Karen Cichowski, PhD of Bringham and Women's Hospital and Harvard Medical School
Gerard Evan PhD, FRS, FMedSci of University of Cambridge
Myc and Ras are two of the most commonly deregulated genes in human cancer. Nonetheless, we still lack a clear understanding of how these cancer-causing genes work, how they cooperate during tumor development, or how we may convert our biological insights into therapies. This Forbeck Foundation Forum brought together thought leaders from both the Myc and Ras fields to address critical questions about these two cancer-causing genes and to think outside the conventional biopharmacological box. Specifically, we discussed 1) how these two oncogenic pathways converge to drive specific cancers, 2) the broad metabolic, transcriptional, and proteomic consequences of Ras and Myc activation, 3) novel approaches to identify targetable vulnerabilities in Ras and Myc driven tumors, and 4) new strategies to pharmacologically target Myc and Ras. Importantly, this meeting resulted in a significant cross-fertilization of ideas between these investigators and stimulated numerous collaborations.
Gerard Evan: How Ras and Myc cooperate to drive cancer
Dr. Evans opened the Forum by discussing how Ras and Myc cooperate to promote different types of cancer. Using sophisticated genetically engineered mouse models Dr. Evans has shown that Ras and Myc drive cancer in virtually all tissues. Here Dr. Evans extensively discussed how oncogenic Ras and Myc function by hacking the local regenerative programs of each tissue. He noted that in normal cells Ras and Myc are critical for driving proliferation and controlling tissue regeneration. During this session he compellingly argued that the distinct phenotypes of different cancers driven by Ras and Myc reflect the unique, tissue-specific differences in regeneration programs. For example, in the lung Ras and Myc induce highly angiogenic and vascular tumors, whereas pancreatic tumors are desomoplasmic and avascular. This concept provides important insight into how the oncogenic functions of Ras and Myc represent exaggerated perturbations of the normal functions of these genes and has important implications for therapeutic development.
Channing Der: Convergence of Ras and Myc in pancreatic cancer
Dr. Der continued with this theme by discussing one of the molecular mechanisms by which Ras and Myc converge in pancreatic cancer. Specifically, he described how KRAS controls MYC protein stability through a novel mechanism involving specific kinases not previously implicated in this process. Importantly, he argued that targeting these newly identified protein kinases may represent a novel therapeutic strategy for pancreatic cancer. He also showed that oncogenic Ras and Myc (and conversely their suppression) phenocopy each other and trigger the same defects in mitochondrial function and metabolic processes, further illustrating the convergence of these two important oncogenic pathways.
Rosalie Sears: Ras signaling controls temporal and spatial activity of Myc to direct cell behavior
As noted by Dr. Der, many studies have shown that Ras induces signals that regulate Myc stability, DNA binding, and transcriptional activity. However, the mechanism by which these events dictate specific transcriptional outputs in cancer is poorly understood. Here Dr. Sears described how a critical regulatory protein, Pin1 controls both the temporal dynamics of Myc and co-activator binding to enhance the transcription of specific target genes. She also discussed the provocative observation that Pin1 also controls Myc’s subcellular localization near nuclear pores and adjacent to specific pro-growth and pro-migration genes that are involved in wound healing and cancer. Accordingly, these regulatory mechanisms reveal new potential strategies to interfere with the transcriptional programs that underlie cancer.
Mara Sherman (Scholar): Microenvironmental regulation of MYC transcriptional programs in pancreatic cancer
One unique aspect of pancreatic cancer is that the stromal component comprises 50-90% of tumor volume. Therefore, Dr. Sherman has been exploring the mechanistic interaction between tumor cells and stromal microenvironment as a means of potentially identifying new therapeutic vulnerabilities. At this Forum she discussed how microenvironmental and tumor cell-autonomous networks converge on a key transcriptional regulator: MYC. She convincingly demonstrated that oncogenic KRAS requires cooperative signals from the tumor microenvironment to maximally activate MYC and drive PDAC progression. She then showed how the fibroinflammatory microenvironment specifically alters MYC subnuclear localization and chromatin interaction, and consequently the MYC transcriptional program. Finally, she identified fibroblast growth factor 1 (FGF1) as a potential stroma-derived factor that may modulate these effects on MYC. Notably, while KRAS and MYC are presently undruggable, identification of novel factors that collaborate with these oncoproteins may lead to new therapeutic options for PDAC patients.
Andrew Aguirre (Scholar): Functional Genetic Approaches to Understanding KRAS in Pancreatic Cancer
At this Forum Dr. Aguirre described the various genetic approaches that he is utilizing to better understand and target KRAS mutant pancreatic cancer. First, he discussed how he has been using CRISPR-Cas9 negative selection screens to identify new KRAS synthetic lethal partners. Importantly, he has already discovered several genes, that when suppressed, result in cell death only in the presence KRAS mutation. Second, he described a unique saturation mutagenesis approach to identify novel structure-function relationships in the KRAS gene/protein that may ultimately lead to new therapeutic angles for targeting KRAS. Finally, he outlined a genetic approach to understand non-cell autonomous signaling between KRAS-mutant cancer cells and surrounding stromal cells in a genetically engineered mouse model of pancreatic cancer, with the goal of identifying KRAS-driven intercellular signaling pathways that may be therapeutically tractable. Each of these approaches has already provided unique insight.
Karen Cichowski: Developing rational combination therapies for Ras-driven cancers
Because oncogenic Ras itself is not yet “druggable”, Dr. Cichowski has been working to identify new/additional therapeutic vulnerabilities in various Ras-driven cancers. Her ultimate goal is to develop novel combination therapies by co-targeting key Ras effector pathways along with cooperating or converging pathways. At this Forum she discussed three new promising therapeutic approaches that have been extremely effective in promoting tumor regression in various animal models. Interestingly, these distinct combinations function by either 1) suppressing converging signaling pathways, 2) attenuating epigenetic regulators, or 3) inhibiting cancer-protective pathways. By dissecting the precise mechanism by which these combinations act, she has uncovered new nodal points of convergence between these distinct pathways and has identified new therapeutic vulnerabilities in Ras driven tumors. These findings are currently being translated into clinical trials.
Dafna Bar-Sagi: Exploiting Oncogenic Ras-dependent tumor cell vulnerabilities to design new therapeutics
A long-standing goal of Dr. Bar-Sagi’s work has been to identify strategies by which the oncogenic potential of mutant Ras can be compromised. As such, she has been intensively studying the biological processes that are critical for pancreatic tumor initiation and progression, with the idea that these processes may represent tumor cell specific vulnerabilities. At this Forum, she discussed several Ras-dependent processes that control 1) metabolic adaptation, 2) immune evasion, 3) stress resistance and 4) proliferative capacity in pancreatic cancer. Notably, the specific findings she discussed not only provide important insight into the mechanisms that drive tumor development, but also suggest that the therapeutic manipulation of these pathways may lead to effective approaches for treating pancreatic cancer.
Kimberly Stegmaier: Targeting MYCN-driven Neuroblastoma
Dr. Stegmaier extensively discussed neuroblastoma, the most common extracranial solid tumor diagnosed in children. Because MYCN amplification is common in these tumors, she has been focusing on identifying therapeutic vulnerabilities in this subtype of neuroblastoma. At this Forum she described several ongoing chemical and functional genomic approaches. First, she has leveraged genome-scale CRISPR-Cas9 screening in 80 pediatric cancer cell lines and the Pediatric Cancer Dependency Map to identify new dependencies in MYCN-amplified disease. Specifically, she identified the Core Regulatory transcriptional Circuitry (CRC) as a specific vulnerability in neuroblastoma and identified a therapeutic approach to repress CRC genes in vitro and in vivo. She also identified and validated a number of epigenetic dependencies emerging from this Pediatric Dependency Map data. Finally, using a chemical screening approach she found that MYCN-amplified neuroblastoma is highly sensitive to BET bromodomain inhibitors, and determined that these compounds repress MYCN expression. Dr. Stegmaier is currently performing ORF, CRISPR, and drug synergy screens to identify mechanisms of resistance to, and synergistic drug combinations with, BET inhibitors in neuroblastoma. Together, these studies have defined epigenetic and transcriptional vulnerabilities in MYCN-amplified neuroblastoma with potential translational implications.
Bruno Amati: Transcriptional programs and therapeutic targets in MYC-driven lymhomas
One of the most debated issues in the field has been whether Myc acts as a selective regulator of growth-associated gene expression programs or a general activator (or “amplifier”) of all active genes. Dr. Amati began by discussing how he believes that the former is true and summarized his recent work addressing mechanisms of Myc-regulated transcription. However, he also described how he has been profiling Myc-regulated transcriptional programs during tumor progression and/or regression in transgenic models of Myc-driven B-cell lymphoma and Hepatocellular Carcinoma. He has now used this insight to conduct an in vivo shRNA dropout screen targeting over 240 of these Myc-activated mRNAs and identified components of the mitochondrial ribosome in his lymphoma model. He then showed that pharmacological inhibition of mitochondrial translation with the antibiotic Tigecycline was synthetic-lethal with Myc activation, impaired tumor cell survival in vitro and extended life-span in lymphoma-bearing mice. Finally, he discussed his work assessing the potential of Tigecycline as a therapeutic agent in double-hit lymphoma (DHL), an aggressive subset of diffuse large B-cell lymphoma. Notably, he uncovered a strong anti-tumoral synergy between Tigecycline and the Bcl-2 inhibitor Venetoclax (ABT-199) in mouse models: two agents currently in the clinic for other indications. As such, he has identified a novel Myc-induced metabolic dependency that can be targeted by common antibiotics, opening new therapeutic perspectives in Myc-associated tumors.
Martin Eilers: Transcriptional amplifier versus gene-specific regulator versus Biocenter
Current views explain the oncogenic properties of MYC proteins and their ability to co-operate with RAS in oncogenic transformation either by their ability to regulate a - somewhat broader than usual - group of specific target genes or by their ability to amplify all ongoing transcription. In this Forum Dr. Eilers discussed a possible alternative to these models. Specifically, he provided compelling evidence suggesting that MYC is required to maintain core promoter functionality in a stressed environment (e.g. in a rapidly growing tumor under nutrient-poor or hypoxic conditions). Based on this model, Dr. Eilers suggests that regulation of the process of transcription itself (e.g. pause and release), rather than changes in steady state levels of target gene mRNAs, accounts for the oncogenic properties of MYC. These findings represent an important paradigm shift in how we think MYC functions.
Kevin Haigis: Context dependence of KRAS oncogenicity
KRAS is the most frequently mutated oncogene in cancer however different mutations (e.g. weaker or stronger) are present in different tumor types. Because of this Dr. Haigis has postulated that cancer-associated K-RAS signaling is dependent upon (1) the tissue of origin of the cancer, (2) the exact mutant form of K-RAS that is expressed, and (3) the other mutations that have occurred in the cancer. To investigate unique tumor and allele-specific functions of KRAS he has been integrating mouse modeling and multiplexed mass spectrometry with bioinformatics and systems biology to understand how context alters the oncogenic signaling output of mutant K-RAS. At this forum Dr. Haigis showed that there are dramatically different effects of K-RAS mutations in different tumor types (colon versus pancreatic cancers), and that various mutant alleles affect phosphorylation and tissue homeostasis in distinct ways. He is currently using this approach to identify and validate context-specific therapeutic strategies for KRAS mutant cancers.
Doug Green: c-myc and T cell metabolism
Dr. Green led a very provocative discussion about c-Myc function in T cells. Specifically, he has found that during the first 24 hours of T cell activation, c-Myc is involved in programming the metabolic changes necessary for rapid T cell proliferation. Interestingly, he suggests that this occurs as a consequence of the subcellular polarization of TORC1, and consequently c-Myc translation at the TCR contact site. He showed that upon the first division, both TORC1 activity and c-Myc are asymetrically associated with the proximal daughter cell. As such, the maintenance of high c-Myc levels in first division daughter cells is sustained by amino acid levels and TORC1 activity resulting in increased proliferation and enhanced glycolysis. In contrast, the TORC1lowMyclow daughter cells preferentially contribute to the memory T cell pool. Importantly, perturbation of TORC1 activity or c-Myc levels alters these outcomes. Together these findings have revealed a unique function of c-Myc in T cells, that may ultimately reveal important insight into tumor immunosurveillance.
Dave Tuveson: KRAS activates Nix-mediated mitophagy to alter cellular metabolism and promote pancreatic cancer
Nearly all cases of pancreatic ductal adenocarcinoma (PDAC) harbor activating activating mutations in the KRAS gene. Therefore Dr. Tuveson has been striving to identify biological changes induced by oncogenic KRAS, in order to identify potentially therapeutic vulnerabilities in pancreatic cancer. In this forum be discussed his work on metabolic changes triggered by KRAS. Specifically he found that endogenous levels of oncogenic Kras suppress mitochondrial content through a mitophagy program mediated by Bnip3l/Nix. Interestingly, he found that Nix levels increase upon KrasG12D activation in vitro and in vivo and that Nix expression induces a metabolic program consistent with increased aerobic glycolysis. Moreover, he showed that loss of Nix in mouse and human pancreatic cancer cell lines leads to an increase in mitochondrial mass, a decrease in proliferation in glucose-limited conditions, and an increase in maximal respiration. Finally, using a genetically engineered mouse model of Kras-mutant pancreatic cancer, he showed that Nix ablation significantly delays tumor progression in vivo. Collectively, his results have uncovered a role for Nix in promoting pancreatic cancer development and suggest new targetable vulnerabilities in pancreatic cancer cells.
Donita Brady (Scholar): Tracing copper utilization by the MAPK pathway: implications for cancer cell metabolic processes
It is well established that transition metals such as copper are critical for normal physiology and development. However, Dr. Brady has recently uncovered an unexpected link between dietary copper, the Ras pathway and cancer. Specifically, she has shown that copper directly binds MEK kinases and enhances ERK signaling. Consequently, she found that genetic or chemical suppression of copper levels substantially suppresses the growth of BRAF mutant melanomas. Based on these findings the therapeutic utility of drugs that reduce copper availability are now being explored. However, she has also discovered that copper affects the activity of ULK1 and 2 kinases and accordingly autophagy in KRAS mutant pancreatic cancer cells. Moreover, she found that copper chelators sensitize these cells to low nutrients, mTOR inhibitors, specific chemotherapeutic agents, and autophagy inhibitors. She is currently using this insight to better understand the function of copper in regulating autophagy and is investigating whether insights can be applied to develop therapeutic strategies for KRAS mutant pancreatic cancer.
W. Clay Gustafson (Scholar): Blocking myc proteins through allosteric inhibition of AURK A
Pharmacologic targeting of MYC proteins has long been a holy grail of cancer therapeutics, but MYC is widely considered undruggable. Consequently, the overall goal of Dr. Gustafson’s work is to elucidate and exploit MYC biology to find novel mechanisms to drug MYC proteins in cancer. In this Forum he discussed his ongoing efforts to destabilize the MYC protein, specifically MYCN in neuroblastoma. MYCN is known to be stabilized by the mitotic regulator Aurora Kinase A, in a kinase independent manner. Therefore Dr. Gustafson identified a new small molecule inhibitor of AURKA that potently drives degradation of MYCN protein in models of neuroblastoma, medulloblastoma, and neuroendocrine prostate cancer. Importantly, his laboratory also solved the co-crystal structure of this drug bound to AURKA, thus demonstrating the molecular changes that promote its destabilization. Notably this agent also drives degradation of MYC (c-MYC) in liver cancer. He discussed how his laboratory is now further developing conformation-disrupting AURKA inhibitors and is beginning to explore their function in other cancer contexts and in resistance mechanisms. Exploiting this paradigm of conformation disruption, he is also interested in developing conformation disrupting inhibitors directed towards additional members of the MYC and MYCN interactome.
Steve Fesik: Therapeutic targeting of MYC
The Myc and Ras proteins have long been considered “undruggable”. However, the focus of Dr. Fesik’s work is to develop new therapeutic agents that target both of these oncogenes by using fragment-based approaches and structure-based design. In this Forum he discussed several promising new agents that bind and destabilize either Myc or Ras. Current studies are underway to determine the molecular mechanism by which these agents function and their specificity. Regardless, the discovery of these agents, along with Dr. Westovers’ below, provide strong support for the notion that these proteins may be effectively targeted one day.
Ken Westover: Direct targeting of RAS oncoproteins
Dr. Westover has been focusing his efforts on developing Ras inhibitors. He has now synthesized an agent that targets the KRAS G12C mutation, which is the most common mutation in lung cancer. Specifically, he developed an irreversible inhibitor that effectively competes with millimolar concentrations of GTP and GDP for the nucleotide binding site. He also solved a high resolution X-Ray crystal structure of G12 C KRAS bound to this drug and found that it binds in a manner similar to GDP. Finally, he showed that this agent effectively discriminates between KRAS G12C and other GTP-binding proteins. Current efforts are aimed at translating this initial tool compound into clinical agents. These findings have generated great optimism in the Ras field about the potential for directly targeting specific mutant Ras alleles.
Conclusions and Outlook
This Forbeck Forum provided the ideal environment to discuss the most current (unpublished) advances and collectively outline the most important “big picture” questions in the field. An additional unique aspect of this meeting was bringing together individuals from both the Ras and Myc fields. While these two oncoproteins clearly converge to drive human cancer, investigators from each of these fields do not have the opportunity to interact very often, and certainly not in such an intimate environment. All of the attendees lauded the format of this Forum and walked away from this meeting with greater insight and new collaborations. For example, Drs. Haigis, Cichowski, and Der have each developed independent collaborations with Dr. Westover. However, at the conclusion of the meeting, at least 12 different collaborations between attendees had been planned.
One of the most exciting aspects of this meeting was the increasing number of new therapeutic approaches and targets that were being discovered. For example, several strategies to target Ras and Myc proteins were presented. These included various structure-driven approaches aimed at directly targeting Ras and Myc, strategies designed to inhibit kinases that regulate oncoprotein stability, as well as epigenetic approaches to suppress expression, to name a few. However, many new therapeutic vulnerabilities in Ras and Myc-driven tumors were also discussed. This has and will continue to lead to the development of an increasing number of novel combination therapies. The goal for the field moving forward is to continue to deconstruct the mechanisms by which Ras and Myc drive tumor development so that we may continue to identify new therapeutic targets, and to begin to bring these new therapeutic approaches to the clinic. This Forbeck Forum has played an important role in cross-fertilizing new ideas between investigators that work in different fields, disciplines, cancer types (and in different parts of the world). As such it has played an invaluable role in shaping our work for years to come.
Andrew Aguirre, MD, PhD
Dana-Farber Cancer Institute
Bruno Amati, PhD
Instituto Italiano di Techologia
Dafna Bar-Sagi, PhD
NYU Langone Medical Center
Donita Brady, PhD
University of Pennsylvania
Karen Cichowski, PhD
Bringham and Women's Hospital and Harvard Medical School
Channing Der, PhD
University of North Carolina
Martin Eilers, PhD
University of Wuerzburg
Gerard Evan, PhD, FRS, FMedSci
University of Cambridge
Gerard Evan, PhD
University of Cambridge
Stephen Fesik, PhD
Doug Green, PhD
St. Jude Children's Research Hospital
W. Clay Gustafson, PhD
University of California, San Francisco
Kevin Hagis, PhD
Harvard Medical School
Rosalie Sears, PhD
Oregon Health & Sciences Institute
Mara Sherman, PhD
Oregon Health & Sciences Institute
Kimberly Stegmaier, MD
Dana-Farber Cancer Institute
David Tuveson, MD
Cold Spring Harbor Laboratory
Ken Westover, MD