The Forbeck Symposium offered a unique environment to vibrantly discuss the latest issues and theories underlying resistance to targeted therapies and the new strategies that may prevent or delay its development in the clinic. Discussions were spirited and launched new collaborations aimed at overcoming this obstacle. Some of the conclusions that achieved consensus are listed below.

1. There is a great need to identify strategies to reduce heterogeneity of initial response to targeted agents
2. There is a great need more potent inhibitors and therapeutic index is a critical determinant for success
3. It is unlikely we will completely cure cancer with one inhibitor, or targeting one target alone
4. Identify dependencies of surviving cells (i.e., resistant cells) to treatment to develop strategies to target them
5. There are a finite number of paths to resistance. This is a solvable problem
6. Dosing scheduling needs to be understood to develop more sophisticated treatment regimens to prevent or delay the emergence of resistance
7. It is imperative to define factors and niches in the microenvironment that favor resistance.

Targeted therapies have emerged as effective treatments for specific subsets of advanced cancers. In particular, impressive results have been observed when cancers harboring specific genetic alterations are matched to selective inhibitors. Examples include RAF inhibitors for melanomas carrying mutations in the BRAF gene, EGFR inhibitors for EGFR mutant lung cancers, ALK inhibitors for EML4-ALK lung cancers, and HER2 inhibitors for breast cancers with amplication (multiple copies) of the HER2 gene. In each of these cancer/treatment paradigms, the treatments are often highly effective, leading to remarkable remissions that have a profound beneficial impact on patients. These successes have changed the landscape of the diagnosis and treatment of cancer for the foreseeable future.

Despite such remarkable successes, initially sensitive cancers may ultimately become resistant to targeted therapies, often within one year. This type of resistance, often termed acquired resistance, has been a major obstacle preventing initially effective targeted therapies from providing a more lasting and transformative impact on patients. Over the past several years, the scientific community has intensely investigated how cancers develop resistance to targeted therapies. These studies have identified several mechanisms and conceptual frameworks underlying the acquisition of resistance. Many cancers become resistant via secondary mutations in the drug target that abrogate the capacity of the drug to inhibit the target. For example, about half of the EGFR mutant lung cancers that become resistant to EGFR inhibitors develop a specific mutation, T790M, that renders the EGFR inhibitor ineffective. In addition to mutations in the drug target, other mechanisms of resistance have been identified. These include the development of new signaling pathways that bypass the need for continued output from original drug target. Resistance can also emerge when inhibition of the drug target leads to the de-repression of negative feedback signaling loops, leading to activation of survival signals. Other potential mechanisms of resistance include acquired defects in cellular growth arrest and apoptosis, alterations in drug pharmacokinetics, and specific micronvironment niches that may limit the efficacy of targeted therapies.

In addition to the challenges of overcoming a specific mechanism of resistance, there are additional obstacles in overcoming resistance in a patient. These include the accumulating data there are different mechanisms of resistance in distinct populations of cancer cells in a single patient. To have a profound effect on overcoming resistance to cancer, there will be an increasing need to monitor and mathematically model the emergence of different populations of cancer cells with distinct resistance mechanisms and to utilize multi-drug cocktails to eliminate the emergence of resistance.

The 2013 Forum provided a unique opportunity to discuss the current state of the biology of resistance, and how we can apply this knowledge to develop new therapeutic approaches for cancer patients. Leading investigators, including a wide range of experts from different disciplines ranging from mathematicians to clinicians, assembled to discuss the latest insights and develop new collaborations and strategies to combat resistance. Below, are some highlights from discussions led by the participants.

Bruce Chabner. Dr. Chabner traced the history of research regarding the mechanism of action of, and resistance to, antifolates. This class of drugs was the second type of chemotherapy to display antitumor effectiveness, following closely behind alkylating agents in the late 1940’s. Many of the lessons learned have provided general principles of cancer pharmacology and resistance. He described experiments demonstrating its target of inhibition, dihydrofolate reductase, its multiple sites of action on folate dependent enzymes after its conversion to a polyglutamate, its transport into cells via the reduced folate transporter, and its induction (selection) of drug-resistant cells that display mutant DHFR, amplified DHFR, loss of transporter, or loss of polyglutamation. He related these mechanisms of resistance to similar findings in drug resistant mutants selected by molecularly targeted drugs such as receptor tyrosine kinase inhibitors. Dr. Chabner also pointed out that leukemias were cured only when multiple agents that were independently toxic to leukemia cells were combined.

Jennifer Grandis. Dr. Grandis focused on a approaches to increase the efficacy of EGFR inhibitors for the treatment of head and neck cancers. Studies to date have demonstrated that targeting EGFR with the monoclonal antibody cetuximab, in combination with radiation or chemotherapy, improves the survival of HNSCC patients. These findings led to the FDA approval of cetuximab for HNSCC treatment where it is the only approved molecular targeting agent. However, despite ubiquitous EGFR expression in HNSCC tumors, only a subset of patients respond to cetuximab- containing therapies. The mechanisms of primary or acquired resistance to cetuximab are incompletely understood and predictive biomarkers are lacking. Thus, Dr. Grandis generated preclinical models of cetuximab resistance in an attempt to elucidate candidate biomarkers that can then be tested in the clinic. Receptor cross talk is increasingly recognized as a potential mechanism of acquired therapeutic resistance. To date, several possible pathways that may contribute to the limited responses seen with cetuximab therapy have been identified. These include the truncated EGFR, EGFR variant III (EGFRvIII), activation of a C-terminal fragment of HER2 (611-CTF), signaling through p70S6Kinase, and/or activation of PI3K, among others. Src family kinases and HGF-c-MET signaling have also been implicated in EGFR tyrosine kinase inhibitor resistance in HNSCC. To model resistance effectively in the laboratory, Dr. Grandis and her colleagues have developed robust preclinical models including heterotopic tumorgrafts derived from patients who progress on cetuximab treatment. The acquisition of high quality tissue specimens before cetuximab treatment and in the setting of cetuximab resistance facilitates validation of potential mechanisms in preclinical models to be tested in clinical specimens. These efforts are coupled to clinical trials aimed to overcome resistance. The heterogeneous genetic underpinnings of HNSCC underscore the likelihood that any individual targeting strategy will be most beneficial in a subgroup of patients. Prospective identification of cetuximab sensitive tumors is likely to improve outcome. EGFR TKIs have not proven effective in unselected HNSCC populations, and Dr. Grandis showed how emerging characterization of exceptional responders to EGFR inhibitors improves our ability to restrict the use these agents to individuals most likely to benefit.

Kris Wood. Dr. Wood was elected as a Forbeck Scholar and presented his innovative research to determine how cancers become resistant to targeted therapies. Cancer cells can activate diverse signaling pathways to overcome the cytotoxic effects of drugs. Blocking these resistance pathways may lead to improved therapies, but for many drugs the pathways that drive resistance are unknown. To address this problem, Dr. Wood created and screened a library of engineered pathway-activating mutant cDNAs to identify those whose expression leads to enhanced survival of cancer cells in the presence of various targeted and cytotoxic chemotherapies. Using this approach, Dr. Wood’s team has uncovered both biomarkers of therapeutic response as well as combination drugging strategies that convert resistant tumors to a drug sensitive state. These findings articulate a systematic approach for identifying the signaling pathways controlling oncogenic and other biological phenotypes that may be useful in a range of discovery contexts.

Yael Mosse. Despite improvements in cancer outcomes over the past several decades, neuroblastoma remains a leading cause of childhood cancer deaths, even with dramatic increases in treatment intensity. In addition, the 30-40% of children who do survive are typically left with significant long-term side effects, many of which can be life threatening. In this era of more rational therapies, substantial efforts are being made to identify optimal targets for each type of cancer. Dr. Mosse’s team discovered that activating mutations within the tyrosine kinase domain of the ALK oncogene are the major cause of hereditary neuroblastoma. Discovery of activating mutations in the intact ALK gene as the major cause of hereditary neuroblastoma provided the first example of a pediatric cancer caused by germline mutations in an oncogene. The additional occurrence of somatically acquired ALK-activating mutations has provided additional and compelling rationale for targeting this oncogenic RTK therapeutically. Through these findings, ALK has emerged as the first tractable oncogene for targeted therapy in neuroblastoma. Rapid clinical translation of findings with ALK in neuroblastoma prompted a phase 1 trial of crizotinib in patients with recurrent or refractory cancer. Results from this study highlighted the differential sensitivity to ALK kinase inhibition of ALK-translocated versus ALK-mutated disease. The results also underlined the need for further detailed investigation of ALK mutations in order to optimize clinical application of ALK inhibitors in neuroblastoma. With this goal, the Mosse group analyzed the spectrum of ALK mutations, and their clinical significance, in a large representative series of neuroblastoma cases. They established structure/function relationships for these mutations and developed computational approaches to predict the effect of novel ALK mutations, and to be able to make upfront predictions about which patients will respond to crizotinib or other ALK inhibitors and which mutations impart de novo resistance to direct ALK kinase inhibition. The findings described here allow physician scientist to formulate molecular diagnostic screening recommendations for newly diagnosed neuroblastoma patients, which will be important as ALK inhibitors for childhood cancer are evaluated in clinical trials.

Kristopher Sarosiek. Dr. Sarosiek was elected as a Forbeck Scholar and presented his innovative research to determine how to manipulate “cell death” programs to increase the efficacy of cancer treatments. Apoptosis is a highly regulated form of cell death that is critical for maintenance of homeostasis as well as the anti-tumor activity of many chemotherapeutic agents, both cytotoxic and targeted. Dr. Sarosiek’s team recently reported that the level of apoptotic priming within a primary tumor is a major determinant of whether a patient will respond to classical chemotherapies. Specifically, cancers that are close to the threshold of apoptosis, or “primed for death,” are sensitive to chemotherapies while those that are far from the threshold of apoptosis, or “unprimed,” are resistant. Tumors can be unprimed by expressing an excess of unbound anti-apoptotic proteins that are poised to neutralize any pro-apoptotic signaling that the cell encounters due to damage or stress. Dr. Sarosiek’s team is working to identify and characterize the key upstream factors that modulate apoptotic priming within cancers. With a better understanding of what makes tumors primed or unprimed for apoptosis, they may be able to pharmacologically target these factors and improve patient outcomes.

Michael Gottesman. As our understanding of the complexity of mechanisms that leads to cancer grows, so does our appreciation of the myriad ways in which cancer resists treatment with chemotherapy. The development of targeted cancer therapy has allowed us to define mutations in targets that result in resistance, as well as downstream alterations in growth-promoting pathways that circumvent targeted therapies. In addition, resistance to chemotherapy can arise from alterations in cellular pharmacology, including decreased uptake and increased efflux of drugs, and altered metabolism or intracellular distribution. Finally, to avoid toxic effects of chemotherapy, some cancers change “lifestyle” as seen with epithelial to mesenchymal transition (EMT), or activation of alternative growth promoting pathways. The study of cultured cancer cells may be a poor model for understanding drug resistance in patients, since these cells have adapted to monolayer growth in tissue culture and express many drug-resistance genes that might not be clinically relevant. Because cancers are inherently heterogeneous, any cancer can exhibit one or more of the known (or unknown) mechanisms of resistance, so it is difficult to predict a priori for a single cancer what mechanism is the most likely to occur. Dr. Gottesman is investigating how individualized molecular analysis of cancers at presentation and during acquisition of resistance mechanisms will be needed to develop effective ways to circumvent resistance.

Bob Kerbel. A long-standing problem in experimental oncology therapeutics is the tendency of preclinical therapy models in mice to over predict anticancer therapeutic activity. A frequent finding is a highly encouraging if not spectacular result in mice that is eventually followed by complete failure in clinical trials, especially at the randomized phase III level. There remains a tendency to evaluate drugs in the setting of established primary tumors, whether transplanted or spontaneously arising. In contrast, the vast majority of clinical trials involve patients with metastatic disease, where achieving significant success – at least when treating advanced metastatic disease – is a much more significant challenge. To address this gap, the Kerbel lab began developing models of advanced spontaneous metastatic disease, where the primary tumor has been surgically resected. Several results, while limited, appear promising with respect to increased clinical relevance and translation using these models. For example, prior studies of the antiangiogenic drug, sunitinib, a TKI targeting VEGF receptors, among others, was shown over a decade ago to be highly active in mice when treating breast cancers primarily grown in multiple models as primary established tumors. Subsequently, four different phase III randomized trials were undertaken evaluating sunitinib alone or in combination with different chemotherapy drugs in women with metastatic breast cancer. All these trials failed – thus constituting a rather discouraging and spectacular example of over prediction of drug activity in mice. Thus, breast cancer appears to be an example of an intrinsically sunitinib resistant tumor type, but this was not reflected in the prior preclinical studies. However, when the Kerbel group evaluated sunitinib alone or with chemotherapy in metastatic breast cancer models research resection of the primary orthotopic transplant tumor, no activity in the drug was observed (E. Guerin et al Cancer Research, 2013). The basis of the seemingly intrinsic resistant phenotype of metastatic breast cancer to sunitinib can now be evaluated using metastasis models. Preliminary results indicate robust angiogenesis is present in primary tumors, but absent in lung metastases; instead the metastases appear to be capitalizing on lung vessel co-option ie exploiting the abundant normal pulmonary vasculature, thereby obviating the need for extensive new blood vessel formation for expansion of tumor mass. In contrast, various chemotherapy regimens involving repetitive, low doses of chemotherapy drugs designed to minimize toxicity have been found to be highly active when treating advanced metastatic disease, especially combined with the targeted agents such as an antibody targeting the VEGF pathway. Given these aforementioned results the Kerbel lab is also evaluating mechanisms of acquired resistance to antiangiogenic drugs, and other types of drug, using models of metastasis rather than models involving treatment of primary tumors only. In summary, undertaking preclinical therapy studies in mice with advanced visceral metastatic disease has the potential of providing results having increased clinical/translational potential, and may constitute a promising strategy to study the basis of resistance (especially intrinsic resistance) to anticancer drugs, including various classes/types targeted agent.

Rakesh Jain. Dr. Jain showed that the blood and lymphatic vasculature, and the extracellular matrix associated with tumors are abnormal, which together create a hostile tumor microenvironment – characterized by hypoxia, low pH, high interstitial fluid pressure, high solid stresees. He then discussed how these abnormalities fuel malignant properties of a tumor while preventing treatments from reaching and attacking tumor cells. He proposed that if we could “normalize” the tumor microenvironment, we should be able to overcome treatment-resistance and improve the treatment outcome. He discussed two strategies to realize this goal: normalization of tumor vessels using anti-angiogenic agents and normalization of tumor matrix using anti-hypertensive drugs.

Specifically, he showed how judicious use of antiangiogenic agents—originally designed to starve tumors—could transiently “normalize” tumor vasculature, alleviate hypoxia, increase delivery of drugs and anti-tumor immune cells, and improve the outcome of various therapies in number of animal models. He also presented clinical evidence in support of this concept – two trials of antiangiogenics in newly diagnosed as well as recurrent glioblastoma patients. These trials revealed that the patients whose tumor blood perfusion/oxygenation increased in response to cediranib – a pan-VEGFR TKI – survived 6-9 months longer than those whose blood perfusion/oxygenation did not increase.

In parallel, by imaging collagen and measuring perfusion in tumors in vivo, his team demonstrated that the extracellular matrix compresses blood vessels and impedes drug and oxygen delivery in desmoplastic tumors (e.g., pancreatic ductal adenocarcinoma, hepatocellular carcinoma, a sub-set of breast cancers). They subsequently discovered that widely prescribed anti-hypertensive drugs – angiotensin receptor blockers – are capable of “normalizing” the extracellular matrix, opening compressed tumor vessels, and improving the delivery and efficacy of molecular and nanomedicine. This finding offers new hope for improving treatment in highly fibrotic tumors and has led to a clinical trial at MGH on losartan and chemotherapy in pancreatic ductal adenocarcinomas (NCT01821729).

Joan Brugge. Dr. Brugge discussed work from her laboratory in collaboration with Gordon Mills describing how treatment with therapies that target genes that drive ovarian and breast cancer tumor cells induce an adaptive/compensatory responses that confer protection from cytotoxicity. This response occurs selectively in subpopulations of tumor and involves an extracellular matrix-dependent upregulation and activation of proteins that protect cells from death induced by the cancer therapy. In addition, she described tumors in otherenvironments where non-tumor cells produce factors that protect the tumors from cytoxicity of the therapies. She then led a discussion of the challenges in overcoming therapy resistance due adaptive responses of tumor cells, factors form the tumor microenvironment that confer protection, as well as genetic and epigenetic alterations with the tumor cells. She proposed that targeting underlying programs that integrate signals from all of the resistance mechanism represents a worthwhile approaches to overcome resistance.

Vito Quaranta. Dr. Quaranta discussed a novel metric to evaluate in vitro response of cultured cell lines to targeted therapy, the Drug Induced Proliferation (DIP) rate. With this metric, a cancer cell line drug response is richly represented as a clonal distribution of DIP rates, which encapsulates dynamics (since rates are time-dependent) and single-cell heterogeneity of response in a cell population. This is a major departure from current drug response assessment based on averaged and single time-point IC50 assays. Quaranta showed data supporting the concept that parameters from in vitro DIP rate distributions could be used to populate mathematical models that predict the in vivo time course of targeted drug treatment (depth of response and time to rebound). Simulations from these models reveal that clonal lineages with positive DIP rates appear to be a reservoir for rebound to therapy, opening potential avenues to circumvent it. The DIP rate approach may be a significant step towards fulfilling the long sought-after goal of predicting in vivo drug response from in vitro assays, in order to streamline clinical trials and facilitate personalized treatment.

Jeffrey Rosen. The Rosen lab does not study the mechanisms of resistance to specific targeted therapies per se. Rather they have discovered an intimate relationship between epithelial to mesenchymal transition (EMT), cancer stem cells, and the claudin-low subtype of breast cancer representing a cell state with aggressive and therapeutic resistant properties. The claudin-low subtype is a recently identified rare molecular subtype of human breast cancer. Like basal-like tumors, these tumors are generally triple (ER, PR, HER2) negative and important therapeutically as there are currently no targeted agents directed at them. Studies comparing paired human breast cancer core biopsies before and after chemotherapy demonstrated that the gene signature from these residual (i.e, chemotherapy resistant) tumors identified the EMT/CSC enriched claudin-low and metaplastic subtypes. An independently derived core EMT interactome gene expression signature was also associated with these tumor subtypes. Furthermore, post–treatment residual tumors contained a higher fraction of claudin-low cells, also enriched with EMT/CSC properties, consistent with their therapeutic resistance. Claudin-low tumors express low levels of tight and adherens junction genes including claudin 3 and E-cadherin, and high levels of markers associated with epithelial-mesenchymal transition (EMT) including the EMT inducers Snail, Twist, Zeb1, and Zeb2. Currently, it is unknown whether the presence of the EMT phenotype is due to intrinsic genetic and epigenetic differences within a population of cells or a response to extrinsic factors emanating from the tumor microenvironment. However, the resulting intratumoral heterogeneity represents one of the major factors involved in tumor relapse and recurrence.

Studies using a basal-like p53 null genetically engineered model have revealed the importance of paracrine interactions including canonical Wnt signaling between different tumor cell subpopulations that influence tumor propagation. In a subset of the p53 null basal-like tumors, the TIC population can be identified using a canonical Wnt pathway reporter as well as by cell surface markers, and display altered DNA double-stranded break repair compared to the bulk tumor cells. The altered DDR is due in part to increased cell survival and non-homologous end joining activity. Treatment of this GEM model with an AKT/TORC1/TORC2 inhibitor Perifosine was able to sensitize these tumors to radiation-induced DNA damage. A similar response to radiation treatment was observed following mild hyperthermia induced using gold nanoparticles. Finally, we have shown that GEM models can be used to predict drug responsiveness in human cancers.

Cory Johannessen. Dr. Joannessen was elected as a Forbeck Scholar and presented his innovative research to determine how melanocyte differentiation impacts sensitivity to targeted therapy. To systematically address how cancers become resistant to targeted therapies, the Johannessen lab has employed a near-genome-scale cDNA/ORF expression library to comprehensively identify genes capable of inducing drug resistance to RAF, MEK, ERK, or combined RAF/MEK inhibitors in BRAFV600E melanoma. These studies nominated a diverse array of previously unappreciated resistance genes, pathways and cellular processes. More specifically, Johannessen uncovered a membrane-to-nucleus, lineage-restricted melanocytic signaling network not previously associated with drug resistance. These findings suggested that histone deacetylase inhibitors might contribute to an efficacious combinatorial treatment strategy. In fact, when these agents were combined with MAPK-pathway inhibitors, drug resistance was abrogated.

Collectively, these data suggest that oncogenic dysregulation of a lineage dependency or reinstatement of MAPK-pathway transcriptional output can cause resistance to RAF/MEK/ERK inhibition. In this context, resistance may be overcome by combining signaling- and chromatin-directed therapeutics. Moreover, these studies suggest that the application of genome-scale functional approaches that characterize anticancer drug resistance, together with directed experimental and clinical studies, may offer a general framework for discovery and clinical prioritization of novel therapeutic regimens.

Jeffrey Engelman. Dr. Engelman shared is laboratory and clinical efforts to understand how cancers become resistant to targeted therapies in the clinic. He shared his team’s efforts in which patient tumors are biopsied upon the development of resistance. These biopsies are interrogated for the resistance mechanisms using genetic and molecular studies. In addition, Dr. Engelman described how resistant cancers harvested directly from patients can be grown as tissue culture models and in mice. These novel laboratory models are interrogated directly using high-throughput screens to discover resistance mechanisms and identify newer, promising therapeutic strategies. Dr. Engelman shared the results of these efforts and highlighted the challenges that we face. He shared his results that individual patients may simultaneously harbor different resistant clones requiring distinct treatments. He also led a discussion that pointed out competing theories of how resistant clones emerge: 1) they pre-exist before treatment and are simply selected for by the treatment and 2) they are product of a multi-step evolution that begins with cells that initially survive treatment, and these surviving cells subsequently develop other molecular events (e.g., genetic mutations) leading to a fully resistant phenotype. The concepts underlying future therapeutic regimens that would effectively account for these findings were discussed.

Alberto Bardelli. The advent of the EGFR-targeted monoclonal antibodies cetuximab and panitumumab paved the way to the individualized treatment of metastatic colorectal cancer (mCRC). In the last 5 years it has become evident that mCRCs respond differently to EGFR-targeted agents and that the tumor-specific response has a genetic basis. After the initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of anti-EGFR therapies. Understanding the molecular bases of secondary resistance to cetuximab and panitumumab is required to design additional therapeutic options. Dr. Bardelli’s lab discovered that molecular alterations in KRAS,NRAS,BRAF and MET are causally associated with the onset of acquired resistance to anti-EGFR blockade in colorectal cancers. Dr. Bardelli optimized a diagnostic platform to identify resistance-associated genetic alterations in the blood of patients (liquid biopsy) months before radiographic documentation of disease progression. Preclinical models of relapse including cell lines and patient-derived xenografts (xenopatients) allowed Dr. Bardelli to assess new lines of therapy. Overall Dr. Bardelli’s results provide the rationale for delaying or reversing resistance to anti EGFR therapies in mCRCs and support the initiation of innovative – molecularly driven- clinical trials.

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