Catherine H. Wilson

Myc Cooperates with Ras by Programming Inflammation and Immune Suppression

Summary The two oncogenes KRas and Myc cooperate to drive tumorigenesis, but the mechanism underlying this remains unclear. In a mouse lung model of KRasG12D-driven adenomas, we find that co-activation of Myc drives the immediate transition to highly proliferative and invasive adenocarcinomas marked by highly inflammatory, angiogenic, and immune-suppressed stroma. We identify epithelial-derived signaling molecules CCL9 and IL-23 as the principal instructing signals for stromal reprogramming. CCL9 mediates recruitment of macrophages, angiogenesis, and PD-L1-dependent expulsion of T and B cells. IL-23 orchestrates exclusion of adaptive T and B cells and innate immune NK cells. Co-blockade of both CCL9 and IL-23 abrogates Myc-induced tumor progression. Subsequent deactivation of Myc in established adenocarcinomas triggers immediate reversal of all stromal changes and tumor regression, which are independent of CD4+CD8+ T cells but substantially dependent on returning NK cells. We show that Myc extensively programs an immune suppressive stroma that is obligatory for tumor progression.

Myc Expression Drives Aberrant Lipid Metabolism in Lung Cancer

MYC-mediated pathogenesis in lung cancer continues to attract interest for new therapeutic strategies. In this study, we describe a transgenic mouse model of KRAS-driven lung adenocarcinoma that affords reversible activation of MYC, used here as a tool for lipidomic profiling of MYC-dependent lung tumors formed in this model. Advanced mass spectrometric imaging and surface analysis techniques were used to characterize the spatial and temporal changes in lipid composition in lung tissue. We found that normal lung tissue was characterized predominantly by saturated phosphatidylcholines and phosphatidylglycerols, which are major lipid components of pulmonary surfactant. In contrast, tumor tissues displayed an increase in phosphatidylinositols and arachidonate-containing phospholipids that can serve as signaling precursors. Deactivating MYC resulted in a rapid and dramatic decrease in arachidonic acid and its eicosanoid metabolites. In tumors with high levels of MYC, we found an increase in cytosolic phospholipase A2 (cPLA2) activity with a preferential release of membrane-bound arachidonic acid, stimulating the lipoxygenase (LOX) and COX pathways also amplified by MYC at the level of gene expression. Deactivating MYC lowered cPLA2 activity along with COX2 and 5-LOX mRNA levels. Notably, inhibiting the COX/5-LOX pathways in vivo reduced tumor burden in a manner associated with reduced cell proliferation. Taken together, our results show how MYC drives the production of specific eicosanoids critical for lung cancer cell survival and proliferation, with possible implications for the use of COX and LOX pathway inhibitors for lung cancer therapy. Cancer Res; 76(16); 4608-18. ©2016 AACR.

The kinetics of ER fusion protein activation in vivo

Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified oestrogen receptor (ERTAM) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ERTAM fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterized ERTAM fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long-term activation of ERTAM fusion proteins in vivo.

Determination of the physiological and pathological roles of E2F3 in adult tissues

While genetically engineered mice have made an enormous contribution towards the elucidation of human disease, it has hitherto not been possible to tune up or down the level of expression of any endogenous gene. Here we describe compound genetically modified mice in which expression of the endogenous E2f3 gene may be either reversibly elevated or repressed in adult animals by oral administration of tetracycline. This technology is, in principle, applicable to any endogenous gene, allowing direct determination of both elevated and reduced gene expression in physiological and pathological processes. Applying this switchable technology to the key cell cycle transcription factor E2F3, we demonstrate that elevated levels of E2F3 drive ectopic proliferation in multiple tissues. By contrast, E2F3 repression has minimal impact on tissue proliferation or homeostasis in the majority of contexts due to redundancy of adult function with E2F1 and E2F2. In the absence of E2F1 and E2F2, however, repression of E2F3 elicits profound reduction of proliferation in the hematopoietic compartments that is rapidly lethal in adult animals.

Abstract 535: Deregulated Myc is an immunosuppressive switch

Early tumor evolution through sustained oncogene activity selectively bypasses the engagement of cell-intrinsic tumor-suppressor signaling and cell-extrinsic microenvironmental restrictions. How the immune system may be involved is virtually unknown. We describe here the contributions of a conditional and reversible low-level expression of Myc in a mouse model of KrasG12D-driven non-small cell lung cancer. Deregulated Myc activity results in highly expansive tumors that appear embedded in inflamed regions and leads to a rapid reduction of mouse survival. Myc activation imposes an immediate switch to an immunosuppressive and pro-angiogenic microenvironment, facilitated through IL23- and CCL9-associated recruitment of PD-L1 loaded macrophages and local exclusion of T-lymphocytes. Reversibly, blocking the activity of Myc-driven IL23 and CCL9 expression or withdrawal of deregulated Myc activity in tumors established by oncogene cooperation results in tumor cell death and regression, associated with a collapse of the established microenvironmental changes and re-engagement of cytotoxic T-cells. During oncogene cooperation with Ras, deregulated Myc directs a sufficient and necessary switch to a microenvironment that shields tumor growth and expansion from immune suppression. Citation Format: Roderik M. Kortlever, Nicole M. Sodir, Catherine H. Wilson, Deborah L. Burkhart, Lamorna Swigart, Trevor D. Littlewood, Gerard I. Evan. Deregulated Myc is an immunosuppressive switch. [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 535.

Abstract B01: An allelic series of deregulated c-Myc expression interrogates the oncogenicity of distinct Myc levels in lung adenocarcinoma

A plethora of transgenic mouse models have found that c-Myc induces tumors inefficiently and requires cooperating mutations. Acute activation of high levels of Myc induces cellular proliferation, tempered by intrinsic tumor suppression via apoptosis. We hypothesized that low, near physiological, levels of deregulated Myc that fail to invoke tumor suppression pathways, would more effectively initiate sporadic tumors. We previously described the Rosa26lslMycER (R26MER) allele in which deregulated c-Myc is expressed at an endogenous-like level following expression of Cre-recombinase. This c-Myc is fused to a modified estrogen receptor, such that Myc function is dependent upon administration of tamoxifen. We have now generated a Rosa26CAGlslMycER (R26(C)MER) allele, in which the addition of the CAG enhancer has increased the amount of MycER driven by this locus 10-fold. Genetic combination of these alleles generates for the first time mice with tightly controlled and defined levels of switchable Myc protein. Lung cancer is the leading cause of cancer death worldwide and is commonly associated with elevated Myc levels. To determine the role of deregulated Myc expression in lung adenocarcinoma, intranasally administered Adenoviral Cre-recombinase was used to induce sporadic c-MycER expression at different levels in lung epithelial cells, and tamoxifen diet was used to activate c-MycER function. Whereas acute low levels of Myc induced proliferation, high levels of Myc induced both proliferation and apoptosis. Contrary to our hypothesis that Myc-induced apoptosis would delay tumorigenesis, mice with high Myc expression exhibited shorter tumor latency and decreased survival compared to mice with lower levels of expression. The observation that acute Myc activation stimulates p53 activity suggests that the Arf-p53 pathway would be selected against during tumorigenesis. We observed that the hyperplastic clusters of cells observed following Myc activation in R26(C)MER mice were enhanced in mice lacking functional Arf protein. However at later time points their number gradually decreased and the clusters remained small, suggesting that further mutations are necessary for tumorigenesis. Of the tumors that develop following long-term Myc activation, 40% carried activating mutations in Kras. We therefore crossed our R26(C)MER mice to the KrasLSL-G12D allele to investigate the mechanism of this oncogene cooperation. Endogenous levels of oncogenic Kras cooperated dramatically with high levels of Myc, inducing rapid growth of lesions in spite of apparent activation of the p53 tumor suppressor pathway and frequent apoptosis. We then hypothesized that Kras provided increased survival via the Akt pathway to counter Myc-induced apoptosis, as has been previously suggested. Indeed, we found that inhibition of PI3K through treatment with PF-04691502 increased the amount of apoptosis in R26(C)MER/(C)MER;KrasLSL-G12D/+ mice. Importantly, a more dramatic effect was observed when the same lesions were treated with a MEK inhibitor, PD 0325901. Despite having no effect on apoptosis, treatment with PD 0325901 significantly reduced proliferation in these tumors, clearing early lesions within a week of treatment, presumably by allowing Myc-induced apoptosis to overcome the residual proliferation. Consistent with this notion, MEK inhibition of human lung cancer cell lines is more effective at inducing apoptosis in the presence of higher levels of Myc protein. These data suggest that MEK inhibition may have a potent therapeutic role in lung adenocarcinomas that express high levels of Myc. Citation Format: Deborah L. Burkhart, Catherine H. Wilson, Megan Bywater, Roderik Kortlever, Jinyang Li, Mark J. Arends, Trevor D. Littlewood, Gerard I. Evan. An allelic series of deregulated c-Myc expression interrogates the oncogenicity of distinct Myc levels in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B01.

Abstract PR02: Myc-Ras cooperation can overwhelm tumor suppressive mechanisms within lung adenocarcinomas

The ability of Myc and Ras to cooperate during tumorigenesis was first observed over thirty years ago. Numerous mechanisms have been implicated in their collaboration. For instance, Myc can act to circumvent Ras-induced growth arrest. Alternatively, Ras has been shown to prevent Myc-induced apoptosis by activating the anti-apoptotic kinase Akt. Early experiments in primary rodent cells demonstrated that neither Ras nor Myc alone was sufficient for transformation, whereas when expressed together, transformation occurred. However, these transformation events were rare, suggesting additional co-operating mutations were also required. These and other investigations ultimately led to the concept of ‘intrinsic tumor suppression’, whereby the effects of activation of an individual oncogene is limited by their dual potential to invoke cell cycle arrest or apoptosis, which limit their other oncogenic functions. The dogma suggests that cancers only arise in oncogene expressing cells when anti-apoptotic/cell arrest mutations are acquired and the disruption of these pathways leads to net proliferation. There is increasing evidence that the activation of intrinsic tumor suppressive mechanisms depend on increased oncogenic flux resulting from changes in the level of oncogene expression. For example, high expression of oncogenic Ras results in premature growth arrest in MEFs, whereas an endogenous level does not. To address this phenomenon with regards to Myc, we previously generated a novel knock-in mouse ( R26 c-MycER ) in which the c-MycER TAM protein was inserted into the relatively ubiquitous, albeit low expressing Rosa26 locus. In contrast to high levels of Myc expressed in classical transgenic models, low-level c-Myc expression did not trigger intrinsic tumor suppression mechanisms in most tissues, suggesting that low levels of oncogenic Myc may be a more efficient initiator of oncogenesis than over expressed Myc, as it might not require cooperating loss of tumor suppressive pathways. We have now created a R26 CAG-c-MycER allele that expresses a 5-10 fold higher constitutive expression of MycER TAM than the R26 c-MycER allele. Together with R26 c-MycER mice we have been able to generate an allelic series of mice that allow the control, in a spatial and temporal fashion, the activity of de-regulated c-Myc at low, medium and high levels. When we sporadically activated c-MycER TAM within the lung epithelial cells, only very few high-level expressing c-MycER TAM and none of the low-level mice developed tumors within 18 weeks suggesting Myc alone is insufficient for tumorigenesis irrespective of oncogenic level. However, in the presence of an endogenous level of oncogenic K-Ras G12D , lung adenomas are evident in mice expressing both low and high levels of c-Myc. Mice expressing the highest levels of c-Myc developed adenocarcinomas with extremely short latency and have a greatly reduced survival when compared to mice with lower levels of c-Myc. As expected, low level c-Myc did not activate intrinsic tumor suppression via the Arf/p53 pathways within K-Ras driven lesions and these tumors grew faster than lesions with oncogenic K-Ras alone. However, tumors expressing high levels of c-Myc did engage a number of key tumor suppressors and apoptosis increased as a function of c-Myc level, despite the continued growth and extremely short latency of the adenomas/adenocarcinomas in these mice. Thus, when combined with oncogenic K-Ras, high c-Myc cooperation results in rapid tumorigenesis whereby tumors are still able to grow prolifically despite the apparent activation of the surveillance mechanisms. Further analysis is currently underway to determine the specific survival advantage oncogenic K-Ras offers within this system. This abstract is also presented as Poster B20. Citation Format: Catherine H. Wilson, Deborah L. Burkhart, Jinyang Li, Trevor D. Littlewood, Gerard I. Evan. Myc-Ras cooperation can overwhelm tumor suppressive mechanisms within lung adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR02. doi: 10.1158/1557-3125.RASONC14-PR02

Abstract 2608: Dissecting Myc inhibition as a cancer therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The MYC family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors are critical for cell growth, proliferation and apoptosis. c-Myc is deregulated in most, if not all, cancers making it an attractive target for cancer therapy. Previously we have shown that Myc inhibition by a mutant c-Myc derivative, Omomyc, leads to tumor regression in several cancer models, notably in murine KRasG12D-driven lung adenocarcinomas. The mechanism by which Omomyc expression leads to tumor regression has not been fully elucidated, nor do we know the biochemical properties of a small molecule that could replicate Omomycs activity. Therefore, we designed two novel Omomyc mutants (ΔMyc and MMO) that are hypothesised not to interact with the full Omomyc interactome but are still able to inhibit Myc transactivation. We subsequently created human lung adenocarcinoma cell lines (A549s) and transgenic mice which inducibly express Omomyc, ΔMyc and MMO. Unexpectedly Omomyc and ΔMyc inhibited proliferation in vitro but not MMO. In vivo, expression of the mutants has been detected and we are currently assessing their effect on KRasG12D-driven adenocarcinomas. Citation Format: Nicholas J. H. Salisbury, Catherine H. Wilson, Dan Lu, Trevor D. Littlewood, Gerard I. Evan. Dissecting Myc inhibition as a cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2608. doi:10.1158/1538-7445.AM2014-2608