Emerging Trends in Cancer Drug Discovery-From Drugging the "Undruggable" to Overcoming Resistance.

Technology advancement and the courage to challenge dogma have been key elements that have continuously shifted druggability limits. We illustrate this notion with several recent cancer drug-discovery examples, while also giving an outlook on the opportunities offered by newer modalities such as chemically induced proximity and direct targeting of RNA. Treatment resistance is a major impediment to the goal of durable efficacy and cure, but the confluence of new biological insights, novel drug modalities, and drug combinations is predicted to enable transformative progress in this decade and beyond.

Thinking Differently about Cancer Treatment Regimens.

Most experimental cancer drugs ultimately fail during the course of clinical development, contributing to the high cost of the few that are granted regulatory approval. Moreover, approved drugs often deliver only modest clinical benefit to patients with advanced disease due to the development of resistance. Here, we discuss opportunities we consider promising to overcome drug resistance associated with interactions between signaling pathways and the presence of multiple coexisting cell states within tumors with distinct vulnerabilities. We highlight how understanding drug-resistance mechanisms can enable innovative treatment regimens that deliver longer-lasting benefit to patients.

Unprovoked Stabilization and Nuclear Accumulation of the Naked Mole-Rat p53 Protein.

The naked mole-rat is a subterranean rodent, approximately the size of a mouse, renowned for its exceptional longevity (>30 years) and remarkable resistance to cancer. To explore putative mechanisms underlying the cancer resistance of the naked mole-rat, we investigated the regulation and function of the most commonly mutated tumor suppressor, TP53, in the naked mole-rat. We found that the p53 protein in naked mole-rat embryonic fibroblasts (NEFs) exhibits a half-life more than ten times in excess of the protein's characterized half-life in mouse and human embryonic fibroblasts. We determined that the long half-life of the naked mole-rat p53 protein reflects protein-extrinsic regulation. Relative to mouse and human p53, a larger proportion of naked mole-rat p53 protein is constitutively localized in the nucleus prior to DNA damage. Nevertheless, DNA damage is sufficient to induce activation of canonical p53 target genes in NEFs. Despite the uniquely long half-life and unprecedented basal nuclear localization of p53 in NEFs, naked mole-rat p53 retains its canonical tumor suppressive activity. Together, these findings suggest that the unique stabilization and regulation of the p53 protein may contribute to the naked mole-rat's remarkable resistance to cancer.

Targeting microtubules sensitizes drug resistant lung cancer cells to lysosomal pathway inhibitors.

Oncogene-addicted cancers are predominantly driven by specific oncogenic pathways and display initial exquisite sensitivity to designer therapies, but eventually become refractory to treatments. Clear understanding of lung tumorigenic mechanisms is essential for improved therapies. Methods: Lysosomes were analyzed in EGFR-WT and mutant cells and corresponding patient samples using immunofluorescence or immunohistochemistry and immunoblotting. Microtubule organization and dynamics were studied using immunofluorescence analyses. Also, we have validated our findings in a transgenic mouse model that contain EGFR-TKI resistant mutations. Results: We herein describe a novel mechanism that a mutated kinase disrupts the microtubule organization and results in a defective endosomal/lysosomal pathway. This prevents the efficient degradation of phosphorylated proteins that become trapped within the endosomes and continue to signal, therefore amplifying downstream proliferative and survival pathways. Phenotypically, a distinctive subcellular appearance of LAMP1 secondary to microtubule dysfunction in cells expressing EGFR kinase mutants is seen, and this may have potential diagnostic applications for the detection of such mutants. We demonstrate that lysosomal-inhibitors re-sensitize resistant cells to EGFR tyrosine-kinase inhibitors (TKIs). Identifying the endosome-lysosome pathway and microtubule dysfunction as a mechanism of resistance allows to pharmacologically intervene on this pathway. Conclusions: We find that the combination of microtubule stabilizing agent and lysosome inhibitor could reduce the tumor progression in EGFR TKI resistant mouse models of lung cancer.

A Single-Step, High-Dose Selection Scheme Reveals Distinct Mechanisms of Acquired Resistance to Oncogenic Kinase Inhibition in Cancer Cells.

Despite the remarkable clinical efficacy demonstrated by molecularly targeted cancer therapeutics, the benefits are typically temporary due to the emergence of acquired drug resistance. This has spurred a massive effort by the cancer research community to identify mechanisms used by cancer cells to evade treatment. Among the various methodologies developed and employed to identify such mechanisms, the most commonly used approach has been to model acquired resistance by exposing cancer cells in culture to gradually increasing concentrations of drug over an extended period of time. Here, we employed a less commonly used variation on this approach, wherein resistant cells are selected by immediately exposing cancer cells to a continuous, high concentration of drug. Using this approach, we isolated clones representing three distinct mechanisms of resistance to inhibition of MET kinase activity from a single clonally derived cancer cell line. The emergent clones had acquired resistance through engagement of alternative receptor tyrosine kinases either through upregulation of FGF3 or HBEGF or increased MAPK signaling through an activating V600E mutation in BRAF. Importantly, these mechanisms were not identified using the conventional "ramp-up" approach in previous studies that employed the same cell line. These results suggest that the particular nature of the selection scheme employed in cell culture modeling studies can determine which potential resistance mechanisms are identified and which ones may be missed, highlighting the need for careful consideration of the specific approach used to model resistance in cultured cells. SIGNIFICANCE: Through modeling resistance to MET kinase inhibition in cultured cancer cells using single-step, high-dose selection, these findings highlight that the specific nature of the selection protocol impacts which resistance mechanisms are identified.See related commentary by Floros et al., p. 25.

Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.

The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.

Adaptive Resistance to Dual BRAF/MEK Inhibition in BRAF-Driven Tumors through Autocrine FGFR Pathway Activation.

PURPOSE: Combined MAPK pathway inhibition using dual BRAF and MEK inhibitors has prolonged the duration of clinical response in patients with BRAFV600E-driven tumors compared with either agent alone. However, resistance frequently arises. EXPERIMENTAL DESIGN: We generated cell lines resistant to dual BRAF/MEK inhibition and utilized a pharmacologic synthetic lethal approach to identify a novel, adaptive resistance mechanism mediated through the fibroblast growth factor receptor (FGFR) pathway. RESULTS: In response to drug treatment, transcriptional upregulation of FGF1 results in autocrine activation of FGFR, which potentiates extracellular signal-regulated kinases (ERK) activation. FGFR inhibition overcomes resistance to dual BRAF/MEK inhibitors in both cell lines and patient-derived xenograft (PDX) models. Abrogation of this bypass mechanism in the first-line setting enhances tumor killing and prevents the emergence of drug-resistant cells. Moreover, clinical data implicate serum FGF1 levels in disease prognosis. CONCLUSIONS: Taken together, these results describe a new, adaptive resistance mechanism that is more commonly observed in the context of dual BRAF/MEK blockade as opposed to single-agent treatment and reveal the potential clinical utility of FGFR-targeting agents in combination with BRAF and MEK inhibitors as a promising strategy to forestall resistance in a subset of BRAF-driven cancers.

Regulation of autophagy, NF-κB signaling, and cell viability by miR-124 in KRAS mutant mesenchymal-like NSCLC cells.

KRAS mutant non-small cell lung cancer (NSCLC) may be classified into epithelial or mesenchymal subtypes. Despite having the same "driver" mutation, mesenchymal NSCLCs are less responsive than are epithelial NSCLCs to inhibition of the RAS pathway. Identifying alternative networks that promote survival specifically in mesenchymal NSCLC may lead to more effective treatments for this subtype. Through their numerous targets in cellular signaling pathways, noncoding microRNAs (miRNAs) often function as tumor suppressors or oncogenes. In particular, some miRNAs regulate the epithelial-mesenchymal transition (EMT). We derived an EMT-related miRNA signature by profiling the abundance of miRNAs in a panel of epithelial (KE) or mesenchymal (KM) KRAS mutant NSCLC cell lines. This signature revealed a number of suppressed miRNAs in KM cell lines, including members of the miR-200 family, which can suppress tumor progression by inhibiting EMT. Reconstituting KM cells with one of these miRNAs, miR-124, disrupted autophagy and decreased cell survival by reducing the abundance of p62, which is both an adaptor for selective autophagy and a regulator of the transcription factor nuclear factor κB (NF-κB). Suppression of p62 by miR-124 correlated with reduced abundance of the autophagy activator beclin 1 (BECN1), the ubiquitin ligase TRAF6, and the NF-κB subunit RELA/p65. The abundance of miR-124 inversely correlated with the expression of BECN1 and TRAF6 in patient NSCLC samples. These findings reveal how the loss of miR-124 promotes cell survival networks in the aggressive mesenchymal subtype of KRAS mutant NSCLC, which might lead to improved subtype-selective therapeutic strategies for patients.

Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug Exposure.

Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over long interspersed repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.

Cotargeting of MEK and PDGFR/STAT3 Pathways to Treat Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal human diseases and remains largely refractory to available drug treatments. Insufficient targeting of the known oncogenic drivers and activation of compensatory feedback loops and inability to prevent metastatic spread contribute to poor prognosis for this disease. The KRAS-driven MEK pathway is mutationally activated in most pancreatic cancers and is an important target for therapeutics. Using a two-dimensional monolayer culture system as well as three-dimensional spheroid culture system, we conducted a screen of a large panel of anticancer agents and found that MAP2K (MEK) inhibitors were most effective in targeting PDAC spheroids in comparison with monolayer cultures. Combination treatment with an MEK inhibitor and the multikinase inhibitor ponatinib was effective in targeting pancreatic cancer cells both in monolayer and spheroids by effectively blocking signaling via the PDGFRα and MEK kinases, while also preventing the activation of STAT3- and S6-mediated compensatory feedback loops in cancer cells. Furthermore, using xenograft models, we demonstrate that cotreatment with a MEK inhibitor and ponatinib causes significant tumor regression. PDAC patient samples also provided evidence of increased STAT3 activation in PDAC tumors and MAPK1 (ERK) activation in liver metastases, implicating STAT3 and ERK as key drivers in primary tumors and metastases, respectively. These results reveal a combination drug treatment strategy that may be effective in pancreatic cancer. Mol Cancer Ther; 16(9); 1729-38. ©2017 AACR.

Ras-MEK Signaling Mediates a Critical Chk1-Dependent DNA Damage Response in Cancer Cells.

Cancer cell line profiling to identify previously unrecognized kinase dependencies revealed a novel nonmutational dependency on the DNA damage response checkpoint kinase Chk1. Although Chk1 is a promising therapeutic target in p53-deficient cancers, we found that Ras-MEK signaling engages Chk1 in a subset of osteosarcoma, ovarian, and breast cancer cells to enable their survival upon DNA damage, irrespective of p53 mutation status. Mechanistically, Ras-MEK signaling drives Chk1 expression and promotes cancer cell growth that produces genotoxic stress that requires Chk1 to mediate a response to the consequent DNA damage. Reciprocally, Chk1 engages a negative feedback loop to prevent hyperactivation of Ras-MEK signaling, thereby limiting DNA damage. Furthermore, exogenous DNA damage promotes Chk1 dependency, and pharmacologic Chk1 inhibition combined with genotoxic chemotherapy potentiates a DNA damage response and tumor cell killing. These findings reveal a mechanism-based diagnostic strategy to identify cancer patients that may benefit from Chk1-targeted therapy. Mol Cancer Ther; 16(4); 694-704. ©2017 AACR.

Proline Starvation Induces Unresolved ER Stress and Hinders mTORC1-Dependent Tumorigenesis.

The role of essential amino acids in metabolic reprogramming of cancer cells is now well established, whereas the role of non-essential amino acids (NEAAs) in malignancy remains less clear. Here, we have identified an important role for the NEAA proline in the tumorigenic potential of a subset of cancer cells. By profiling a large panel of cancer cell lines, we observed that proline consumption and expression of proline biosynthesis enzymes were well correlated with clonogenic and tumorigenic potential. Moreover, proline starvation or inhibition of proline biosynthesis enzymes impaired clonogenic/tumorigenic potential. Cancer cells exhibiting dependency on exogenous proline displayed hyperactivation of the mTORC1-mediated 4EBP1 signaling axis, as well as unresolved ER stress. Exogenous proline alleviated ER stress and promoted cellular homeostasis and clonogenicity. Increased dependence on proline may therefore define a specific vulnerability in some cancers that can be exploited by proline depletion.

Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs.

Acquired resistance to cancer drug therapies almost always occurs in advanced-stage patients even following a significant response to treatment. In addition to mutational mechanisms, various non-mutational resistance mechanisms have now been recognized. We previously described a chromatin-mediated subpopulation of reversibly drug-tolerant persisters that is dynamically maintained within a wide variety of tumour cell populations. Here we explore a potential role for microRNAs in such transient drug tolerance. Functional screening of 879 human microRNAs reveals miR-371-3p as a potent suppressor of drug tolerance. We identify PRDX6 (peroxiredoxin 6) as a key target of miR-371-3p in establishing drug tolerance by regulating PLA2/PKCα activity and reactive oxygen species. PRDX6 expression is associated with poor prognosis in cancers of multiple tissue origins. These findings implicate miR-371-3p as a suppressor of PRDX6 and suggest that co-targeting of peroxiredoxin 6 or modulating miR-371-3p expression together with targeted cancer therapies may delay or prevent acquired drug resistance.

Reproducible pharmacogenomic profiling of cancer cell line panels.

The use of large-scale genomic and drug response screening of cancer cell lines depends crucially on the reproducibility of results. Here we consider two previously published screens, plus a later critique of these studies. Using independent data, we show that consistency is achievable, and provide a systematic description of the best laboratory and analysis practices for future studies.

mTORC1-Dependent Metabolic Reprogramming Underlies Escape from Glycolysis Addiction in Cancer Cells.

Although glycolysis is substantially elevated in many tumors, therapeutic targeting of glycolysis in cancer patients has not yet been successful, potentially reflecting the metabolic plasticity of tumor cells. In various cancer cells exposed to a continuous glycolytic block, we identified a recurrent reprogramming mechanism involving sustained mTORC1 signaling that underlies escape from glycolytic addiction. Active mTORC1 directs increased glucose flux via the pentose phosphate pathway back into glycolysis, thereby circumventing a glycolysis block and ensuring adequate ATP and biomass production. Combined inhibition of glycolysis and mTORC1 signaling disrupted metabolic reprogramming in tumor cells and inhibited their growth in vitro and in vivo. These findings reveal novel combinatorial therapeutic strategies to realize the potential benefit from targeting the Warburg effect.

Communication in Drug Development: "Translating" Scientific Discovery.

The discovery and development of new medicines that promote human health and potentially extend natural life remains a remarkably challenging endeavor. In this Commentary, we identify key elements of communication required to successfully translate promising biological findings to novel approved drug therapies and discuss the attendant challenges and opportunities.