IGF-binding proteins secreted by cancer-associated fibroblasts induce context-dependent drug sensitization of lung cancer cells.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are often linked to drug resistance. Here, we found that coculture with CAFs or culture in CAF-conditioned medium unexpectedly induced drug sensitivity in certain lung cancer cell lines. Gene expression and secretome analyses of CAFs and normal lung-associated fibroblasts (NAFs) revealed differential abundance of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), which promoted or inhibited, respectively, signaling by the receptor IGF1R and the kinase FAK. Similar drug sensitization was seen in gefitinib-resistant, EGFR-mutant PC9GR lung cancer cells treated with recombinant IGFBPs. Conversely, drug sensitivity was decreased by recombinant IGFs or conditioned medium from CAFs in which IGFBP5 or IGFBP6 was silenced. Phosphoproteomics and receptor tyrosine kinase (RTK) array analyses indicated that exposure of PC9GR cells to CAF-conditioned medium also inhibited compensatory IGF1R and FAK signaling induced by the EGFR inhibitor osimertinib. Combined small-molecule inhibition of IGF1R and FAK phenocopied the CAF-mediated effects in culture and increased the antitumor effect of osimertinib in mice. Cells that were osimertinib resistant and had MET amplification or showed epithelial-to-mesenchymal transition also displayed residual sensitivity to IGFBPs. Thus, CAFs promote or reduce drug resistance in a context-dependent manner, and deciphering the relationship between the differential content of CAF secretomes and the signaling dependencies of the tumor may reveal effective combination treatment strategies.

CDK4/6 inhibition synergizes with inhibition of P21-Activated Kinases (PAKs) in lung cancer cell lines.

Theoretically, small molecule CDK4/6 inhibitors (CDK4/6is) represent a logical therapeutic option in non-small cell lung cancers since most of these malignancies have wildtype RB, the key target of CDKs and master regulator of the cell cycle. Unfortunately, CDK4/6is are found to have limited clinical activity as single agents in non-small cell lung cancer. To address this problem and to identify effective CDK4/6i combinations, we screened a library of targeted agents for efficacy in four non-small cell lung cancer lines treated with CDK4/6 inhibitors Palbociclib or Abemaciclib. The pan-PAK (p21-activated kinase) inhibitor PF03758309 emerged as a promising candidate with viability ratios indicating synergy in all 4 cell lines and for both CDK4/6is. It is noteworthy that the PAKs are downstream effectors of small GTPases Rac1 and Cdc42 and are overexpressed in a wide variety of cancers. Individually the compounds primarily induced cell cycle arrest; however, the synergistic combination induced apoptosis, accounting for the synergy. Surprisingly, while the pan-PAK inhibitor PF03758309 synergizes with CDK4/6is, no synergy occurs with group I PAK inhibitors FRAX486 or FRAX597. Cell lines treated only with Ribociclib, FRAX486 or FRAX597 underwent G1/G0 arrest, whereas combination treatment with these compounds predominantly resulted in autophagy. Combining high concentrations of FRAX486, which weakly inhibits PAK4, and Ribociclib, mimics the autophagy and apoptotic effect of PF03758309 combined with Ribociclib. FRAX597, a PAKi that does not inhibit PAK4 did not reduce autophagy in combination with Ribociclib. Our results suggest that a unique combination of PAKs plays a crucial role in the synergy of PAK inhibitors with CDK4/6i. Targeting this unique PAK combination, could greatly improve the efficacy of CDK4/6i and broaden the spectrum of cancer treatment.

Divergent Polypharmacology-Driven Cellular Activity of Structurally Similar Multi-Kinase Inhibitors through Cumulative Effects on Individual Targets.

Despite recent successes of precision and immunotherapies there is a persisting need for novel targeted or multi-targeted approaches in complex diseases. Through a systems pharmacology approach, including phenotypic screening, chemical and phosphoproteomics, and RNA-seq, we elucidated the targets and mechanisms underlying the differential anticancer activity of two structurally related multi-kinase inhibitors, foretinib, and cabozantinib, in lung cancer cells. Biochemical and cellular target validation using probe molecules and RNAi revealed a polypharmacology mechanism involving MEK1/2, FER, and AURKB, which were each more potently inhibited by foretinib than cabozantinib. Based on this, we developed a synergistic combination of foretinib with barasertib, a more potent AURKB inhibitor, for MYC-amplified small-cell lung cancer. This systems pharmacology approach showed that small structural changes of drugs can cumulatively, through multiple targets, result in pronounced anticancer activity differences and that detailed mechanistic understanding of polypharmacology can enable repurposing opportunities for cancers with unmet medical need.

p53-Pirh2 Complex Promotes Twist1 Degradation and Inhibits EMT.

Epithelial-mesenchymal transition (EMT) is a critical process involved in cancer metastasis and chemoresistance. Twist1 is a key EMT-inducing transcription factor, which is upregulated in multiple types of cancers and has been shown to promote tumor cell invasiveness and support tumor progression. Conversely, p53 is a tumor suppressor gene that is frequently mutated in cancers. This study demonstrates the ability of wild-type (WT) p53 to promote the degradation of Twist1 protein. By forming a complex with Twist1 and the E3 ligase Pirh2, WT p53 promotes the ubiquitination and proteasomal degradation of Twist1, thus inhibiting EMT and maintaining the epithelial phenotype. The ability of p53 to induce Twist1 degradation is abrogated when p53 is mutated. Consequently, the loss of p53-induced Twist1 degradation leads to EMT and the acquisition of a more invasive cancer phenotype.Implication: These data provide new insight into the metastatic process at the molecular level and suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to curtail cancer progression.

Functional Proteomics and Deep Network Interrogation Reveal a Complex Mechanism of Action of Midostaurin in Lung Cancer Cells.

Lung cancer is associated with high prevalence and mortality, and despite significant successes with targeted drugs in genomically defined subsets of lung cancer and immunotherapy, the majority of patients currently does not benefit from these therapies. Through a targeted drug screen, we found the recently approved multi-kinase inhibitor midostaurin to have potent activity in several lung cancer cells independent of its intended target, PKC, or a specific genomic marker. To determine the underlying mechanism of action we applied a layered functional proteomics approach and a new data integration method. Using chemical proteomics, we identified multiple midostaurin kinase targets in these cells. Network-based integration of these targets with quantitative tyrosine and global phosphoproteomics data using protein-protein interactions from the STRING database suggested multiple targets are relevant for the mode of action of midostaurin. Subsequent functional validation using RNA interference and selective small molecule probes showed that simultaneous inhibition of TBK1, PDPK1 and AURKA was required to elicit midostaurin's cellular effects. Immunoblot analysis of downstream signaling nodes showed that combined inhibition of these targets altered PI3K/AKT and cell cycle signaling pathways that in part converged on PLK1. Furthermore, rational combination of midostaurin with the potent PLK1 inhibitor BI2536 elicited strong synergy. Our results demonstrate that combination of complementary functional proteomics approaches and subsequent network-based data integration can reveal novel insight into the complex mode of action of multi-kinase inhibitors, actionable targets for drug discovery and cancer vulnerabilities. Finally, we illustrate how this knowledge can be used for the rational design of synergistic drug combinations with high potential for clinical translation.

EGFR Mediates Responses to Small-Molecule Drugs Targeting Oncogenic Fusion Kinases.

Oncogenic kinase fusions of ALK, ROS1, RET, and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1+ lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naïve cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion-positive lung cancer cells, mouse models, and human clinical specimens before the onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to cotarget EGFR to reduce the risks of developing drug resistance. Cancer Res; 77(13); 3551-63. ©2017 AACR.

Novel approach for the detection of intraperitoneal micrometastasis using an ovarian cancer mouse model.

Patients with epithelial ovarian cancer have the best overall survival when maximal surgical effort is accomplished. However, despite numerous technological advances, surgery still relies primarily on white-light reflectance and the surgeon's vision. As such, micrometastases are usually missed and most patients clinically classified as a complete responder eventually recur and succumb to the disease. Our objective is to develop optical enhancers which can aid in the visualization of ovarian cancer micrometastasis. To this end we developed a nanoparticle (NP) platform, which is specifically targeted to the tumor microenvironment. Targeting is achieved by coating FDA-approved PLGA-PEG NP with the peptide sequence RGD, which binds with high affinity to αVß3 integrins present in both the tumor-associated neovasculature and on the surface of ovarian cancer cells. Administration of the NP platform carrying fluorescent dyes to mice bearing intraperitoneal ovarian cancer allowed visualization of tumor-associated vasculature and its contrast against normal blood vessels. More importantly, we demonstrate the visualization of intraperitoneal ovarian cancer micrometastasis as small as 100 µm with optimal resolution. Finally, we demonstrate that the fluorescent dye cargo was able to penetrate intra-tumorally. Such modality could be used to allow microscopic surgical debulking to assure maximal surgical effort.

Chemoproteomics Reveals Novel Protein and Lipid Kinase Targets of Clinical CDK4/6 Inhibitors in Lung Cancer.

Several selective CDK4/6 inhibitors are in clinical trials for non-small cell lung cancer (NSCLC). Palbociclib (PD0332991) is included in the phase II/III Lung-MAP trial for squamous cell lung carcinoma (LUSQ). We noted differential cellular activity between palbociclib and the structurally related ribociclib (LEE011) in LUSQ cells. Applying an unbiased mass spectrometry-based chemoproteomics approach in H157 cells and primary tumor samples, we here report distinct proteome-wide target profiles of these two drug candidates in LUSQ, which encompass novel protein and, for palbociclib only, lipid kinases. In addition to CDK4 and 6, we observed CDK9 as a potent target of both drugs. Palbociclib interacted with several kinases not targeted by ribociclib, such as casein kinase 2 and PIK3R4, which regulate autophagy. Furthermore, palbociclib engaged several lipid kinases, most notably, PIK3CD and PIP4K2A/B/C. Accordingly, we observed modulation of autophagy and inhibition of AKT signaling by palbociclib but not ribociclib.

Clostridium perfringens enterotoxin C-terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy-resistant ovarian cancer.

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.

Murine model for non-invasive imaging to detect and monitor ovarian cancer recurrence.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

Multiple blocks in the engagement of oxidative phosphorylation in putative ovarian cancer stem cells: implication for maintenance therapy with glycolysis inhibitors.

Survival rate in ovarian cancer has not improved since chemotherapy was introduced a few decades ago. The dismal prognosis is mostly due to disease recurrence where majority of the patients succumb to the disease. The demonstration that tumors are comprised of subfractions of cancer cells displaying heterogeneity in stemness potential, chemoresistance, and tumor repair capacity suggests that recurrence may be driven by the chemoresistant cancer stem cells. Thus to improve patient survival, novel therapies should eradicate this cancer cell population. We show that in contrast to the more differentiated ovarian cancer cells, the putative CD44+/MyD88+ ovarian cancer stem cells express lower levels of pyruvate dehydrogenase, Cox-I, Cox-II, and Cox-IV, and higher levels of UCP2. Together, this molecular phenotype establishes a bioenergetic profile that prefers the use of glycolysis over oxidative phosphorylation to generate ATP. This bioenergetic profile is conserved in vivo and therefore a maintenance regimen of 2-deoxyglucose administered after Paclitaxel treatment is able to delay the progression of recurrent tumors and decrease tumor burden in mice. Our findings strongly suggest the value of maintenance with glycolysis inhibitors with the goal of improving survival in ovarian cancer patients.

Phenotypic modifications in ovarian cancer stem cells following Paclitaxel treatment.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite initial responsiveness, 80% of EOC patients recur and present with chemoresistant and a more aggressive disease. This suggests an underlying biology that results in a modified recurrent disease, which is distinct from the primary tumor. Unfortunately, the management of recurrent EOC is similar to primary disease and does not parallel the molecular changes that may have occurred during the process of rebuilding the tumor. We describe the characterization of unique in vitro and in vivo ovarian cancer models to study the process of recurrence. The in vitro model consists of GFP+/CD44+/MyD88+ EOC stem cells and mCherry+/CD44-/MyD88- EOC cells. The in vivo model consists of mCherry+/CD44+/MyD88+ EOC cells injected intraperitoneally. Animals received four doses of Paclitaxel and response to treatment was monitored by in vivo imaging. Phenotype of primary and recurrent disease was characterized by quantitative polymerase chain reaction (qPCR) and Western blot analysis. Using the in vivo and in vitro models, we confirmed that chemotherapy enriched for CD44+/MyD88+ EOC stem cells. However, we observed that the surviving CD44+/MyD88+ EOC stem cells acquire a more aggressive phenotype characterized by chemoresistance and migratory potential. Our results highlight the mechanisms that may explain the phenotypic heterogeneity of recurrent EOC and emphasize the significant plasticity of ovarian cancer stem cells. The significance of our findings is the possibility of developing new venues to target the surviving CD44+/MyD88+ EOC stem cells as part of maintenance therapy and therefore preventing recurrence and metastasis, which are the main causes of mortality in patients with ovarian cancer.