A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.

Accumulating evidence implicates heterogeneity within cancer cell populations in the response to stressful exposures, including drug treatments. While modeling the acute response to various anticancer agents in drug-sensitive human tumor cell lines, we consistently detected a small subpopulation of reversibly "drug-tolerant" cells. These cells demonstrate >100-fold reduced drug sensitivity and maintain viability via engagement of IGF-1 receptor signaling and an altered chromatin state that requires the histone demethylase RBP2/KDM5A/Jarid1A. This drug-tolerant phenotype is transiently acquired and relinquished at low frequency by individual cells within the population, implicating the dynamic regulation of phenotypic heterogeneity in drug tolerance. The drug-tolerant subpopulation can be selectively ablated by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents, potentially yielding a therapeutic opportunity. Together, these findings suggest that cancer cell populations employ a dynamic survival strategy in which individual cells transiently assume a reversibly drug-tolerant state to protect the population from eradication by potentially lethal exposures.

Systematic identification of genomic markers of drug sensitivity in cancer cells.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

Epidermal growth factor receptor mutations in lung cancer.

The development and clinical application of inhibitors that target the epidermal growth factor receptor (EGFR) provide important insights for new lung cancer therapies, as well as for the broader field of targeted cancer therapies. We review the results of genetic, biochemical and clinical studies focused on somatic mutations of EGFR that are associated with the phenomenon of oncogene addiction, describing 'oncogenic shock' as a mechanistic explanation for the apoptosis that follows the acute treatment of susceptible cells with kinase inhibitors. Understanding the genetic heterogeneity of epithelial tumours and devising strategies to circumvent their rapid acquisition of resistance to targeted kinase inhibitors are essential to the successful use of targeted therapies in common epithelial cancers.

A common signaling cascade may underlie "addiction" to the Src, BCR-ABL, and EGF receptor oncogenes.

"Oncogene addiction" describes an unexplained dependency of cancer cells on a particular cellular pathway for survival or proliferation. We report that differential attenuation rates of prosurvival and proapoptotic signals in oncogene-dependent cells contribute to cell death following oncogene inactivation. Src-, BCR-ABL-, and EGF receptor-dependent cells exhibit a similar profile of signal attenuation following oncogene inactivation characterized by rapid diminution of phospho-ERK, -Akt, and -STAT3/5, and a delayed accumulation of the proapoptotic effector phospho-p38 MAPK. These findings implicate a transient imbalance in survival and apoptotic oncogenic outputs in the apoptotic response to oncogene inactivation. Moreover, these observations implicate a common profile of signal attenuation for multiple oncogenes and suggest that "addiction" associated with apoptosis reflects an active rather than a passive process.

Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR.

Somatic activating mutations in EGFR identify a subset of non-small cell lung cancer that respond to tyrosine kinase inhibitors. Acquisition of drug resistance is linked to a specific secondary somatic mutation, EGFR T790M. Here we describe a family with multiple cases of non-small cell lung cancer associated with germline transmission of this mutation. Four of six tumors analyzed showed a secondary somatic activating EGFR mutation, arising in cis with the germline EGFR mutation T790M. These observations implicate altered EGFR signaling in genetic susceptibility to lung cancer.

Inhibition of gamma-secretase activity inhibits tumor progression in a mouse model of pancreatic ductal adenocarcinoma.

BACKGROUND & AIMS: The Notch signaling pathway is required for the expansion of undifferentiated pancreatic progenitor cells during embryonic development and has been implicated in the progression of pancreatic ductal adenocarcinoma (PDAC). The interaction of Notch ligands with their receptors promotes a gamma-secretase-dependent cleavage of the Notch receptor and release of the Notch intracellular domain, which translocates to the nucleus and activates transcription. We investigated the role of this pathway in PDAC progression. METHODS: We tested the effects of a gamma-secretase inhibitor (GSI) that blocks Notch signaling in PDAC cell lines and a genetically engineered mouse model of PDAC (Kras p53 L/+ mice). RESULTS: Notch signaling was activated in PDAC precursors and advanced tumors. The GSI inhibited the growth of premalignant pancreatic duct-derived cells in a Notch-dependent manner. Additionally, in a panel of over 400 human solid tumor-derived cell lines, PDAC cells, as a group, were more sensitive to the GSI than any other tumor type. Finally, the GSI completely inhibited tumor development in the genetically engineered model of invasive PDAC (P < .005, chi2 test; compared with mice exposed to vehicle). CONCLUSIONS: These results suggest that Notch signaling is required for PDAC progression. Pharmacologic targeting of this pathway offers therapeutic potential in this treatment-refractory malignancy.

ErbBs in lung cancer.

Lung cancer remains the leading cause of cancer deaths worldwide, and advanced stage disease is largely refractory to conventional chemotherapy. Thus, there is an important need for alternative treatment strategies, and the ErbB proteins have emerged as potentially important therapeutic drug targets in this setting, apparently reflecting a state of "oncogene addiction" in some lung tumors. In this review, we discuss the recent identification of mutations that promote activation of ErbB family proteins in a subset of lung cancers, and the development of selective inhibitors of these proteins that have demonstrated clinical efficacy. We also discuss the problem of drug resistance, which severely limits the clinical utility of such agents, and has prompted intense efforts to better understand molecular mechanisms underlying drug resistance as well as strategies to overcome or prevent such resistance.

Identification of genotype-correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling.

Kinase inhibitors constitute an important new class of cancer drugs, whose selective efficacy is largely determined by underlying tumor cell genetics. We established a high-throughput platform to profile 500 cell lines derived from diverse epithelial cancers for sensitivity to 14 kinase inhibitors. Most inhibitors were ineffective against unselected cell lines but exhibited dramatic cell killing of small nonoverlapping subsets. Cells with exquisite sensitivity to EGFR, HER2, MET, or BRAF kinase inhibitors were marked by activating mutations or amplification of the drug target. Although most cell lines recapitulated known tumor-associated genotypes, the screen revealed low-frequency drug-sensitizing genotypes in tumor types not previously associated with drug susceptibility. Furthermore, comparing drugs thought to target the same kinase revealed striking differences, predictive of clinical efficacy. Genetically defined cancer subsets, irrespective of tissue type, predict response to kinase inhibitors, and provide an important preclinical model to guide early clinical applications of novel targeted inhibitors.

Drug-target identification from total cellular lysate by drug-induced conformational changes.

Identification of drug targets is a key step in the development of novel pharmaceuticals. To this end, chemical probes or affinity matrices are often used, requiring substantial structure-activity relationship (SAR) studies. Here we report on the development of a novel technique for drug-target identification from total cellular lysate conducted independently of SAR information. This technique relies on binding of a drug to its target inducing a conformational change in target protein, thereby altering its susceptibility to proteolysis and resulting in specific degradation in some cases or in protection of target protein in others. As proof of concept, three drugs with identified targets were used. First, incubation of cellular lysates with okadaic acid elicited a specific protective effect on its target, protein phosphatase 2A catalytic subunit. Second, specific protection from exogenous protease of FKBP12 by FK506 and Hsp90 fragments by radicicol were observed. We then used the method to validate the targets of UCS15A, an Src signaling inhibitor. UCS15A induced proteolysis of a number of proteins, one of which was identified as Sam68. These studies suggest that the technology may be generally useful for identification and validation of drug targets.

Reduced Erlotinib sensitivity of epidermal growth factor receptor-mutant non-small cell lung cancer following cisplatin exposure: a cell culture model of second-line erlotinib treatment.

PURPOSE: Epidermal growth factor receptor (EGFR) kinase inhibitors induce dramatic clinical responses in a subset of non-small cell lung cancer (NSCLC) patients with advanced disease, and such responses are correlated with the presence of somatic activating mutations within the EGFR kinase domain. Consequently, one of these inhibitors, erlotinib, has been Food and Drug Administration-approved as a second- or third-line treatment for chemotherapy-refractory advanced NSCLC. However, responses are typically relatively short-lived due to acquired drug resistance, prompting studies to determine whether first-line treatment with EGFR inhibitors could provide greater clinical benefit. NSCLC-derived cell lines have provided a powerful system for modeling EGFR mutation-correlated sensitivity to EGFR inhibitors and for modeling mechanisms of acquired drug resistance that are observed clinically. EXPERIMENTAL DESIGN: In a cell culture model of an erlotinib-sensitive EGFR-mutant NSCLC cell line, we tested the hypothesis that prior exposure to platinum agents, a standard component of NSCLC chemotherapy treatment, affects the subsequent response to erlotinib. RESULTS: Indeed, NSCLC cells initially selected for growth in cisplatin exhibit 5-fold reduced sensitivity to erlotinib, even after propagating the cisplatin-treated cells in the absence of cisplatin for several months. This lingering effect of cisplatin exposure appears to reflect changes in PTEN tumor suppressor activity and persistent EGFR-independent signaling through the phosphatidylinositol 3-kinase/AKT survival pathway. CONCLUSIONS: These preclinical findings suggest that first-line chemotherapy treatment of EGFR-mutant NSCLCs may reduce the benefit of subsequent treatment with EGFR kinase inhibitors and should prompt further clinical investigation of these inhibitors as a first-line therapy in NSCLC.

High-throughput lung cancer cell line screening for genotype-correlated sensitivity to an EGFR kinase inhibitor.

Human cancer cell lines that can be propagated and manipulated in culture have proven to be excellent models for studying many aspects of gene function in cancer. In addition, they can provide a powerful system for assessing the molecular determinants of sensitivity to anticancer drugs. They have also been used in recent studies to identify genomic alterations and gene expression patterns that provide important insights into the genetic features that distinguish the properties of tumor cells associated with similar histologies. We have established a large repository of human tumor cell lines (>1000) corresponding to a wide variety of tumor types, and we have developed a methodology for profiling the collection for sensitivity to putative anticancer compounds. The rationale for examining tumor cell lines on this relatively large scale reflects accumulating evidence indicating that there is substantial genetic heterogeneity among human tumor cells-even those derived from tumors of similar histologies. Thus, to develop an accurate picture of the molecular determinants of tumorigenesis and response to therapy, it is essential to study the nature of such heterogeneity in a relatively large sample set. Here, we describe the methodologies used to conduct such screens and we describe a "proof-of-concept" screen using the EGFR kinase inhibitor, erlotinib (Tarceva), with a panel of lung cancer lines to demonstrate a correlation between EGFR mutations and drug sensitivity.

"Oncogenic shock": explaining oncogene addiction through differential signal attenuation.

"Oncogene addiction" describes the curious acquired dependence of tumor cells on an activated oncogene for their survival and/or proliferation, a phenomenon that has important implications for the success of targeted cancer therapies. However, the mechanisms explaining oncogene addiction remain elusive. We propose that "addiction" may be an illusion generated as a consequence of differential attenuation rates of prosurvival and proapoptotic signals emanating from an oncoprotein acutely following its inactivation. According to this model, which we call "oncogenic shock," prosurvival signals dissipate quickly on oncoprotein inactivation whereas proapoptotic signals linger sufficiently long to commit the cell to an apoptotic death. This mechanism may contribute to the rapid and dramatic clinical responses observed in some cancer patients treated with selective tyrosine kinase inhibitors and could yield additional drug targets that determine the balance of signaling outputs from activated oncoproteins.

UCS15A, a novel small molecule, SH3 domain-mediated protein-protein interaction blocking drug.

Protein-protein interactions play critical regulatory roles in mediating signal transduction. Previous studies have identified an unconventional, small-molecule, Src signal transduction inhibitor, UCS15A. UCS15A differed from conventional Src-inhibitors in that it did not alter the levels or the tyrosine kinase activity of Src. Our studies suggested that UCS15A exerted its Src-inhibitory effects by a novel mechanism that involved the disruption of protein-protein interactions mediated by Src. In the present study we have examined the ability of UCS15A to disrupt the interaction of Src-SH3 with Sam68, both in vivo and in vitro. This ability of UCS15A was not restricted to Src-SH3 mediated protein-protein interactions, since the drug was capable of disrupting the in vivo interactions of Sam68 with other SH3 domain containing proteins such as Grb2 and PLCgamma. In addition, UCS15A was capable of disrupting other typical SH3-mediated protein-protein interactions such as Grb2-Sos1, cortactin-ZO1, as well as atypical SH3-mediated protein-protein interactions such as Grb2-Gab1. However, UCS15A was unable to disrupt the non-SH3-mediated protein-protein interactions of beta-catenin, with E-cadherin and alpha-catenin. In addition, UCS15A had no effect on the SH2-mediated interaction between Grb2 and activated Epidermal Growth Factor receptor. Thus, the ability of UCS15A, to disrupt protein-protein interactions appeared to be restricted to SH3-mediated protein-protein interactions. In this regard, UCS15A represents the first example of a non-peptide, small molecule agent capable of disrupting SH3-mediated protein-protein interactions. In vitro analyses suggested that UCS15A did not bind to the SH3 domain itself but rather may interact directly with the target proline-rich domains.

Synthetic inhibitors of proline-rich ligand-mediated protein-protein interaction: potent analogs of UCS15A.

The proline-rich motif in proteins is known to function as a ligand sequence that binds to protein modules such as SH3, WW, and several other protein interaction domains. These proline-rich ligand-mediated protein-protein interactions (abbreviated PLPI) are important in many signaling pathways that are involved in various diseases. Our previous studies showed that UCS15A, produced by Streptomyces species, inhibited PLPI. Here we report on synthetic analogs of UCS15A that show more potent activity than UCS15A in inhibiting PLPI. A synthetic analog, compound 2c, blocked in vitro PLPI of Sam68-Fyn-SH3 as well as in vivo PLPI of Grb2-Sam68 and Grb2-Sos1. Activation of MEK was also inhibited by compound 2c. Unlike UCS15A, compound 2c was an order of magnitude less cytotoxic and did not cause morphological changes in treated cells.

Development of radicicol analogues.

Radicicol, a macrocyclic antibiotic produced by fungi, was originally isolated many years ago, and was described as tyrosine kinase inhibitor. We also rediscovered radicicol as an inhibitor of signal transduction of oncogene products, such as K-ras and v-Src, using yeast and mammalian cell-based assays. In a study of mechanisms of action, it was revealed that radicicol depletes the Hsp90 client signaling molecules in cells, and thus inhibit the signal transduction pathway. In addition, direct binding of radicicol to the N-terminal ATP/ADP binding site of Hsp90 was shown, and thus radicicol has been recognized as a structurally unique antibiotic that binds and inhibits the molecular chaperone Hsp90. Although radicicol itself has little or no activity in animals because of instability in animals, its oxime derivatives showed potent antitumor activities against human tumor xenograft models. Hsp90 client proteins were depleted and apoptosis was induced in the tumor specimen treated with radicicol oxime derivatives. Taken together, these results suggest that the antitumor activity of radicicol oxime derivatives is mediated by binding to Hsp90 and destabilization of Hsp90 client proteins in the tumor. Among Hsp90 clients, we focused on ErbB2 and Bcr-Abl as examples of important targets of Hsp90 inhibitors. Radicicol oxime showed potent antitumor activity against ER negative/ErbB2 overexpressing breast cancer and Bcr-Abl expressing CML. Putative mechanisms of action and future directions of radicicol oxime against these kinds of tumor are discussed.

Inhibition of Casein Kinase 1 Alpha Prevents Acquired Drug Resistance to Erlotinib in EGFR-Mutant Non-Small Cell Lung Cancer.

Patients with lung tumors harboring activating mutations in the EGF receptor (EGFR) show good initial treatment responses to the EGFR tyrosine kinase inhibitors (TKI) erlotinib or gefitinib. However, acquired resistance invariably develops. Applying a focused shRNA screening approach to identify genes whose knockdown can prevent and/or overcome acquired resistance to erlotinib in several EGFR-mutant non-small cell lung cancer (NSCLC) cell lines, we identified casein kinase 1 α (CSNK1A1, CK1α). We found that CK1α suppression inhibits the NF-κB prosurvival signaling pathway. Furthermore, downregulation of NF-κB signaling by approaches independent of CK1α knockdown can also attenuate acquired erlotinib resistance, supporting a role for activated NF-κB signaling in conferring acquired drug resistance. Importantly, CK1α suppression prevented erlotinib resistance in an HCC827 xenograft model in vivo. Our findings suggest that patients with EGFR-mutant NSCLC might benefit from a combination of EGFR TKIs and CK1α inhibition to prevent acquired drug resistance and to prolong disease-free survival.

A new structural class of proteasome inhibitors identified by microbial screening using yeast-based assay.

A yeast-based growth interference assay was developed utilizing a yeast strain in which expression of Xenopus cyclin A1 was induced to elevate cell division cycle 28 (Cdc28) kinase activity. Since the hyperactivation of Cdc28 kinase in yeast results in a growth-arrest phenotype, compounds which could rescue the cyclin A1-induced growth arrest might be potential new, antitumor drug candidates acting on the cyclin-dependent, kinase-mediated, cell cycle regulation pathway. In the course of our microbial screening program, the new Streptomyces metabolites, belactosins, were identified. As reported previously, belactosin A induced cell cycle arrest at G2/M phase in human cancer cells. However, the molecular mechanism of action was unknown. We herein demonstrate the proteasome inhibition by belactosin A. Belactosin A did not inhibit yeast Cdc28 kinase and human cyclin-dependent kinase in vitro. On the other hand, it inhibited the chymotrypsin-like activity of the rabbit 20S proteasome. From the initial SAR studies, we identified a hydrophobic belactosin A derivative, KF33955, which exhibited a 100-fold greater growth-inhibitory activity against HeLa S3 cells than belactosin A, presumably due to its higher cell permeability. The biochemical analysis using KF33955 suggested that the proteasome inhibitory activity of KF33955 were irreversible and required the beta-lactone moiety to inhibit the proteasome. KF33955 increased the intracellular levels of protein ubiquitination in NIH3T3 cells. In addition, KF33955 treatment resulted in the accumulation of known proteasome substrates in HeLa S3 cells. These results identify belactosin A as a useful lead compound to target proteasome for the treatment of disease whose etiology is dependent on the unregulated ubiquitin-proteasome pathway.

A phase I study of erlotinib and hydroxychloroquine in advanced non-small-cell lung cancer.

INTRODUCTION: This investigator-initiated study explores the safety, maximum tolerated dose, clinical response, and pharmacokinetics of hydroxychloroquine (HCQ) with and without erlotinib in patients with advanced non-small-cell lung cancer. METHODS: Patients with prior clinical benefit from an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor were randomized to HCQ or HCQ plus erlotinib in a 3 + 3 dose-escalation schema. RESULTS: Twenty-seven patients were treated, eight with HCQ (arm A) and 19 with HCQ plus erlotinib (arm B). EGFR mutations were detected in 74% of the patients and 85% had received two or more prior therapies. Arm A had no dose-limiting toxicities, but the maximum tolerated dose was not reached as this arm closed early to increase overall study accrual. In arm B, one patient each experienced grade 3 rash, nail changes, skin changes, nausea, dehydration, and neutropenia; one had grade 4 anemia; and one developed fatal pneumonitis, all considered unrelated to HCQ. There were no dose-limiting toxicities, therefore the highest tested dose for HCQ with erlotinib 150 mg was 1000 mg daily. One patient had a partial response to erlotinib/HCQ, for an overall response rate of 5% (95% confidence interval, 1-25). This patient had an EGFR mutation and remained on therapy for 20 months. Administration of HCQ did not alter the pharmacokinetics of erlotinib. CONCLUSIONS: HCQ with or without erlotinib was safe and well tolerated. The recommended phase 2 dose of HCQ was 1000 mg when given in combination with erlotinib 150 mg.

A chemical biology approach to developing STAT inhibitors: molecular strategies for accelerating clinical translation.

STAT transcription factors transduce signals from the cell surface to the nucleus, where they regulate the expression of genes that control proliferation, survival, self-renewal, and other critical cellular functions. Under normal physiological conditions, the activation of STATs is tightly regulated. In cancer, by contrast, STAT proteins, particularly STAT3 and STAT5, become activated constitutively, thereby driving the malignant phenotype of cancer cells. Since these proteins are largely dispensable in the function of normal adult cells, STATs represent a potentially important target for cancer therapy. Although transcription factors have traditionally been viewed as suboptimal targets for pharmacological inhibition, chemical biology approaches have been particularly fruitful in identifying compounds that can modulate this pathway through a variety of mechanisms. STAT inhibitors have notable anti-cancer effects in many tumor systems, show synergy with other therapeutic modalities, and have the potential to eradicate tumor stem cells. Furthermore, STAT inhibitors identified through the screening of chemical libraries can then be employed in large scale analyses such as gene expression profiling, RNA interference screens, or large-scale tumor cell line profiling. Data derived from these studies can then provide key insights into mechanisms of STAT signal transduction, as well as inform the rational design of targeted therapeutic strategies for cancer patients.

Exploiting the balance between life and death: targeted cancer therapy and "oncogenic shock".

Rational approaches to targeted cancer therapy have begun to predominate the pipelines of oncology drug development. Our rapidly increasing understanding of the "wiring" of tumor cells and the vulnerabilities of such cells that can potentially be exploited through targeted treatments has opened up enormous opportunities for improved therapies. Accumulating evidence suggests that many of these vulnerabilities reflect states of dependency or "addiction" that are unique to cancer cells (versus normal cells). Such addiction can arise due to a strict dependency on a single activated oncogene, a cell lineage-specific factor, or even to a non-oncogene, and identifying these "Achilles' heels" within individual tumors remains an important challenge to the development of targeted therapies. Recent technology advances that facilitate high-throughput genomic analysis of tumor specimens and genome-wide RNA interference screening in cancer cell lines are key among the newly developed tools that are beginning to reveal novel context-dependent therapeutic targets, and the rapidly increasing application of these technologies by a large number of laboratories will undoubtedly lead to more effective cancer therapies in the near future. Here, we review the various forms of cancer cell addiction and their relevance to the discovery of novel therapeutic targets.