A novel pan-PI3K inhibitor KTC1101 synergizes with anti-PD-1 therapy by targeting tumor suppression and immune activation.

BACKGROUND: Phosphoinositide 3-kinases (PI3Ks) are critical regulators of diverse cellular functions and have emerged as promising targets in cancer therapy. Despite significant progress, existing PI3K inhibitors encounter various challenges such as suboptimal bioavailability, potential off-target effects, restricted therapeutic indices, and cancer-acquired resistance. Hence, novel inhibitors that overcome some of these challenges are needed. Here, we describe the characterization of KTC1101, a novel pan-PI3K inhibitor that simultaneously targets tumor cell proliferation and the tumor microenvironment. Our studies demonstrate that KTC1101 significantly increases the anti-PD-1 efficacy in multiple pre-clinical mouse models. METHODS: KTC1101 was synthesized and characterized employing chemical synthesis, molecular modeling, Nuclear Magnetic Resonance (NMR), and mass spectrometry. Its target specificity was confirmed through the kinase assay, JFCR39 COMPARE analysis, and RNA-Seq analysis. Metabolic stability was verified via liver microsome and plasma assays, pharmacokinetics determined by LC-MS/MS, and safety profile established through acute toxicity assays to determine the LD50. The antiproliferative effects of KTC1101 were evaluated in a panel of cancer cell lines and further validated in diverse BALB/c nude mouse xenograft, NSG mouse xenograft and syngeneic mouse models. The KTC1101 treatment effect on the immune response was assessed through comprehensive RNA-Seq, flow cytometry, and immunohistochemistry, with molecular pathways investigated via Western blot, ELISA, and qRT-PCR. RESULTS: KTC1101 demonstrated strong inhibition of cancer cell growth in vitro and significantly impeded tumor progression in vivo. It effectively modulated the Tumor Microenvironment (TME), characterized by increased infiltration of CD8+ T cells and innate immune cells. An intermittent dosing regimen of KTC1101 enhanced these effects. Notably, KTC1101 synergized with anti-PD-1 therapy, significantly boosting antitumor immunity and extending survival in preclinical models. CONCLUSION: KTC1101's dual mechanism of action-directly inhibiting tumor cell growth and dynamically enhancing the immune response- represents a significant advancement in cancer treatment strategies. These findings support incorporating KTC1101 into future oncologic regimens to improve the efficacy of immunotherapy combinations.

Cytochrome P450 2J2 is required for the natural compound austocystin D to elicit cancer cell toxicity.

Austocystin D is a natural compound that induces cytochrome P450 (CYP) monooxygenase-dependent DNA damage and growth inhibition in certain cancer cell lines. Cancer cells exhibiting higher sensitivity to austocystin D often display elevated CYP2J2 expression. However, the essentiality and the role of CYP2J2 for the cytotoxicity of this compound remain unclear. In this study, we demonstrate that CYP2J2 depletion alleviates austocystin D sensitivity and DNA damage induction, while CYP2J2 overexpression enhances them. Moreover, the investigation into genes involved in austocystin D cytotoxicity identified POR and PGRMC1, positive regulators for CYP activity, and KAT7, a histone acetyltransferase. Through genetic manipulation and analysis of multiomics data, we elucidated a role for KAT7 in CYP2J2 transcriptional regulation. These findings strongly suggest that CYP2J2 is crucial for austocystin D metabolism and its subsequent cytotoxic effects. The potential use of austocystin D as a therapeutic prodrug is underscored, particularly in cancers where elevated CYP2J2 expression serves as a biomarker.

Discovery of novel DNA-damaging agents through phenotypic screening for DNA double-strand break.

DNA double-strand breaks (DSBs) seriously damage DNA and promote genomic instability that can lead to cell death. They are the source of conditions such as carcinogenesis and aging, but also have important applications in cancer therapy. Therefore, rapid detection and quantification of DSBs in cells are necessary for identifying carcinogenic and anticancer factors. In this study, we detected DSBs using a flow cytometry-based high-throughput method to analyze γH2AX intensity. We screened a chemical library containing 9600 compounds and detected multiple DNA-damaging compounds, although we could not identify mechanisms of action through this procedure. Thus, we also profiled a representative compound with the highest DSB potential, DNA-damaging agent-1 (DDA-1), using a bioinformatics-based method we termed "molecular profiling." Prediction and verification analysis revealed DDA-1 as a potential inhibitor of topoisomerase IIα, different from known inhibitors such as etoposide and doxorubicin. Additional investigation of DDA-1 analogs and xenograft models suggested that DDA-1 is a potential anticancer drug. In conclusion, our findings established that combining high-throughput DSB detection and molecular profiling to undertake phenotypic analysis is a viable method for efficient identification of novel DNA-damaging compounds for clinical applications.

Invivo anti-cancer activity of 10-methyl-aplog-1, a simplified analog of aplysiatoxin, and its possible signaling pathway associated with G1 arrest.

Naturally occurring protein kinase C (PKC) activators such as phorbol esters, teleocidins, and aplysiatoxins, have the potential to become anti-cancer agents, since they are anti-proliferative against specific cancer cell lines in vitro. However, their potent tumor-promoting and proinflammatory activities have hampered their clinical uses. Recently, we developed 10-methyl-aplog-1 (1), a simplified analog of tumor-promoting debromoaplysiatoxin (DAT), which retained anti-proliferative activity comparable to DAT, but induced neither tumorigenesis nor inflammation on mouse skin. Our previous study suggested that PKCα and δ were involved in the cell line-selective anti-proliferative activity of 1, but the downstream signaling of PKC isozymes remained unknown. In this study, we confirmed that 1 inhibited the growth of three aplog-sensitive cancer cell lines (NCI-H460, HCC-2998, and HBC-4) without severe side effects in mice xenograft models. In addition, in vitro analysis using A549, one of the aplog-sensitive cell lines in vitro, revealed that PKCα induced PP2A-mediated attenuation of the Akt/S6 signaling axis. Since S6 inhibition in A549 was reported to result in G1 arrest, this pathway could be involved in the PKCα-dependent anti-proliferative activity of 1.

Structural Determination, Total Synthesis, and Biological Activity of Iezoside, a Highly Potent Ca2+-ATPase Inhibitor from the Marine Cyanobacterium Leptochromothrix valpauliae.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein on the endoplasmic reticulum (ER) that transports Ca2+ from the cytosol into the ER. As its function is associated with various biological phenomena, SERCA has been recognized as a promising druggable target. Here, we report the second-strongest SERCA-inhibitory compound known to date, which we isolated from the marine cyanobacterium Leptochromothrix valpauliae and named iezoside (1). The structure of iezoside (1) is fundamentally different from that of any other SERCA inhibitor, and its potency is the strongest among marine natural products (Ki 7.1 nM). In this article, we report our comprehensive analysis of iezoside (1), which covers its isolation, structural characterization supported by density functional theory (DFT) calculations and statistical analysis, total synthesis, and clarification of the mode of action of its potent antiproliferative activity (IC50 6.7 ± 0.4 nM against HeLa cells).

Synthesis of Acetogenin Analogs Comprising Pyrimidine Moieties Linked by Amine Bonds and Their Inhibitory Activity against Human Cancer Cell Lines.

Here, we synthesized three acetogenin analogs containing pyrimidine moieties linked by amine bonds, which represent the skeleton structure of pyrimidifen, a mitochondrial complex I-inhibiting insecticide. Replacing the pyrimidine moiety linked by the amine bond remarkably enhanced growth-inhibitory activity of the analogs against several human cancer cell lines. Moreover, these analogs selectively and potently inhibited the growth of these human cancer cell lines regardless of the pyrimidine substituents. Furthermore, COMPARE analyses suggested that these analogs inhibited cancer growth by inhibiting mitochondrial complex I. Our study provides insights into the design of acetogenin analogs as novel antitumor agents.

Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor.

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.

(S)-Erypoegin K, an isoflavone isolated from Erythrina poeppigiana, is a novel inhibitor of topoisomerase IIα: Induction of G2 phase arrest in human gastric cancer cells.

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has a single chiral carbon in its structure and exists naturally as a racemic mixture. Our previous study showed (S)-erypoegin K selectively exhibits potent anti-proliferative and apoptosis-inducing activity against human leukemia HL-60 cells. To identify the target molecule of (S)-erypoegin K, we employed the human cancer cell panel analysis (termed JFCR39) coupled with a drug sensitivity database of pharmacologically well-characterized drugs for comparison using the COMPARE algorithm. (S)-erypoegin K exhibited a similar profile to that of etoposide, suggesting the molecular target for erypoegin K may be topoisomerase II (Topo II). Subsequent experiments using purified human Topo IIα established that the (S)-isomer selectively stabilizes the cleavage complex composed of double-stranded plasmid DNA and the enzyme. Moreover, (S)-erypoegin K inhibited decatenation of kinetoplast DNA. Molecular docking studies clearly indicated specific binding of the (S)-isomer to the active site of Topo IIα involving hydrogen bonds that help stabilize the cleavage complex. (S)-erypoegin K displayed potent cytotoxic activity against two human gastric cancer cells GCIY and MKN-1 with IC50 values of 0.270 and 0.327 µM, respectively, and induced enzyme activities of caspase 3 and 9. Cell cycle analysis showed marked cell cycle arrest at G2 phase in both cell lines. (S)-erypoegin K also displayed significant antitumor activity toward GCIY xenografted mice. The present study suggests (S)-erypoegin K acts as a Topo II inhibitor to block the G2/M transition of cancer cells.

Serum IL-8 level as a candidate prognostic marker of response to anti-angiogenic therapy for metastatic colorectal cancer.

PURPOSE: Liver metastasis (LM) is associated with poor prognosis in patients with metastatic colorectal cancer (mCRC). Here, we investigated the prognostic utility of several serum factors in mCRC patients with or without LM who were treated with anti-angiogenic agents in first-line (FL) or salvage-line (SL) settings. METHODS: A combined cohort of 125 patients was analyzed in this single institute pooled analysis: FL cohort receiving bevacizumab (n = 71) and SL cohort receiving regorafenib (n = 54). Blood samples were obtained at baseline (BL) and during treatment, and serum factors were measured by ELISA. Overall survival (OS) was analyzed using Kaplan-Meier curves, the log-rank test, and Cox proportional hazard regression methods. RESULTS: In univariate analysis of the combined cohort, right-sided CRC, primary unresected tumor, wild-type KRAS, LM, ≥ 2 metastatic sites, and SL were associated with shorter OS; in multivariable analysis, LM and SL remained significant. Serum angiopoietin-2 (Ang-2) levels ≥ 2190.3 pg/ml and interleukin (IL)-8 levels ≥ 15.1 pg/ml at BL were significantly associated with LM. Using these cut-off values, patients with higher Ang-2 or IL-8 levels at BL had shorter OS than those with lower BL levels (Ang-2: hazard ratio [HR] 2.57, 95% confidence interval [CI] 1.47-4.51, P = 0.001; IL-8: HR 4.31, 95%CI 2.11-8.79, P < 0.001). High serum IL-8 level remained a significant predictor of shorter OS in multivariable analysis (HR 3.24, 95%CI 1.47-7.16, P = 0.004). CONCLUSION: Circulating IL-8 and Ang-2 levels are associated with LM in mCRC patients. IL-8 may be a prognostic marker of response to anti-angiogenic therapy, regardless of the treatment timing.

Effects of side chain length of 10-methyl-aplog-1, a simplified analog of debromoaplysiatoxin, on PKC binding, anti-proliferative, and pro-inflammatory activities.

10-Methyl-aplog-1 (1), a simplified analog of debromoaplysiatoxin, exhibits a high binding affinity for protein kinase C (PKC) isozymes and potent antiproliferative activity against several cancer cells with few adverse effects. A recent study has suggested that its phenol group in the side chain is involved in hydrogen bonding and CH/π interactions with the binding cleft-forming loops in the PKCδ-C1B domain. To clarify the effects of the side chain length on these interactions, four analogs of 1 with various lengths of side chains (2-5) were prepared. The maximal PKC binding affinity and antiproliferative activity were observed in 1. Remarkably, the introduction of a bromine atom into the phenol group of 2 increased not only these activities but also proinflammatory activity. These results indicated that 1 has the optimal side chain length as an anticancer seed. This conclusion was supported by docking simulations of 1-5 to the PKCδ-C1B domain.

Structure-Activity Relationships of Thiophene Carboxamide Annonaceous Acetogenin Analogs: Shortening the Alkyl Chain in the Tail Part Significantly Affects Their Growth Inhibitory Activity against Human Cancer Cell Lines.

In a previous study, we found that the thiophene carboxamide solamin analog, which is a mono-tetrahydrofuran annonaceous acetogenin, showed potent antitumor activity through the inhibition of mitochondrial complex I. In this study, we synthesized analogs with short alkyl chains instead of the n-dodecyl group in the tail part. We evaluated their growth inhibitory activities against human cancer cell lines. We found that the alkyl chain in the tail part plays an essential role in their activity.

Epidermal growth factor receptor mRNA expression: A potential molecular escape mechanism from regorafenib.

Regorafenib has improved the survival of patients with refractory metastatic colorectal cancer (mCRC), yet the mechanisms of inherited or acquired resistance are not well understood. A total of 50 patients with refractory mCRC were enrolled. Circulating tumor cell (CTC) enumeration was carried out at baseline, day 21 after initiation of regorafenib, and at the time of progression of disease (PD) using the CellSearch System (Veridex LLC, NJ, USA). Poly(A) mRNA was extracted from CTCs, and gene expression of epithelial and epithelial-mesenchymal transition markers was analyzed by a multiplex-PCR based DNA Chip. Patients with fewer than 3 CTCs at baseline and day 21 had a longer progression-free survival than those with 3 or more CTCs (3.3 vs 2.0 months, P = .008 and 3.3 vs 2.0 months, P = .004, respectively). Patients with fewer than 3 CTCs at baseline and day 21 had a longer overall survival (OS) than those with 3 or more CTCs (10.0 vs 4.6 months, P < .001 and 8.7 vs 3.8 months, P = .003, respectively). In multivariable analysis, CTC counts remained significantly associated with OS at baseline and day 21 (P = .019 and P = .028). Circulating tumor cell EGFR gene expression was upregulated at day 21 and/or PD in 64% of patients. Patients had significantly increased EGFR expression at PD compared to baseline (P = .041) and at day 21 and/or PD compared to baseline (P = .004). Our findings suggest that CTC count and EGFR expression could be useful markers of regorafenib efficacy and outcomes. Upregulation of CTC EGFR expression might be a molecular escape mechanism under regorafenib therapy.

High-throughput screening in colorectal cancer tissue-originated spheroids.

Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.

Stereoisomer-Specific Induction of G2/M Phase Arrest and Apoptosis by 9-(E,Z)-Hydroxyoctadecadienoic Acid in Mouse Lymphoma Cells.

Hydroxyoctadecadienoic acids (HODEs) are generated by oxidation of linoleic acid in vivo and thought to mediate various pathophysiological responses. In this study, we examined the effects of HODEs on EL4 mouse lymphoma cell growth and found that 9-(E,Z)-HODE inhibited EL4 cell growth in a dose-dependent manner, whereas no such growth inhibition was observed with other isomers (9-(E,E)-, 13-(Z,E)-, or 13-(E,E)-HODE), suggesting that the growth-inhibitory effect of HODEs was stereospecific. Analysis by flow cytometry (FACS) with annexin V and propidium iodide (PI) staining showed that 9-(E,Z)-HODE induced apoptosis with G2/M phase arrest. We next examined the growth inhibition profile of 9-(E,Z)-HODE against a panel of 39 human cancer cell lines (JFCR39). The fingerprint of growth inhibition by 9-(E,Z)-HODE exhibited a high degree of similarity to that by MLN4924, an inhibitor of NEDD8-activating enzyme. The intracellular NEDD8 (ubiquitin-like protein) expression in EL4 cells was decreased by the treatment with 9-(E,Z)-HODE as assessed by immunoblotting and flow cytometry. In conclusion, 9-(E,Z)-HODE specifically induced G2/M phase arrest and apoptosis, and the decrease of NEDD8 expression might be involved in this effect.

Target Identification of Yaku'amide B and Its Two Distinct Activities against Mitochondrial FoF1-ATP Synthase.

Yaku'amide B (1b) is a structurally unique tetradecapeptide bearing four ß,ß-dialkylated α,ß-unsaturated amino acid residues. Growth-inhibitory profile of 1b against a panel of 39 human cancer cell lines is distinct from those of clinically used anticancer drugs, suggesting a novel mechanism of action. We achieved total syntheses of chemical probes based on 1b and elucidated the cellular target and mode of action of 1b. Fluorescent (3, 4) and biotinylated (5, 6) derivatives of 1b were prepared for cell imaging studies and pull-down assays, respectively. In addition, the unnatural enantiomer of 1b ( ent-1b) and its fluorescent probe ( ent-3) were synthesized for control experiments. Subcellular localization analysis using 3 and 4 showed that 1b selectively accumulates in the mitochondria of MCF-7 human breast cancer cells. Pull-down assays with 6 revealed FoF1-ATP synthase as the major target protein of 1b. Consistent with these findings, biochemical activity assays showed that 1b inhibits ATP production catalyzed by mitochondrial FoF1-ATP synthase. Remarkably, 1b was also found capable of enhancing the ATP hydrolytic activity of FoF1-ATP synthase. On the other hand, ent-1b inhibits ATP synthesis more weakly than does 1b and does not affect ATP hydrolysis, suggesting the stereospecific requirement for the characteristic multimodal functions of 1b. These findings corroborate that 1b causes growth arrest in MCF-7 cells by inhibiting ATP production and enhancing ATP hydrolysis, thereby depleting the cellular ATP pool. This study provides, for the first time, a structural basis for the design and development of anticancer agents exploiting the novel mode of action of 1b.

Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles.

Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.

M-COPA, a novel Golgi system disruptor, suppresses apoptosis induced by Shiga toxin.

Shiga toxin (Stx) is a main virulence factor of Stx-producing Escherichia coli (STEC) that contributes to diarrhea and hemorrhagic colitis and occasionally to fatal systemic complications. Therefore, the development of an antidote to neutralize Stx toxicity is urgently needed. After internalization into cells, Stx is transferred to the Golgi apparatus via a retrograde vesicular transport system. We report here that 2-methylcoprophilinamide (M-COPA), a compound that induces disassembly of the Golgi apparatus by inactivating ADP-ribosylation factor 1 (Arf1), suppresses Stx-induced apoptosis. M-COPA inhibited transport of Stx from the plasma membrane to the Golgi apparatus and suppressed degradation of anti-apoptotic proteins and the activation of caspases. These findings suggest that inhibition of Stx retrograde transport by M-COPA could be a novel approach to suppress Stx toxicity.

Loss of the Phenolic Hydroxyl Group and Aromaticity from the Side Chain of Anti-Proliferative 10-Methyl-aplog-1, a Simplified Analog of Aplysiatoxin, Enhances Its Tumor-Promoting and Proinflammatory Activities.

Aplysiatoxin (ATX) is a protein kinase C (PKC) activator with potent tumor-promoting activity. In contrast, 10-methyl-aplog-1 (1), a simplified analog of ATX, was anti-proliferative towards several cancer cell lines without significant tumor-promoting and proinflammatory activities. To determine the effects of the phenolic group on the biological activities of 1, we synthesized new derivatives (2, 3) that lack the phenolic hydroxyl group and/or the aromatic ring. Compound 2, like 1, showed potent anti-proliferative activity against several cancer cell lines, but little with respect to tumor-promoting and proinflammatory activities. In contrast, 3 exhibited weaker growth inhibitory activity, and promoted inflammation and tumorigenesis. The binding affinity of 3 for PKCδ, which is involved in growth inhibition and apoptosis, was several times lower than those of 1 and 2, possibly due to the absence of the hydrogen bond and CH/π interaction between its side chain and either Met-239 or Pro-241 in the PKCδ-C1B domain. These results suggest that both the aromatic ring and phenolic hydroxyl group can suppress the proinflammatory and tumor-promoting activities of 1 and, therefore, at least the aromatic ring in the side chain of 1 is indispensable for developing anti-cancer leads with potent anti-proliferative activity and limited side effects. In accordance with the binding affinity, the concentration of 3 necessary to induce PKCδ-GFP translocation to the plasma membrane and perinuclear regions in HEK293 cells was higher than that of 1 and 2. However, the translocation profiles for PKCδ-GFP due to induction by 1-3 were similar.

Basal expression of insulin-like growth factor 1 receptor determines intrinsic resistance of cancer cells to a phosphatidylinositol 3-kinase inhibitor ZSTK474.

Drug resistance often critically limits the efficacy of molecular targeted drugs. Although pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) is an attractive therapeutic strategy for cancer therapy, molecular determinants for efficacy of PI3K inhibitors (PI3Kis) remain unclear. We previously identified that overexpression of insulin-like growth factor 1 receptor (IGF1R) contributed to the development of drug resistance after long-term exposure to PI3Kis. In this study, we examined the involvement of basal IGF1R expression in intrinsic resistance of drug-naïve cancer cells to PI3Kis and whether inhibition of IGF1R overcomes the resistance. We found that cancer cells highly expressing IGF1R showed resistance to dephosphorylation of Akt and subsequent antitumor effect by ZSTK474 treatment. Knockdown of IGF1R by siRNAs facilitated the dephosphorylation and enhanced the drug efficacy. These cells expressed tyrosine-phosphorylated insulin receptor substrate 1 at high levels, which was dependent on basal IGF1R expression. In these cells, the efficacy of ZSTK474 in vitro and in vivo was improved by its combination with the IGF1R inhibitor OSI-906. Finally, we found a significant correlation between the basal expression level of IGF1R and the inefficacy of ZSTK474 in an in vivo human cancer panel, as well as in vitro. These results suggest that basal IGF1R expression affects intrinsic resistance of cancer cells to ZSTK474, and IGF1R is a promising target to improve the therapeutic efficacy. The current results provide evidence of combination therapy of PI3Kis with IGF1R inhibitors for treating IGF1R-positive human cancers.

Comprehensive transcriptomic analysis of molecularly targeted drugs in cancer for target pathway evaluation.

Targeted therapy is a rational and promising strategy for the treatment of advanced cancer. For the development of clinical agents targeting oncogenic signaling pathways, it is important to define the specificity of compounds to the target molecular pathway. Genome-wide transcriptomic analysis is an unbiased approach to evaluate the compound mode of action, but it is still unknown whether the analysis could be widely applicable to classify molecularly targeted anticancer agents. We comprehensively obtained and analyzed 129 transcriptomic datasets of cancer cells treated with 83 anticancer drugs or related agents, covering most clinically used, molecularly targeted drugs alongside promising inhibitors of molecular cancer targets. Hierarchical clustering and principal component analysis revealed that compounds targeting similar target molecules or pathways were clustered together. These results confirmed that the gene signatures of these drugs reflected their modes of action. Of note, inhibitors of oncogenic kinase pathways formed a large unique cluster, showing that these agents affect a shared molecular pathway distinct from classical antitumor agents and other classes of agents. The gene signature analysis further classified kinome-targeting agents depending on their target signaling pathways, and we identified target pathway-selective signature gene sets. The gene expression analysis was also valuable in uncovering unexpected target pathways of some anticancer agents. These results indicate that comprehensive transcriptomic analysis with our database (http://scads.jfcr.or.jp/db/cs/) is a powerful strategy to validate and re-evaluate the target pathways of anticancer compounds.