Oncogene Addiction Research Articles

Abstract B30: Inhibition of eIF4E/MNK axis by novel MNKDAs reduces breast cancer tumor growth, migration, invasion and metastasis

Abstract Triple-negative breast cancer (TNBC) represents a molecularly heterogeneous subgrouping of high-risk breast tumors characterized by aggressive behavior, poor prognosis and lack of targeted therapeutic options. The translation initiation factor eIF4E, an oncogene essential for cap-dependent translation of specific mRNAs critical for cell division, cell survival, angiogenesis, and metastasis is commonly overexpressed in primary breast cancers including TNBC. eIF4E therapeutic targeting is considered novel and significant because tumor cells specifically overexpress eIF4E and develop an oncogene addiction to eIF4E, rendering the cells vulnerable to eIF4E inhibition, whereas normal cells are relatively insensitive. Phosphorylation of eIF4E at serine-209 is critical for the oncogenic activity of eIF4E, and it has been suggested that chemical compounds that prevent the phosphorylation of eIF4E could act as effective anticancer drugs. Herein, we report that a novel inhibitor of peIF4E function (Novel Retinamides, NRs), safely and potently suppresses breast tumor formation. Administration of novel retinamides (mnk degrading agents: MNKDAs) blocked the growth of primary breast tumors in MDA-MB-231 xenograft model, TNBC patient derived xenografts (PDX), and reduced the development of lung metastases in an invasive model. Mechanistically, NRs resulted in the degradation of MNKs (MNK1 and MNK2), a key kinase chiefly responsible for phosphorylating and activating eIF4E. This in turn hampered peIF4E involved translation initiation and activation of genes associated with cell proliferation and survival. Furthermore, MNK degradation by NRs also suppressed epithelial-to-mesenchymal transition, a process crucial for metastasis as evident by increased expression of E-cadherin and reduced expression of N-cadherin, fibronectin and vimentin; snail, slug, twist, fibronectin, claudin, lamin, TCF/ZEB-1, beta catenin, MMPs and vimentin. Taken together, the preclinical evidence of tumor regression and metastasis in TNBC models suggest that NRs offers considerable promise as new therapeutic candidate to target TNBC and metastatic breast cancer. Citation Format: Senthilmurugan Ramalingam, Lalji Gediya, Vidya Ramamurthy, Yong Choi, Rena Lapidus, Njar Vincent. Inhibition of eIF4E/MNK axis by novel MNKDAs reduces breast cancer tumor growth, migration, invasion and metastasis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B30.

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Ras oncogene-independent activation of RALB signaling is a targetable mechanism of escape from NRAS(V12) oncogene addiction in acute myeloid leukemia

Somatic mutations that lead to constitutive activation of NRAS and KRAS proto-oncogenes are among the most common in human cancer and frequently occur in acute myeloid leukemia (AML). An inducible NRAS(V12)-driven AML mouse model has established a critical role for continued NRAS(V12) expression in leukemia maintenance. In this model genetic suppression of NRAS(V12) expression results in rapid leukemia remission, but some mice undergo spontaneous relapse with NRAS(V12)-independent (NRI) AMLs providing an opportunity to identify mechanisms that bypass the requirement for Ras oncogene activity and drive leukemia relapse. We found that relapsed NRI AMLs are devoid of NRAS(V12) expression and signaling through the major oncogenic Ras effector pathways, phosphatidylinositol-3-kinase and mitogen-activated protein kinase, but express higher levels of an alternate Ras effector, Ralb, and exhibit NRI phosphorylation of the RALB effector TBK1, implicating RALB signaling in AML relapse. Functional studies confirmed that inhibiting CDK5-mediated RALB activation with a clinically relevant experimental drug, dinaciclib, led to potent RALB-dependent antileukemic effects in human AML cell lines, induced apoptosis in patient-derived AML samples in vitro and led to a 2-log reduction in the leukemic burden in patient-derived xenograft mice. Furthermore, dinaciclib potently suppressed the clonogenic potential of relapsed NRI AMLs in vitro and prevented the development of relapsed AML in vivo. Our findings demonstrate that Ras oncogene-independent activation of RALB signaling is a therapeutically targetable mechanism of escape from NRAS oncogene addiction in AML.

Novel Strategies to Discover Effective Drug Targets in Metabolic and Immune Therapy for Glioblastoma.

Glioblastoma multiforme is a common primary brain tumor, which exhibits an imbalance between glioma cell growth and glucose metabolism. Recent discoveries have found that the multiple pathways and downstream genes involved in the dysregulated metabolic pathway allow tumor to manifest and progress, which is critical to patients with glioblastoma associated with significant systemic and immunosuppression. Moreover, immune microenvironment is considered a major obstacle to generating an effective antitumor immune response. Therefore, identification of patient-specific tumor antigens through highly personalized approach, and effective combination with other therapeutic modalities such as molecular agents targeting tumor metabolic oncogene addiction and potent host immune modulators, may provide targets for more effective therapeutic strategies for glioblastoma. In this review, we aim to highlight the most recent findings regarding glucose uptake and proliferation, cell mobility and to expand our investigations and more comprehensively examine different aspects of glucose metabolism in glioblastoma, such as pentose phosphate pathway (PPP) and its enzymes, metabolic modulation of genetics and epigenetics and key metabolic regulators, importantly, tumor cell-induced glucose deprivation inhibits T-cell glycolysis and immunogenic functions. Furthermore, this review will concentrate on how to discover effective drug targets to regulate glucose metabolism in tumor and T cell growth for future glioblastoma therapies, and the challenges faced by the field of metabolism in tumor immune microenviroment.

Adaptive Reactivation of Signaling Pathways As a Novel Mechanism of Resistance to JAK Inhibitors in Ph-like ALL

Background:Ph-like ALL was first described in 2009 and was accepted as a novel B-ALL subgroup by the WHO in 2016. Ph-like ALL is characterized by different chromosomal rearrangements involving CRLF2, ABL1, JAK2, and EPOR. In addition, mutations in PAX5, IKZF1, and JAK2 are very common. The gene expression profile of this disease is very similar to Ph+ ALL which is characterized by the BCR-ABL1 oncogene. We have previously shown that Ph-like ALL is a high risk disease with a poor prognosis and constitutive activation of cytokine receptor signaling pathway (Tasian et al., submitted). Our current study is focusing on redundant signaling activation in Ph-like ALL that allows the cells to survive cytokine receptor inhibition.Results:We and others have shown that Ph-like ALL have constitutive activation of the cytokine receptor signaling pathway evident by high expression levels of pSTAT5. Xenotransplantation models of Ph-like ALL show a decrease in leukemia burden when treated with Ruxolitinib a JAK1/2 inhibitor. However, all mice ultimately succumb to the disease, demonstrating that Ph-like ALL does not demonstrate oncogene addiction. To understand these results, we have initially used Ph-like ALL cell lines. Ruxolitinib treatment of such cell lines decreases survival but does not eliminate all viable cells. In addition, Ruxolitinib leads to down regulation of pSTAT5, pSTAT3, pAKTT308 as well as pAKTS473. To further study the mechanism of resistance to JAK inhibition we performed a time course experiments and found that as early as 48-72 hours after the initial JAK inhibitor treatment Ph-like ALL cell are able to reactivate pSTAT5 and pAKT. This effect was not due to drug inactivation. Based on this observation, we hypothesized that adaptive signaling through PI3 kinase protects Ph-like ALL cells from cell death. Interestingly, PI3K inhibition (Idelalisib) alone had only a minor effect on cell viability and cell proliferation and the Ph-like ALL cells were able to reactivate the signaling network within 48-72h. Dual treatment with JAK1/2 and PI3 kinase inhibition significantly increased the apoptotic effect and stopped proliferation. In addition, the dual treatment induced a long lasting inhibitory effect with no signs of pAKT reactivation. Strikingly, combining JAK and PI3K inhibition with Methotrexate an antimetabolite that is used for the treatment of ALL patients significantly increased the apoptotic effect of the combinatory treatment. Overall, these results demonstrate that adaptive signaling with reactivation of the PI3K signaling cascade protects Ph-like ALL cells from inhibition of JAK kinases and suggests that dual kinase inhibitor therapy may increase response in the clinic. Experiments studying how reactivation occurs and the in vivo response to dual JAK-PI3K inhibition are in progress.Clinical relevance:Older patients (>40 years) often do not qualify for induction chemotherapy as they do not tolerate high doses of chemotherapy regimens and are therefore in clinical need of alternative treatment regimens that can improve their clinical outcome. While children with ALL currently have a 5-year survival rate of 90% adults especially older adults have a very poor clinical outcome with survival rates of about 40%. We therefore combined JAK1/2 and PI3K inhibitory treatments with methotrexate a frequently used antimetabolite that is well tolerated by older patients. Strikingly, the combinatory treatment had a strong effect on cell viability and represents so far the first study using specific signaling inhibitors combined with an antimetabolite treatment in Ph-like ALL.Conclusion:These findings identify that Ph-like ALL are able to reestablish their signaling networks. Based on our finding it is important to target more than one signaling pathways to fully inhibit cell proliferation and cell survival. We propose to treat Ph-like ALL patients with a combinatory treatment of JAK1/2 and PI3K inhibitor and currently used antimetabolite drugs to reduce the risk of relapse and to improve the clinical outcome of patients with Ph-like ALL. DisclosuresPerl:Seattle Genetics: Consultancy; Actinium Pharmaceuticals, Inc.: Research Funding.

Conflicting Signals for Cancer Treatment.

Next-generation sequencing technologies have provided us with a precise description of the mutational burden of cancers, making it possible to identify targetable oncogene addictions. However, the emergence of resistant clones is an inevitable limitation of therapies targeting these addictions. Alternative approaches to cancer treatment are therefore required. We propose here a novel approach, based on the notion of conflicting signals and on a phenotypic description of cancer cells. "Phenotype" is an inherently complex notion that we describe in the conceptual framework of the epigenetic landscape, with a view to bridging the gap between theory and practice at the patient's bedside. By passing from theory to the description of several examples, we will illustrate how this approach can facilitate data analysis and the design of new strategies for cancer treatment. Cancer Res; 76(23); 6768-73. ©2016 AACR.

High Energy Particle Radiation-associated Oncogenic Transformation in Normal Mice: Insight into the Connection between Activation of Oncotargets and Oncogene Addiction.

Concerns on high-energy particle radiation-induced tumorigenic transformation of normal tissue in astronauts, and in cancer patients undergoing radiotherapy, emphasizes the significance of elucidating the mechanisms involved in radiogenic transformation processes. Mostly used genetically modified or tumor-prone models are less reliable in determining human health risk in space or protracted post-treatment normal tissue toxicity. Here, in wild type C57BL/6 mice, we related the deregulation of distinctive set of tissue-specific oncotargets in major organs upon 56Fe (600 MeV/amu; 0.5 Gy/min; 0.8 Gy) particle radiation and compared the response with low LET γ-radiation (137Cs; 0.5 Gy/min; 2 Gy). One of the novel findings is the ‘tissue-independent’ activation of TAL2 upon high-energy radiation, and thus qualifies TAL2 as a potential biomarker for particle and other qualities of radiation. Heightened expression of TAL2 gene transcript, which sustained over four weeks post-irradiation foster the concept of oncogene addiction signaling in radiogenic transformation. The positive/negative expression of other selected oncotargets that expresses tissue-dependent manner indicated their role as a secondary driving force that addresses the diversity of tissue-dependent characteristics of tumorigenesis. This study, while reporting novel findings on radiogenic transformation of normal tissue when exposed to particle radiation, it also provides a platform for further investigation into different radiation quality, LET and dose/dose rate effect in healthy organs.

The Synthetic β-Nitrostyrene Derivative CYT-Rx20 Inhibits Esophageal Tumor Growth and Metastasis via PI3K/AKT and STAT3 Pathways.

The β-nitrostyrene family have been implicated for anti-cancer property. However, the pharmacological role of β-nitrostyrene in esophageal cancer remain unclear. Here, a β-nitrostyrene derivative, CYT-Rx20, was synthesized and assessed for its anti-cancer activities and underlying mechanism in esophageal cancer. CYT-Rx20 induced cytotoxicity in esophageal cancer cells by promoting apoptosis through activation of caspase cascade and poly(ADP-ribose) polymerase (PARP) cleavage. Besides, CYT-Rx20 inhibited esophageal cancer cell migration and invasion by regulating the expression of epithelial to mesenchymal transition (EMT) markers. CYT-Rx20 decreased cell viability and migration through suppression of the PI3K/AKT and STAT3 pathways. Of note, the cytotoxicity and anti-migratory effect of CYT-Rx20 were enhanced by co-treatment with SC79 (AKT activator) or colivelin (STAT3 activator), suggesting the dependency of esophageal cancer cells on AKT and STAT3 for survival and migration, an oncogene addiction phenomenon. In xenograft tumor-bearing mice, CYT-Rx20 significantly reduced tumor growth of the implanted esophageal cancer cells accompanied by decreased Ki-67, phospho-AKT, and phospho-STAT3 expression. In orthotopic esophageal cancer mouse model, decreased tumor growth and lung metastasis with reduced Ki-67 and phospho-STAT3 expression were observed in mice treated with CYT-Rx20. Together, our results suggest that CYT-Rx20 is a potential β-nitrostyrene-based anticancer compound against the tumor growth and metastasis of esophageal cancer.

Unveiling changes in the landscape of patient populations in cancer early drug development.

The introduction of new Molecularly Targeted Agents (MTA) has changed the landscape in Early Drug Development (EDD) over the last two decades, leading to an improvement in clinical trial design. Previous Phase 1 (Ph1) studies with cytotoxics focused on safety objectives, only recruiting heavily pre-treated cancer patients, have been left behind. In this review, we will illustrate the slow although unstoppable change that has increasingly been observed in those populations candidate to participate in EDD trials with the advent of MTA. As more evidence regarding oncogene addiction becomes available, molecular-biomarker driven selection has been implemented among Molecularly-Selected Population (MSP) studies. New Window-Of-Opportunity (WOO) and Phase 0 (Ph0) studies have been developed in order to assess whether a MTA produces the hypothetical proposed biological effect. The rising need of getting early pharmacokinetics and pharmacodynamics data has led to the conduction of Healthy Volunteer (HV) studies, in part favoured for the particular and different toxicity profile of these MTA. However, several challenges will need to be addressed in order to boost the implementation of these new clinical trial designs in the forthcoming years. Among the problems to overcome, we would highlight a better coordination effort between centers for ensuring adequate patient accrual among small patient populations and a deepening into the ethics implied in enrolling patients in studies with no therapeutic intent. However, these tribulations will be certainly compensated by the possibility of opening a new horizon of treatment for diseases with dismal prognosis.

Androgen receptor signaling in castration-resistant prostate cancer: a lesson in persistence.

The androgen receptor (AR) signaling axis drives all stages of prostate cancer, including the lethal, drug-resistant form of the disease termed castration-resistant prostate cancer (CRPC), which arises after failure of androgen deprivation therapy (ADT). Persistent AR activity in spite of ADT and the second-generation AR-targeting agents enzalutamide and abiraterone is achieved in many cases by direct alterations to the AR signaling axis. Herein, we provide a detailed description of how such alterations contribute to the development and progression of CRPC. Aspects of this broad and ever-evolving field specifically addressed in this review include: the etiology and significance of increased AR expression; the frequency and role of gain-of-function mutations in the AR gene; the function of constitutively active, truncated forms of the AR termed AR variants and the clinical relevance of alterations to the activity and expression of AR coregulators. Additionally, we examine the novel therapeutic strategies to inhibit these classes of therapy resistance mechanisms, with an emphasis on emerging agents that act in a manner distinct from the current ligand-centric approaches. Throughout, we discuss how the central role of AR in prostate cancer and the constant evolution of the AR signaling axis during disease progression represent archetypes of two key concepts in oncology, oncogene addiction and therapy-mediated selection pressure.

Approaches to modernize the combination drug development paradigm.

Recent advances in genomic sequencing and omics-based capabilities are uncovering tremendous therapeutic opportunities and rapidly transforming the field of cancer medicine. Molecularly targeted agents aim to exploit key tumor-specific vulnerabilities such as oncogenic or non-oncogenic addiction and synthetic lethality. Additionally, immunotherapies targeting the host immune system are proving to be another promising and complementary approach. Owing to substantial tumor genomic and immunologic complexities, combination strategies are likely to be required to adequately disrupt intricate molecular interactions and provide meaningful long-term benefit to patients. To optimize the therapeutic success and application of combination therapies, systematic scientific discovery will need to be coupled with novel and efficient clinical trial approaches. Indeed, a paradigm shift is required to drive precision medicine forward, from the traditional “drug-centric” model of clinical development in pursuit of small incremental benefits in large heterogeneous groups of patients, to a “strategy-centric” model to provide customized transformative treatments in molecularly stratified subsets of patients or even in individual patients. Crucially, to combat the numerous challenges facing combination drug development—including our growing but incomplete understanding of tumor biology, technical and informatics limitations, and escalating financial costs—aligned goals and multidisciplinary collaboration are imperative to collectively harness knowledge and fuel continual innovation.

Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growthin vivo

Mutation of p53 occasionally results in a gain of function, which promotes tumor growth. We asked whether destabilizing the gain-of-function protein would kill tumor cells. Downregulation of the gene reduced cell proliferation in p53-mutant cells, but not in p53-null cells, indicating that the former depended on the mutant protein for survival. Moreover, phenformin and 2-deoxyglucose suppressed cell growth and simultaneously destabilized mutant p53. The AMPK pathway, MAPK pathway, chaperone proteins and ubiquitination all contributed to this process. Interestingly, phenformin and 2-deoxyglucose also reduced tumor growth in syngeneic mice harboring the p53 mutation. Thus, destabilizing mutant p53 protein in order to kill cells exhibiting “oncogene addiction” could be a promising strategy for combatting p53 mutant tumors.

Drugging the addict: non-oncogene addiction as a target for cancer therapy.

Historically, cancers have been treated with chemotherapeutics aimed to have profound effects on tumor cells with only limited effects on normal tissue. This approach was followed by the development of small‐molecule inhibitors that can target oncogenic pathways critical for the survival of tumor cells. The clinical targeting of these so‐called oncogene addictions, however, is in many instances hampered by the outgrowth of resistant clones. More recently, the proper functioning of non‐mutated genes has been shown to enhance the survival of many cancers, a phenomenon called non‐oncogene addiction. In the current review, we will focus on the distinct non‐oncogenic addictions found in cancer cells, including synthetic lethal interactions, the underlying stress phenotypes, and arising therapeutic opportunities.

BIM-mediated apoptosis and oncogene addiction

Oncogene addiction is a phenomenon whereby suppression of a driver oncogene is associated with dramatic tumor regression that has been observed in experimental models and in response to targeted therapies [1]. However, the mechanism by which oncogene inactivation induces this massive reduction in tumor burden is not clear. In tumors addicted to the MYC oncogene, suppression of this oncogene leads to tumor regression that is associated with a marked increase in apoptosis. This at first glance appears to be paradoxical since generally oncogene activation, and MYC activation in particular, is associated with increased apoptosis. Recently, we have described a possible mechanism that may explain why inactivation of pro-apoptotic oncogenes, such as MYC, induce apoptosis [2].

ALK alterations and inhibition in lung cancer.

The advent of precision medicine in non-small cell lung cancer has remarkably altered the direction of research and improved clinical outcomes. The identification of molecular subsets with differential response to targeted therapies began with the identification of epidermal growth factor receptor mutated tumors in subsets of non-small cell lung cancer (NSCLC). Emboldened by unprecedented response rates to kinase inhibitors seen in that subset, the oncologic community searched for other molecular subsets featuring oncogene addiction. An early result of this search was the discovery of NSCLC driven by activating rearrangements of the anaplastic lymphoma kinase (ALK) gene. In an astoundingly brief period following the recognition of ALK-positive NSCLC, details of the biology, clinicopathologic features, development of targeted inhibitors, mechanisms of therapeutic resistance, and new generations of treatment were elucidated. This review summarizes the current understanding of the pathologic features, diagnostic approach, treatment options, resistance mechanisms, and future research areas for ALK-positive NSCLC.

Oncogenic Deregulation of EZH2 as an Opportunity for Targeted Therapy in Lung Cancer.

As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways, leading to epigenetic transformation independent of canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a potent open-source EZH2 inhibitor, JQEZ5, that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer. EZH2 overexpression induces murine lung cancers that are similar to human NSCLC with high EZH2 expression and low levels of phosphorylated AKT and ERK, implicating biomarkers for EZH2 inhibitor sensitivity. Our EZH2 inhibitor, JQEZ5, promotes regression of these tumors, revealing a potential role for anti-EZH2 therapy in lung cancer. Cancer Discov; 6(9); 1006-21. ©2016 AACR.See related commentary by Frankel et al., p. 949This article is highlighted in the In This Issue feature, p. 932.

Identification of Bisindolylmaleimide IX as a potential agent to treat drug-resistant BCR-ABL positive leukemia.

Chronic myeloid leukemia (CML) treatment with BCR-ABL inhibitors is often hampered by development of drug resistance. In a screen for novel chemotherapeutic drug candidates with genotoxic activity, we identified a bisindolylmaleimide derivative, IX, as a small molecule compound with therapeutic potential against CML including drug-resistant CML. We show that Bisindolylmaleimide IX inhibits DNA topoisomerase, generates DNA breaks, activates the Atm-p53 and Atm-Chk2 pathways, and induces cell cycle arrest and cell death. Interestingly, Bisindolylmaleimide IX is highly effective in targeting cells positive for BCR-ABL. BCR-ABL positive cells display enhanced DNA damage and increased cell cycle arrest in response to Bisindolylmaleimide IX due to decreased expression of topoisomerases. Cells positive for BCR-ABL or drug-resistant T315I BCR-ABL also display increased cytotoxicity since Bisindolylmaleimide IX inhibits B-Raf and the downstream oncogene addiction pathway. Mouse cancer model experiments showed that Bisindolylmaleimide IX, at doses that show little side effect, was effective in treating leukemia-like disorders induced by BCR-ABL or T315I BCR-ABL, and prolonged the lifespan of these model mice. Thus, Bisindolylmaleimide IX presents a novel drug candidate to treat drug-resistant CML via activating BCR-ABL-dependent genotoxic stress response and inhibiting the oncogene addiction pathway activated by BCR-ABL.

High-Level Clonal FGFR Amplification and Response to FGFR Inhibition in a Translational Clinical Trial.

FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy. Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients. Cancer Discov; 6(8); 838-51. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Abstract 3498: Increased cellular stress and apoptosis induced by anti-SIAH2 therapy in human cancer cells with oncogenic K-RAS addiction

Abstract Hyperactivated K-RAS oncoproteins are known to drive cell proliferation, tumor growth, and metastasis. Oncogene addiction is often associated with increased ER and mitochondrial stress due to excessive reactive oxygen and nitrogen species (ROS & NROS) production. RING finger E3 ubiquitin ligase, Seven in Absentia homolog 2 (Siah2) is a highly conserved, critical signaling module downstream of the EGFR/HER2/K-RAS pathway. SIAH2 function is required for proper K-RAS signal transduction and has been shown to target key regulators of oxidative stress for ubiquitination and proteasome degradation. However, the mechanisms by which SIAH2 regulates cellular stress and protects against programmed cell death in oncogenic K-RAS-addicted cancer cells are not well understood. Since SIAH2 is upregulated in proliferating tumor cells, we hypothesized that SIAH2-deficient cancer cells may be compromised in their ability to adapt to increased cellular and oxidative stress induced by oncogenic K-RAS pathway blockade. In this study, SIAH2 knockdown was carried out in cancer cells carrying oncogenic K-RAS mutations (A549, MDA-MB-231 and MiaPaCa cells). Triplicates of fresh cell lysates from SIAH2 proficient and SIAH2 deficient cells were then interrogated for altered expression of 19 signaling molecules with known functions in modulating stress response and apoptosis using the PathScan® Stress and Apoptosis Signaling Antibody Array kit. Differences in the protein expression were quantified and validated by Western blots, immunofluorescence (IF) and flow cytometry. We found that oxidative stress was markedly increased, evidenced by mitochondrial aggregations and dysfunction in a subset K-RAS-driven cancer cells, as we blocked SIAH2 function downstream of the oncogenic K-RAS signaling pathway. SIAH2 deficiency in breast cancer (MDA-MB-231) and non-small cell lung adenocarcinoma (A549) cells resulted in decreased ERK1/2 and AKT (S473) phosphorylation, increased ROS production, compromised mitochondria integrity and function, increased Caspase 3 and 7 activity, and PARP 1 cleavage that led to massive cell death observed in these cancer cells. In contrast, the prominent cell death was not observed in SIAH2 deficient pancreatic cancer (MiaPaCa) cells that expressed high levels of Survivin, which is an anti-apoptosis protein. The anti-SIAH2 therapy is highly effective in inducing oxidative stress, altered mitochondrial bioenergetics, and apoptosis in a subset of invasive and metastatic cancer lines expressing insufficient levels of Survivin. Our results suggest that, anti-SIAH2 strategy reveals novel oncogenic K-RAS-dependent cancer cell vulnerability in stress response that can be exploited in future for anti-SIAH2 therapy alone, or in combination with other anti-proliferative treatment to effectively combat KRAS-dependent cancers. Citation Format: Monicah M. Njogu, Caroline Lee, Jamie Eisner, Minglei Bian, Robert Vansciver, Amy H. Tang. Increased cellular stress and apoptosis induced by anti-SIAH2 therapy in human cancer cells with oncogenic K-RAS addiction. [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 3498.

Abstract 69: Identification of new therapeutic targets and molecular predictors of response in oesophageal cancer

Abstract Purpose of the study Oesophageal cancer is the seventh most common cause of cancer related death worldwide. Disease relapse is frequent and treatment options are limited. With the exception of HER-2 and EGFR amplification, there are currently no validated biomarkers to predict response to biological therapies in this disease. The purpose of this study was to identify new biomarker defined therapeutic approaches for patients with oesophageal cancer. Experimental approach We have undertaken a functional screening approach focused on the druggable kinome to generate a first map of the genetic dependencies found in oesophageal cancer. A panel of 19 oesophageal and 130 non-oesophageal cancer cell lines were screened with siRNA targeting 720 kinases to generate functional profiles. By integrating this dataset with exome sequencing and copy number data, we identified key genetic dependencies (KGDs) associated with oesophageal histology (adenocarcinoma or squamous cell carcinoma) or key driver genotypes including amplifications in c-MYC, CCND1, EGFR and ERBB2, as well as loss of functional mutations in SMAD4 and ARID1A. Results Interrogation of the oesophageal functional genomics dataset established oncogene addiction effects including c-MYC, which is amplified in 30% of oesophageal carcinomas. We confirmed that a novel isoform of Bruton agammaglobulinaemia tyrosine kinase gene (BTK-C) was expressed in oesophageal cancer models and that silencing of BTK resulted in selective lethality in c-MYC amplified oesophageal cell lines. To further assess the impact of BTK inhibition in oesophageal cancer, we treated a panel of oesophageal cell lines with increasing doses of ibrutinib, a small molecule tyrosine kinase inhibitor of BTK. Analysis of ibrutinib SF50 revealed that there was preferential sensitivity to ibrutinib in c-MYC amplified oesophageal cell lines. Furthermore, we observed a significant association between mutation of the chromatin remodelling factor gene SMARCA4 and dependency upon the bromodomain protein BRD4. We also identified SMAD4 kinase genetic dependencies including AKT1 and a number of its substrates (FGR, MAP3K3, CHEK1 and WEE1). We noted a densely connected group of kinases that regulate the mitotic cell cycle in the SMAD4 dependency network, and found that SMAD4 mutant cell lines exhibited an increased sensitivity to drugs, including paclitaxel, that target the mitotic checkpoint. Conclusions Our results support the potential of BTK small molecule inhibition as a novel therapeutic strategy in c-MYC amplified oesophageal carcinoma. Loss of function SMAD4 mutations represents a candidate biomarker of response to drugs that target the mitotic checkpoint in oesophageal cancer. Citation Format: Irene Chong, lauren Aronson, David Cunningham, James Campbell, Colm Ryan, Michael Davidson, Ian Chau, Alan Ashworth, Christopher J. Lord. Identification of new therapeutic targets and molecular predictors of response in oesophageal cancer. [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 69.