Loss Of Tumor Suppressor Research Articles

Loss of RasGAP Tumor Suppressors Underlies the Aggressive Nature of Luminal B Breast Cancers.

The lack of insight into mechanisms that underlie the aggressive behavior of luminal B breast cancers impairs treatment decisions and therapeutic advances. Here, we show that two RasGAP tumor suppressors are concomitantly suppressed in aggressive luminal B tumors and demonstrate that they drive metastasis by activating RAS and NF-κB. Cancer Discov; 7(2); 202-17. ©2016 AACR.See related commentary by Sears and Gray, p. 131This article is highlighted in the In This Issue feature, p. 115.

Merlin controls the repair capacity of Schwann cells after injury by regulating Hippo/YAP activity.

Loss of the Merlin tumor suppressor and activation of the Hippo signaling pathway play major roles in the control of cell proliferation and tumorigenesis. We have identified completely novel roles for Merlin and the Hippo pathway effector Yes-associated protein (YAP) in the control of Schwann cell (SC) plasticity and peripheral nerve repair after injury. Injury to the peripheral nervous system (PNS) causes a dramatic shift in SC molecular phenotype and the generation of repair-competent SCs, which direct functional repair. We find that loss of Merlin in these cells causes a catastrophic failure of axonal regeneration and remyelination in the PNS. This effect is mediated by activation of YAP expression in Merlin-null SCs, and loss of YAP restores axonal regrowth and functional repair. This work identifies new mechanisms that control the regenerative potential of SCs and gives new insight into understanding the correct control of functional nerve repair in the PNS.

Targeting HIF-2 α in clear cell renal cell carcinoma: A promising therapeutic strategy.

The loss of the Von Hippel-Lindau tumor suppressor (VHL) is a key oncogenic event in the vast majority of patients with clear cell renal cell carcinoma (ccRCC). With the loss of the VHL protein (pVHL) function, the hypoxia inducible factor α (HIF-α) accumulates inside the tumor cell and dimerizes with HIF-β. The HIF-α/HIF-β complex transcriptionally activates hundreds of genes promoting the adaptation to hypoxia that is implicated in tumor development. There is growing evidence showing that HIF-2α subunit has a central role in ccRCC over HIF-1α. Thus, efforts have been made to specifically target this pathway. PT2385 and PT2399 are first-in-class, orally available, small molecule inhibitors of HIF-2 that selectively disrupt the heterodimerization of HIF-2α with HIF-1β. Preclinical and clinical data indicate that these new molecules are effective in blocking cancer cell growth, proliferation, and tumor angiogenesis characteristic in ccRCC. Treatment with HIF-2α specific antagonists, either alone or in combination with immunotherapy or other antiangiogenic agents have the potential to transform the therapeutic landscape in this tumor in the future. Herein, we summarize the molecular background behind the use of HIF-2α inhibitors in ccRCC and give an overview of the development of new agents in this setting.

Abstract IA09: Extrinsic and intrinsic force regulate cancer progression and aggression

Abstract Cells experience force and possess mechanotransduction machinery to detect physical cues from their microenvironment and to transduce and biochemically amplify these signals to modulate their differentiation, growth, survival, migration and morphogenesis. Tumors show increased tissue level forces and transformed cells exhibit a perturbed mechanophenotype. We have been studying how cells transduce mechanical cues to regulate their fate and how altered force compromises tissue homeostasis to drive malignancy and metastasis. We found that the ECM in breast, pancreas, skin and glioblastomas is remodeled during malignancy such that the ECM stiffens as a function of tumor progression and aggression. A stiffened ECM compromises tissue differentiation by promoting integrin focal adhesion (FA) assembly that potentiates transmembrane receptor signaling and induces cytoskeletal remodeling and actomyosin contractility. Our findings indicate that high mechanosignaling collaborates with oncogenes including Ras and ErbB2 and reduces the levels of key tumor suppressors including PTEN and BRCA1 to drive transformation. By this means an increase in ECM stiffness promotes the malignant transformation of pre malignant cells and drives tumor cell metastasis, and inhibiting ECM stiffening restores tumor suppressor function and reduces oncogenic signaling to prevent malignancy. We determined that these effects are linked to the cells ability to “tune” their actomyosin tension to the stiffness of their surrounding microenvironment which induces focal adhesion assembly through a vicious feed-forward mechanism. The elevated cell contractility and increase in focal adhesions modifies cell signaling through RhoGTPases modify ERK, PI3 kinase, Myc and Wnt signaling to amplify proliferation and survival, and invasion, to increase the level of factors such as VEGF that drives angiogenesis, to stimulate cytokine and chemokine expression that fosters inflammation and to induce hypoxic reprogramming to drive tumor metastasis and compromise therapeutic efficacy. Interestingly, we showed that oncogenic transformation and the loss of key tumor suppressors per se also elevate cell intrinsic tension and drive tumor progression and aggression by inducing ECM remodeling and stiffening to elevate mechanosignaling. Indeed, we could show that the cancer cells genotype “tunes” the stromal response of the tissue and influences tumor aggression in part by modifying tissue tension. We will present evidence to support a role for both intrinsic and extrinsic mechanical force in solid tumors and discuss how these findings could provide insight to guide and improve cancer therapy (Supported by DOD BCRP BC122990 to VMW, NIH NCI R01CA192914 to VMW, 5 R01 CA085492-11A1 to VMW and HM, 1U54CA163155-01 to VMW and GB, R01CA174929 to VMW and CP, R33 CA183685-01 to VMW and KH and 1U01CA202241-01 to VMW and JG.) Citation Format: Valerie Weaver. Extrinsic and intrinsic force regulate cancer progression and aggression. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr IA09.

SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer.

Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. We use in vitro and in vivo human prostate cancer models to show that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR)-dependent luminal epithelial cells to AR-independent basal-like cells. This lineage plasticity is enabled by the loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2, and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching.

Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance.

Prostate cancer relapsing from antiandrogen therapies can exhibit variant histology with altered lineage marker expression, suggesting that lineage plasticity facilitates therapeutic resistance. The mechanisms underlying prostate cancer lineage plasticity are incompletely understood. Studying mouse models, we demonstrate that Rb1 loss facilitates lineage plasticity and metastasis of prostate adenocarcinoma initiated by Pten mutation. Additional loss of Trp53 causes resistance to antiandrogen therapy. Gene expression profiling indicates that mouse tumors resemble human prostate cancer neuroendocrine variants; both mouse and human tumors exhibit increased expression of epigenetic reprogramming factors such as Ezh2 and Sox2. Clinically relevant Ezh2 inhibitors restore androgen receptor expression and sensitivity to antiandrogen therapy. These findings uncover genetic mutations that enable prostate cancer progression; identify mouse models for studying prostate cancer lineage plasticity; and suggest an epigenetic approach for extending clinical responses to antiandrogen therapy.

Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice

It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing. We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be up-regulated after tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines. We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation. Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

A Novel Somatic Mutation in IKZF1 Is Common in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant for Myelofibrosis

Background:Myeloproliferative neoplasms (MPN) including polycythemia vera, essential thrombocytosis, and primary and secondary myelofibrosis (MF) are characterized by driver mutations in JAK2, CALR, and MPL. Additional somatic variants can be seen. Currently allogeneic hematopoietic stem cell transplant (HCT) is the only treatment with curative potential, but it is associated with a high risk of mortality, exceeding 50% in some patient subsets. The risk of disease progression usually outweighs the risk of transplant in patients with advanced MF. Here, we have evaluated the mutational profile using a targeted next-generation sequencing panel of 54 genes involved in myeloid neoplasms in patients with MF treated with HCT.Patients and Methods:48 patients with a diagnosis of primary (n=18) or secondary (n=30) MF who received a HCT at our center and from whom genomic DNA was available from pre-HCT marrow aspirates were evaluated. DNA was isolated from whole marrow aspirates obtained prior to HCT. Paired-end indexed libraries were sequenced at an average read depth of 1000x on Illumina MiSeq platform. Sequences were aligned to hg19 using the Burrows-Wheeler Aligner, and nucleotide variants were identified using the Genome Analysis Toolkit and Picard tools. Variants were annotated with ANNOVAR. Variants present at >5% variant allele frequency (VAF) with at a minimum of 25 variant reads were selected for further analysis. Variants were called "mutations" if they resulted in (1) non-synonymous changes in amino acid sequence, including nonsense and missense mutations, (2) the variant was known to be associated with hematologic malignancies in COSMIC database, or (3) the variant was present at < 0.5% in 1000 genomes and ExAC databases. Comparisons between clinical variables and mutations were analyzed with chi-square tests, adjusted for multiple testing.Results:The average age of the 48 patients was 55 years (range 23-72) and 52% of patients were female. Disease risk was estimated with DIPSS-Plus (High 33%, INT-2 53%, INT-1 7%, Low 7%). Unfavorable cytogenetics were present in 27% of patients. Donors included a matched sibling donor in 30% (n=14), a matched unrelated donor in 60% (n=29), and an alternative donor in 10% (n=5). At 3 years, overall survival was 52%, 17% of patients had relapsed disease, and 31% had non-relapse mortality.In our cohort, 38% had a mutation in CALR (n=15 type-I and n=3 type-II), 29% in JAK2 (n=14), 0% in MPL, and 33% were triple-negative (n=16; no evidence of CALR, JAK2, or MPL mutation). The average VAF of CALR mutations was 50% (range 20-82%) and JAK2 mutations was 58% (range 25-81%). In addition to driver mutations, 94% of patients had at least one other mutation in 26 of the 54 genes sequenced. On average, patients had 2.1 other mutations (range 0-5 mutations in addition to driver mutation). The most common other mutations were in ASXL1 (31%), IKZF1 (23%), ETV6 (17%), and U2AF1 (17%). The VAF of these other mutations spanned 9-76%.Patients with triple-negative MF were more likely to have primary MF (p=0.019) and platelets <100 (p<0.001), than patients with JAK2 and CALR mutations. Patients with ≥3 other mutations were more likely to have hemoglobin <10 (p=0.02) and a higher rate of relapse (p<0.01) than patients with <3 other mutations. Patients with ASXL1 mutations were more likely to have peripheral blood blasts (p<0.001).Mutations in ASXL1, ETV6, and U2AF1 have been previously reported in myeloid malignancies. Like other groups, we found ASXL1 mutations associated with worse prognosis. Here we report a novel, recurrent mutation in the gene IKZF1. All patients with IKZF1 mutation carried the same insertion of 11 nucleotides leading to an early frameshift and ultimate nonsense mutation (L92fs), which would lead to deletion of the zinc-finger domain. Such a mutation likely leads to loss of IKZF1 tumor suppressor function.Conclusions:Somatic mutations, in addition to driver mutations, are common in patients with MF treated with HCT. Mutation profiling in this patient subset may improve relapse risk prediction. Loss of heterozygosity on chromosome 7 mapping to the IKZF1 locus has been found by others to be associated with leukemic transformation in patients with MPN (Jaeger et al. 2010. Leukemia. 24: 1290). Given their frequency in this high risk MF population, further study of the role of IKZF1 mutations in MPN is warranted. DisclosuresRadich:Novartis: Consultancy, Other: laboratory contract; Bristol-MyersSquibb: Consultancy; TwinStrand: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy.

Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence.

Background:Colorectal cancer (CRC) is the third most common cancer worldwide. Accurately identifying stage II CRC patients at risk for recurrence is an unmet clinical need. KCNQ1 was previously identified as a tumour suppressor gene and loss of expression was associated with poor survival in patients with CRC liver metastases. In this study the prognostic value of KCNQ1 in stage II and stage III colon cancer patients was examined.Methods:KCNQ1 mRNA expression was assessed in 90 stage II colon cancer patients (AMC-AJCCII-90) using microarray gene expression data. Subsequently, KCNQ1 protein expression was evaluated in an independent cohort of 386 stage II and stage III colon cancer patients by immunohistochemistry of tissue microarrays.Results:Low KCNQ1 mRNA expression in stage II microsatellite stable (MSS) colon cancers was associated with poor disease-free survival (DFS) (P=0.025). Loss of KCNQ1 protein expression from epithelial cells was strongly associated with poor DFS in stage II MSS (P<0.0001), stage III MSS (P=0.0001) and stage III microsatellite instable colon cancers (P=0.041). KCNQ1 seemed an independent prognostic value in addition to other high-risk parameters like angio-invasion, nodal stage and microsatellite instability-status.Conclusions:We conclude that KCNQ1 is a promising biomarker for prediction of disease recurrence and may aid stratification of patients with stage II MSS colon cancer for adjuvant chemotherapy.

Chromosomal Instability Estimation Based on Next Generation Sequencing and Single Cell Genome Wide Copy Number Variation Analysis.

Genomic instability is a hallmark of cancer often associated with poor patient outcome and resistance to targeted therapy. Assessment of genomic instability in bulk tumor or biopsy can be complicated due to sample availability, surrounding tissue contamination, or tumor heterogeneity. The Epic Sciences circulating tumor cell (CTC) platform utilizes a non-enrichment based approach for the detection and characterization of rare tumor cells in clinical blood samples. Genomic profiling of individual CTCs could provide a portrait of cancer heterogeneity, identify clonal and sub-clonal drivers, and monitor disease progression. To that end, we developed a single cell Copy Number Variation (CNV) Assay to evaluate genomic instability and CNVs in patient CTCs. For proof of concept, prostate cancer cell lines, LNCaP, PC3 and VCaP, were spiked into healthy donor blood to create mock patient-like samples for downstream single cell genomic analysis. In addition, samples from seven metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. CTCs were enumerated and characterized using the Epic Sciences CTC Platform. Identified single CTCs were recovered, whole genome amplified, and sequenced using an Illumina NextSeq 500. CTCs were then analyzed for genome-wide copy number variations, followed by genomic instability analyses. Large-scale state transitions (LSTs) were measured as surrogates of genomic instability. Genomic instability scores were determined reproducibly for LNCaP, PC3, and VCaP, and were higher than white blood cell (WBC) controls from healthy donors. A wide range of LST scores were observed within and among the seven mCRPC patient samples. On the gene level, loss of the PTEN tumor suppressor was observed in PC3 and 5/7 (71%) patients. Amplification of the androgen receptor (AR) gene was observed in VCaP cells and 5/7 (71%) mCRPC patients. Using an in silico down-sampling approach, we determined that DNA copy number and genomic instability can be detected with as few as 350K sequencing reads. The data shown here demonstrate the feasibility of detecting genomic instabilities at the single cell level using the Epic Sciences CTC Platform. Understanding CTC heterogeneity has great potential for patient stratification prior to treatment with targeted therapies and for monitoring disease evolution during treatment.

Abstract IA21: Mitogenic signaling and the RB/p53 network

Abstract Cells entering the division cycle from a quiescent state (G0) in response to mitogenic cues synthesize one or more D-type cyclins (D1, D2, D3) that assemble in G1 phase with cyclin-dependent kinases CDK4 and/or CDK6. Notably, the transcription, assembly with CDK 4/6, and stability of D-type cyclins are each mitogen-dependent steps. Accumulation of cyclin D-dependent kinases in G1 phase leads to the progressive phosphorylation of the retinoblastoma protein (RB), facilitating transcription of E2F-responsive genes, including cyclins E and A, whose CDK2-dependent functions are normally required in S phase. Moreover, the stoichiometric sequestration of CDK2 inhibitors, p27Kip1 and p21Cip1, by cyclin D-dependent kinases, further enables CDK2 activation as well as SCFSkp2-dependent p27 degradation as cells approach the G1/S boundary. In G0, RB remains unphosphorylated, and E2F-responsive genes are repressed by the DREAM complex, which includes E2F-4/5, at least one retinoblastoma family member (RBL2/p130), and the MuvB complex. An open question is whether CDK4 and CDK6 are responsible, at least in part, for dissolution of the DREAM complex or relief of p130/E2F repression in early G1 phase. Cells in G0 and G1 express the APCCDH1 E3 ubiquitin ligase that prevents accumulation of the mitotic cyclins, A and B, prior to S phase entry. As cells near the G1/S transition, APCCDH1 is inactivated by CDK2 in conjunction with the E2F-responsive early mitotic inhibitor EMI1. Cyclin D-CDK4/6 complexes accumulate maximally in the nucleus at G1/S, where their phosphorylation by GSK-3β triggers their subsequent nuclear export, ubiquitination, and proteasomal degradation in S phase. In continuously cycling cells, Ras signaling in G2 phase restores nuclear cyclin D, enabling its re-accumulation before M phase; cyclin D-CDK4/6 activity in G2/M shortens the subsequent G1 interval and cell cycle length. Mitogen withdrawal, leads to rapid cyclin D turnover and ultimately to G1/G0 arrest. Similar effects can result from oncogene-mediated induction of the specific CDK4/6 inhibitor p16INK4A, or by CDK4/6 inhibitory drugs (CDK4/6i's) that act as chemical p16 mimetics. Notably, continuous CDK4/6 inhibition by CDK4/6i's or p16 can induce reversible (quiescence) or irreversible (senescence) cell cycle exit; understanding the factors that distinguish these outcomes continues to represent a challenge. Although monotherapy with orally available, potent, and specific CDK4/6i's has not generally yielded durable clinical benefits in cancer treatment, these drugs can induce significant progression-free survival with minimal dose limiting side effects (principally clinically manageable neutropenia) when combined with targeted inhibitors of mitogenic signaling pathways that limit the accumulation of D-type cyclins. Perhaps, an “addiction” of cancer cells to mitogenic pathways constitutively activated by oncogenic mutations increases their vulnerability to CDK4/6i's while sparing normal cycling cells. Expression of the CDKN2A/B gene cluster (encoding p16, p15INK4B, and ARF) is driven by an upstream super-enhancer. Canonical super-enhancer histone “marks” (H3K4me1 and H3K27Ac) are detected in cells that are able to express CDKN2A/B in response to hyperproliferative signals but are absent from their reprogrammed iPS derivatives or from ES cells. More than 30 independent GWAS studies have fingered SNPs within the CDKN2A/B super-enhancer region that are associated not only with various cancers but also with common degenerative diseases of aging populations, including coronary artery disease, aortic aneurysm, and type II diabetes. These findings suggest that while downregulation of CDKN2A/B results in a loss of tumor suppression, its increased expression during aging leads to cellular senescence and a loss of tissue regenerative capacity. Citation Format: Charles J. Sherr. Mitogenic signaling and the RB/p53 network. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA21.

A Study of C-MYC, SOX10 and BCL-2 Proteins Expression in Head and Neck Mucosal Melanomas

Background: Head and Neck Mucosal Melanoma (HNMM) is rare, accounting 1% of all melanomas in USA, and 6% in Japan. Melanoma’s development is a complex process involving activation of proto-oncogenes and loss of tumor suppressors. BCL-2 oncogene encodes a family of anti-apoptotic proteins and it is overexpressed in melanomas. On the other hand, proto-oncogene C-MYC is a transcriptional factor and plays crucial role both in driving cell proliferation and promoting apoptosis, its overexpression has been associated to melanoma progression. Moreover, oncogene SOX10 cooperates with other transcription factors to direct the development and differentiation of melanocytes. These three nuclear markers are associated to melanoma’s metastatic risk.Methods: We studied of BCL-2; C-MYC and SOX10 proteins in 19 Formalin Fixed Paraffin Embedded cases of HNMM from Yale School of Medicine and 10 cases from University of São Paulo by Alkalin Phosphatase immunohistochemistry technique.Results: We considered as positive expression when over 25% of tumor cells were immunostained. We observed 27/29 were positive to BCL-2 and 28/29 cases showed SOX10 expression (both markers showing immunostaining in over 75% of tumor cells). However, we noticed variable expression for C-MYC in 15/29 of HNMM cases.Conclusions: According to ours results, we suggest that BCL-2 and SOX10, should be good adjuctive biomarkers for HNMM, however lower expression of C-MYC has not shown to play main role in the HNMM development, thus further molecular biology studies should corroborate this present study.

Costimulatory and coinhibitory immune checkpoint receptors in head and neck cancer: unleashing immune responses through therapeutic combinations.

Head and neck squamous cell carcinoma (HNSCC) represents a model of escape from anti-tumor immunity. The high frequency of p53 tumor suppressor loss in HNSCC leads to genomic instability and immune stimulation through the generation of neoantigens. However, the aggressive nature of HNSCC tumors and significant rates of resistance to conventional therapies highlights the ability of HNSCC to evade this immune response. Advances in understanding the role of co-stimulatory and immune checkpoint receptors in HNSCC-mediated immunosuppression lay the foundation for development of novel therapeutic approaches. This article provides an overview of these co-stimulatory and immune checkpoint pathways, as well as a review of preclinical and clinical evidence supporting the modulation of these pathways in HNSCC. Finally, the synergistic potential of combining these approaches is discussed, along with an update of current clinical trials evaluating combinations of immune-based therapies in HNSCC patients.

Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells

The HVEM (TNFRSF14) receptor gene is among themost frequently mutated genes in germinal center lymphomas. We report that loss of HVEM leads tocell-autonomous activation of B cell proliferationand drives the development of GC lymphomasinvivo. HVEM-deficient lymphoma B cells also induce a tumor-supportive microenvironment marked by exacerbated lymphoid stroma activation and increased recruitment of T follicular helper (TFH) cells. These changes result from the disruption of inhibitory cell-cell interactions between the HVEM and BTLA (B and T lymphocyte attenuator) receptors. Accordingly, administration of the HVEM ectodomain protein (solHVEM(P37-V202)) binds BTLA and restores tumor suppression. To deliver solHVEM to lymphomas invivo, we engineered CD19-targeted chimeric antigen receptor (CAR) Tcells that produce solHVEM locally and continuously. These modified CAR-T cells show enhanced therapeutic activity against xenografted lymphomas. Hence, the HVEM-BTLA axis opposes lymphoma development, and our study illustrates the use of CAR-T cells as "micro-pharmacies" able to deliver an anti-cancer protein.

The role of 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) in metabolic reprogramming of cancer cells

The high rate of glucose breakdown is the fingerprint of cancer. Increased glycolysis allows tumour cells to fulfil their high energetic and biosynthetic demands. Interestingly, however, rather than metabolizing glucose in the oxidative phosphorylation pathway, cancer cells generally use glucose for aerobic glycolysis. This phenomenon is known as the Warburg effect and is considered as one of the most fundamental forms of metabolic reprogramming during cancerogenesis. Changes in the rate of glycolytic activity of cancer cells are caused mainly by the increased expression of glycolytic enzymes as a consequence of activation of oncogenes or loss of tumour suppressors. In addition, the hypoxic tumour environment also triggers upregulation of a series of genes involved in glucose metabolism. Among the metabolic enzymes that are modulated by these factors in cancer cells are the 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatases (PFKFBs), a family of bifunctional enzymes that control the levels of fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. Fru-2,6-P2 strongly activates glucose breakdown in glycolysis through allosteric modulation of the rate-limiting enzyme of glycolysis, phosphofructokinase-1 (PFK-1). Thus far, many studies have reported a correlation between aberrant PFKFB expression level and the grade of tumour aggressiveness, which directly indicates that these enzymes may play a crucial role in cancerogenesis. The objective of this review is to highlight the recent studies on aberrant expression of PFKFBs and its influence on cancer progression.

TLE4 regulation of wnt-mediated inflammation underlies its role as a tumor suppressor in myeloid leukemia

The presence of AML1-ETO (RUNX1-CBF2T1), a fusion oncoprotein resulting from a t(8;21) chromosomal translocation, has been implicated as a necessary but insufficient event in the development of a subset of acute myeloid leukemias (AML). While AML1-ETO prolongs survival and inhibits differentiation of hematopoietic stem cells (HSC), other contributory events are needed for cell proliferation and leukemogenesis. We have postulated that specific tumor suppressor genes keep the leukemic potential of AML1-ETO in check. In studying del(9q), one of the most common concomitant chromosomal abnormalities with t(8;21), we identified the loss of an apparent tumor suppressor, TLE4, that appears to cooperate with AML1-ETO to confer a leukemic phenotype. This study sought to identify the molecular basis of this cooperation. We show that the loss of TLE4 confers proliferative advantage to leukemic cells, simultaneous with an upregulation of a pro- inflammatory signature mediated through aberrant increases in Wnt signaling activity. We further demonstrate that inhibition of cyclooxygenase (COX) activity partly reverses the pro-leukemic phenotype due to TLE4 knockdown, pointing towards a novel therapeutic approach for myeloid leukemia.

Abstract 5268: Seq2C: from sequence to copy number for cancer samples

Abstract Targeted sequencing is used increasingly in clinics to guide therapeutic decisions by measuring mutations, small indels and/or rearrangements in cancer genes. An ability to use the same platform to detect additional oncogene activation (or tumor suppressor loss) through copy number changes could significantly expand the number of patients able to benefit from targeted therapies. Many currently available tools either requires a matched normal, works only for whole genome or exome sequencing, or don't work for PCR-based targeted sequencing. In addition, no tools available to detect breakpoints within a gene from targeted sequencing. A versatile copy number analysis tool from sequencing is needed to maximize the value of the sequencing routinely applied in clinics. Here we presented a novel computational tool, Seq2C (Sequencing To Copy Number), which is versatile to handle various situations and reports aberrations at gene level ready for interpretation. Seq2C works at cohort level and does not require a matched ‘normal’, though it can optionally use one or more normal samples for small (&amp;lt;20) or homogeneous cohorts. Seq2C applies a robust three-step cohort-based normalization to identify and quantify sequencing coverage variability in given regions, which can be sequence or platform dependant. Seq2C works for hybrid or PCR based targeted sequencing, exome and even whole genome sequencing. In targeted sequencing, Seq2C automatically identify and exclude regions that fail to be captured by the assay to prevent false positive calls of deletions, and can accommodate several magnitude differences in PCR efficiency. Another distinct feature differentiating Seq2C from other currently available tools is that Seq2C identifies breakpoints and detects one or more exon deletion or duplication rearrangements within a gene, which is common in tumor suppressors as a mechanism to lose function. In addition, it can also detect potential fusions in genes such as ALK and ERG, where a copy number change is often accompanied with the fusion and a breakpoint can thus be called by Seq2C. Furthermore, it can predict gender from exome or whole genome sequencing. We applied Seq2C to exome sequencing of CCLE cell lines, and showed that it produced gene level copy number data highly correlated to those derived from microarrays, the current gold standard. Interestingly, Seq2C identified one or more exon deletions in several common tumor suppressors, such as TP53, PTEN, CDKN2A, NF1, STK11 and RB1, in cell lines with no known aberrations. They were supported by RNA-Seq data. It also correctly called known ERG fusion in prostate cell line NCI-H660 and identified a previously un-reported EML4-ALK fusion in a pancreatic cancer cell line SNU-324, which is confirmed by RNA-Seq data, suggesting EML4-ALK is not limited to lung cancer. In conclusion, Seq2C is a versatile copy number analysis tool for sequencing and will be useful for cancer research. Seq2C is freely available in GitHub. Citation Format: Zhongwu Lai, Aleksandra Markovets, Jonathan Dry. Seq2C: from sequence to copy number for cancer samples. [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 5268.

Abstract 2023: SOX4 is essential for PTEN-mediated prostate tumorigenesis in vivo

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer mortality in American men, underscoring the significance of unraveling the molecular mechanisms involved in the initiation and progression of the disease. The sex-determining region Y-box 4 (SOX4) gene is overexpressed in many types of human cancers, including prostate cancer, suggesting that SOX4 plays a fundamental role in tumorigenesis. In this study, we demonstrate that SOX4 is critical for PTEN-mediated prostate cancer progression in vivo. We show that homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibits tumor progression initiated by homozygous loss of the Pten tumor suppressor, demonstrating the key role of SOX4 in the development of prostate cancer. Homozygous deletion of Sox4 also reduces the activation of AKT and β-catenin in Pten-null mice, resulting in inhibition of an invasive cancer phenotype. We also show that SOX4 expression is induced by loss of PTEN, and that PI3K-AKT-mTOR signaling activity is critical for SOX4 expression, suggesting a positive feedback loop between SOX4 protein and PI3K-AKT-mTOR activity. Our findings indicate that SOX4 is a critical component of the PTEN-PI3K-AKT pathway in prostate cancer, suggesting that SOX4 may be a promising molecular target for novel combinatorial therapies for both primary and advanced prostate cancers. Citation Format: Birdal Bilir, Adeboye O. Osunkoya, W. Guy Wiles, Soma Sannigrahi, Veronique Lefebvre, Daniel Metzger, Demetri D. Spyropoulos, W. David Martin, Carlos S. Moreno. SOX4 is essential for PTEN-mediated prostate tumorigenesis in vivo. [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 2023.

Abstract 681: Using a genome-editing approach for the stepwise establishment of zebrafish models of pediatric high-grade gliomas and MPNSTs

Abstract By high-throughput sequencing of cancer patient's genomes, driver mutations have recently been described in high-grade pediatric glioma, an aggressive type of brain tumor in children. The most prominent recurrent mutations were found in four genes, namely NF1, TP53, ATRX and H3F3A. NF1 and TP53 are tumor suppressor genes and their loss of function is characteristic for various human malignancies. However, the mechanisms through which precise missense mutations in H3F3A and loss-of-function of ATRX influence tumor biology remain largely unknown. In our project, we employ the genome-editing technology CRISPR-Cas9 to develop a zebrafish model of pediatric high-grade glioma, in order to examine the impact of these mutations on tumor onset and progression. We previously created a zebrafish line that is deficient for tp53 (tp53-/-) and has a loss-of-function mutation in 3 of 4 alleles of nf1 (nf1a+/-, nf1b-/-), which is duplicated in zebrafish. These fish develop high-grade gliomas with low penetrance and malignant peripheral nerve sheath tumors (MPNSTs) with high penetrance. By CRISPR-Cas9 we further incorporated early frameshift mutations of atrx in the background of tp53-/-, nf1a+/-, nf1b-/-. All analyzed fish injected with Cas9-mRNA and CRISPR guide-RNAs (gRNAs) targeting atrx coding sequences showed germline transmission of mutant alleles, of which one third were frameshift mutations. Primary CRISPR-Cas9 atrx-gRNA injected tp53-/-, nf1a+/-, nf1b-/- fertilized eggs developed as mosaics and exhibited increased tumor penetrance and faster onset of MPNSTs and these tumors harbored atrx mutations in 8/10 analyzed samples. This indicates that atrx loss-of-function mutations cooperate with loss of tp53 and nf1 tumor suppressors. Gliomas were not observed in mosaic atrx mutated fish, and these will be reanalyzed in stable atrx mutant lines. Next, we will add previously described missense mutations of h3f3a. To unravel the synergy between oncogenic mutations in atrx and h3f3a in tumor progression, mutant stable lines for both genes will be investigated independently and in a combined manner. The zebrafish model of pediatric high-grade gliomas and MPNSTs that we are developing will be useful to dissect the mechanisms underlying cooperation among driver mutations and for small molecule screens to identify specific inhibitors of cell growth and survival in these malignancies. Citation Format: Felix Oppel, Shuning He, Ting Tao, Mark W. Zimmerman, Adam D. Durbin, Nina Weichert, Dong H. Ki, A Thomas Look. Using a genome-editing approach for the stepwise establishment of zebrafish models of pediatric high-grade gliomas and MPNSTs. [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 681.

Control of apoptosis by Drosophila DCAF12

Regulated Apoptosis (Programmed Cell Death, PCD) maintains tissue homeostasis in adults, and ensures proper growth and morphogenesis of tissues during development of metazoans. Accordingly, defects in cellular processes triggering or executing apoptotic programs have been implicated in a variety of degenerative and neoplastic diseases. Here, we report the identification of DCAF12, an evolutionary conserved member of the WD40-motif repeat family of proteins, as a new regulator of apoptosis in Drosophila. We find that DCAF12 is required for Diap1 cleavage in response to pro-apoptotic signals, and is thus necessary and sufficient for RHG (Reaper, Hid, and Grim)-mediated apoptosis. Loss of DCAF12 perturbs the elimination of supernumerary or proliferation-impaired cells during development, and enhances tumor growth induced by loss of neoplastic tumor suppressors, highlighting the wide requirement for DCAF12 in PCD.