gene assay diagnostic paradigm in clinical practice, could play a role in clinical outcomes and/or mechanisms of resistance. MethodsWe retrospectively probed our institutional lung cancer database for tumors with EGFR kinase domain mutations that were also evaluated by more comprehensive molecular profiling, and evaluated tumor response to EGFR tyrosine kinase inhibitors (TKIs). ResultsOut of 171 EGFR mutated tumor-patient cases, 20 were sequenced using at least a limited comprehensive genomic profiling platform. 50% harbored concurrent TP53 mutation, 10% PIK3CA mutation, 5% PTEN mutation, among others. The response rate to EGFR TKIs, the median progression-free survival (PFS) to TKIs, the percentage of EGFR-T790M TKI resistance and survival had higher trends in EGFR mutant/TP53 wild-type cases when compared to EGFR mutant/TP53 mutant tumors (all p >0.05 without statistical significance); with a significantly longer median PFS in EGFR-exon 19 deletion mutant/TP53 wild-type cancers treated with 1st generation EGFR TKIs (p=0.035). ConclusionsConcurrent mutations, specifically TP53, are common in EGFR mutated lung cancer and may alter clinical outcomes. Additional cohorts will be needed to determine if comprehensive molecular profiling adds clinically relevant information to single gene assay identification in oncogene-driven lung cancers.