Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

Inherent or acquired resistance to sotorasib poses a substantialt challenge for NSCLC treatment. Here, we demonstrate that acquired resistance to sotorasib in isogenic cells correlated with increased expression of integrin ß4 (ITGB4), a component of the focal adhesion complex. Silencing ITGB4 in tolerant cells improved sotorasib sensitivity, while overexpressing ITGB4 enhanced tolerance to sotorasib by supporting AKT-mTOR bypass signaling. Chronic treatment with sotorasib induced WNT expression and activated the WNT/ß-catenin signaling pathway. Thus, silencing both ITGB4 and ß-catenin significantly improved sotorasib sensitivity in tolerant, acquired, and inherently resistant cells. In addition, the proteasome inhibitor carfilzomib (CFZ) exhibited synergism with sotorasib by down-regulating ITGB4 and ß-catenin expression. Furthermore, adagrasib phenocopies the combination effect of sotorasib and CFZ by suppressing KRAS activity and inhibiting cell cycle progression in inherently resistant cells. Overall, our findings unveil previously unrecognized nongenetic mechanisms underlying resistance to sotorasib and propose a promising treatment strategy to overcome resistance.

Recent Advancement in Breast Cancer Research: Insights from Model Organisms-Mouse Models to Zebrafish.

Animal models have been utilized for decades to investigate the causes of human diseases and provide platforms for testing novel therapies. Indeed, breakthrough advances in genetically engineered mouse (GEM) models and xenograft transplantation technologies have dramatically benefited in elucidating the mechanisms underlying the pathogenesis of multiple diseases, including cancer. The currently available GEM models have been employed to assess specific genetic changes that underlay many features of carcinogenesis, including variations in tumor cell proliferation, apoptosis, invasion, metastasis, angiogenesis, and drug resistance. In addition, mice models render it easier to locate tumor biomarkers for the recognition, prognosis, and surveillance of cancer progression and recurrence. Furthermore, the patient-derived xenograft (PDX) model, which involves the direct surgical transfer of fresh human tumor samples to immunodeficient mice, has contributed significantly to advancing the field of drug discovery and therapeutics. Here, we provide a synopsis of mouse and zebrafish models used in cancer research as well as an interdisciplinary 'Team Medicine' approach that has not only accelerated our understanding of varied aspects of carcinogenesis but has also been instrumental in developing novel therapeutic strategies.

Bromodomain and extraterminal domain protein bromodomain inhibitor based cancer therapeutics.

PURPOSE OF REVIEW: Bromodomain and extraterminal domain (BET) proteins are evolutionarily conserved, multifunctional super-regulators that specifically recognize acetyl-lysine on histones and other proteins controlling gene transcription. Several studies show that small molecules targeting these regulators preferentially suppress the transcription of cancer-promoting genes. Consequently, several BET inhibitors reached clinical trials and are in various stages for different kind of malignancies. In this review, we provide a concise summary of the molecular basis and preliminary clinical outcomes of BET inhibitors as anticancer therapeutics. RECENT FINDINGS: Results from early clinical trials with BET inhibitors confirmed their antitumor potential in both hematologic and solid tumours, but the evidence does not support the application of BET inhibitors as a monotherapy for cancer treatment. Treatment-emergent toxicities such as thrombocytopenia and gastrointestinal disorders are also reported. Preclinical data suggest that BET inhibitors may have a promising future in combination with other anticancer agents. SUMMARY: Despite of various challenges, BET inhibitors have high potential in combinatorial therapy and the future development of next-generation inhibitors could be promising. Further studies are needed to determine the predictive biomarkers for therapeutic response, which would translate into the long-term success of BET inhibitors as personalized medicines in cancer treatment.

The Small Molecule BC-2059 Inhibits Wingless/Integrated (Wnt)-Dependent Gene Transcription in Cancer through Disruption of the Transducin ß-Like 1-ß-Catenin Protein Complex.

The central role of ß-catenin in the Wnt pathway makes it an attractive therapeutic target for cancers driven by aberrant Wnt signaling. We recently developed a small-molecule inhibitor, BC-2059, that promotes apoptosis by disrupting the ß-catenin/transducin ß-like 1 (TBL1) complex through an unknown mechanism of action. In this study, we show that BC-2059 directly interacts with high affinity for TBL1 when in complex with ß-catenin. We identified two amino acids in a hydrophobic pocket of TBL1 that are required for binding with ß-catenin, and computational modeling predicted that BC-2059 interacts at the same hydrophobic pocket. Although this pocket in TBL1 is involved in binding with NCoR/SMRT complex members G Protein Pathway Suppressor 2 (GSP2) and SMRT and p65 NFκB subunit, BC-2059 failed to disrupt the interaction of TBL1 with either NCoR/SMRT or NFκB. Together, our results show that BC-2059 selectively targets TBL1/ß-catenin protein complex, suggesting BC-2059 as a therapeutic for tumors with deregulated Wnt signaling pathway. SIGNIFICANCE STATEMENT: This study reports the mechanism of action of a novel Wnt pathway inhibitor, characterizing the selective disruption of the transducin ß-like 1/ß-catenin protein complex. As Wnt signaling is dysregulated across cancer types, this study suggests BC-2059 has the potential to benefit patients with tumors reliant on this pathway.

The novel reversible LSD1 inhibitor SP-2577 promotes anti-tumor immunity in SWItch/Sucrose-NonFermentable (SWI/SNF) complex mutated ovarian cancer.

Mutations of the SWI/SNF chromatin remodeling complex occur in 20% of all human cancers, including ovarian cancer. Approximately half of ovarian clear cell carcinomas (OCCC) carry mutations in the SWI/SNF subunit ARID1A, while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) presents with inactivating mutations of the SWI/SNF ATPase SMARCA4 alongside epigenetic silencing of the ATPase SMARCA2. Loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A), which regulates the chromatin landscape and gene expression by demethylating proteins such as histone H3. Cross-cancer analysis of the TCGA database shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. SCCOHT and OCCC cell lines have shown sensitivity to the reversible LSD1 inhibitor SP-2577 (Seclidemstat), suggesting that SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Moreover, it has been shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of endogenous retroviral elements and may thereby overcome resistance to checkpoint blockade. In this study, we investigated the ability of SP-2577 to promote anti-tumor immunity and T-cell infiltration in SCCOHT and OCCC cell lines. We found that SP-2577 stimulated IFN-dependent anti-tumor immunity in SCCOHT and promoted the expression of PD-L1 in both SCCOHT and OCCC. Together, these findings suggest that the combination therapy of SP-2577 with checkpoint inhibitors may induce or augment immunogenic responses of SWI/SNF-mutated ovarian cancers and warrants further investigation.

Targeting nuclear ß-catenin as therapy for post-myeloproliferative neoplasm secondary AML.

Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear ß-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of ß-catenin or treatment with BC2059 that disrupts binding of ß-catenin to TBL1X (TBL1) depleted nuclear ß-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of ß-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of ß-catenin and BETP antagonists against post-MPN sAML BPCs.

Folding of Fibroblast Growth Factor 1 Is Critical for Its Nonclassical Release.

Fibroblast growth factor 1 (FGF1), a ubiquitously expressed pro-angiogenic protein that is involved in tissue repair, carcinogenesis, and maintenance of vasculature stability, is released from the cells via a stress-dependent nonclassical secretory pathway. FGF1 secretion is a result of transmembrane translocation of this protein. It correlates with the ability of FGF1 to permeabilize membranes composed of acidic phospholipids. Like several other nonclassically exported proteins, FGF1 exhibits ß-barrel folding. To assess the role of folding of FGF1 in its secretion, we applied targeted mutagenesis in combination with a complex of biophysical methods and molecular dynamics studies, followed by artificial membrane permeabilization and stress-induced release experiments. It has been demonstrated that a mutation of proline 135 located in the C-terminus of FGF1 results in (i) partial unfolding of FGF1, (ii) a decrease in FGF1's ability to permeabilize bilayers composed of phosphatidylserine, and (iii) drastic inhibition of stress-induced FGF1 export. Thus, folding of FGF1 is critical for its nonclassical secretion.

Design, Synthesis, and Biological Evaluation of a Series of Anthracene-9,10-dione Dioxime ß-Catenin Pathway Inhibitors.

The Wnt/ß-catenin signaling pathway plays a vital role in cell growth, the regulation, cell development, and the differentiation of normal stem cells. Constitutive activation of the Wnt/ß-catenin signaling pathway is found in many human cancers, and thus, it is an attractive target for anticancer therapy. Specific inhibitors of this pathway have been keenly researched and developed. Cell based screening of compounds library, hit-to-lead optimization, computational and structure-based design strategies resulted in the design and synthesis of a series of anthracene-9,10-dione dioxime series of compounds demonstrated potent inhibition of ß-catenin in vitro (IC50 < 10 nM, 14) and the growth of several cancer cell lines. This article discusses the potential of inhibiting the Wnt/ß-catenin signaling pathway as a therapeutic approach for cancer along with an overview of the development of specific inhibitors.

SIRT1 pathway dysregulation in the smoke-exposed airway epithelium and lung tumor tissue.

Cigarette smoke produces a molecular field of injury in epithelial cells lining the respiratory tract. However, the specific signaling pathways that are altered in the airway of smokers and the signaling processes responsible for the transition from smoking-induced airway damage to lung cancer remain unknown. In this study, we use a genomic approach to study the signaling processes associated with tobacco smoke exposure and lung cancer. First, we developed and validated pathway-specific gene expression signatures in bronchial airway epithelium that reflect activation of signaling pathways relevant to tobacco exposure, including ATM, BCL2, GPX1, NOS2, IKBKB, and SIRT1. Using these profiles and four independent gene expression datasets, we found that SIRT1 activity is significantly upregulated in cytologically normal bronchial airway epithelial cells from active smokers compared with nonsmokers. In contrast, this activity is strikingly downregulated in non-small cell lung cancer. This pattern of signaling modulation was unique to SIRT1, and downregulation of SIRT1 activity is confined to tumors from smokers. Decreased activity of SIRT1 was validated using genomic analyses of mouse models of lung cancer and biochemical testing of SIRT1 activity in patient lung tumors. Together, our findings indicate a role of SIRT1 in response to smoke and a potential role in repressing lung cancer. Furthermore, our findings suggest that the airway gene expression signatures derived in this study can provide novel insights into signaling pathways altered in the "field of injury" induced by tobacco smoke and thus may impact strategies for prevention of tobacco-related lung cancer.

A pharmacogenomic method for individualized prediction of drug sensitivity.

Identifying the best drug for each cancer patient requires an efficient individualized strategy. We present MATCH (Merging genomic and pharmacologic Analyses for Therapy CHoice), an approach using public genomic resources and drug testing of fresh tumor samples to link drugs to patients. Valproic acid (VPA) is highlighted as a proof-of-principle. In order to predict specific tumor types with high probability of drug sensitivity, we create drug response signatures using publically available gene expression data and assess sensitivity in a data set of >40 cancer types. Next, we evaluate drug sensitivity in matched tumor and normal tissue and exclude cancer types that are no more sensitive than normal tissue. From these analyses, breast tumors are predicted to be sensitive to VPA. A meta-analysis across breast cancer data sets shows that aggressive subtypes are most likely to be sensitive to VPA, but all subtypes have sensitive tumors. MATCH predictions correlate significantly with growth inhibition in cancer cell lines and three-dimensional cultures of fresh tumor samples. MATCH accurately predicts reduction in tumor growth rate following VPA treatment in patient tumor xenografts. MATCH uses genomic analysis with in vitro testing of patient tumors to select optimal drug regimens before clinical trial initiation.

Airway PI3K pathway activation is an early and reversible event in lung cancer development.

Although only a subset of smokers develop lung cancer, we cannot determine which smokers are at highest risk for cancer development, nor do we know the signaling pathways altered early in the process of tumorigenesis in these individuals. On the basis of the concept that cigarette smoke creates a molecular field of injury throughout the respiratory tract, this study explores oncogenic pathway deregulation in cytologically normal proximal airway epithelial cells of smokers at risk for lung cancer. We observed a significant increase in a genomic signature of phosphatidylinositol 3-kinase (PI3K) pathway activation in the cytologically normal bronchial airway of smokers with lung cancer and smokers with dysplastic lesions, suggesting that PI3K is activated in the proximal airway before tumorigenesis. Further, PI3K activity is decreased in the airway of high-risk smokers who had significant regression of dysplasia after treatment with the chemopreventive agent myo-inositol, and myo-inositol inhibits the PI3K pathway in vitro. These results suggest that deregulation of the PI3K pathway in the bronchial airway epithelium of smokers is an early, measurable, and reversible event in the development of lung cancer and that genomic profiling of these relatively accessible airway cells may enable personalized approaches to chemoprevention and therapy. Our work further suggests that additional lung cancer chemoprevention trials either targeting the PI3K pathway or measuring airway PI3K activation as an intermediate endpoint are warranted.

Novel cross-talk between three cardiovascular regulators: thrombin cleavage fragment of Jagged1 induces fibroblast growth factor 1 expression and release.

Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast growth factor (FGF) signaling pathways. FGF1, a prototype member of FGF family, lacks a signal peptide and is released through an endoplasmic reticulum-Golgi-independent mechanism. A soluble extracellular domain of the Notch ligand Jagged1 (sJ1) inhibits Notch signaling and induces FGF1 release. Thrombin, a key protease of the blood coagulation cascade and a potent inducer of angiogenesis, stimulates rapid FGF1 release through a mechanism dependent on the major thrombin receptor protease-activated receptor (PAR) 1. This study demonstrates that thrombin cleaves Jagged1 in its extracellular domain. The sJ1 form produced as a result of thrombin cleavage inhibits Notch-mediated CBF1/Suppressor of Hairless [(Su(H)]/Lag-1-dependent transcription and induces FGF1 expression and release. The overexpression of Jagged1 in PAR1 null cells results in a rapid thrombin-induced export of FGF1. These data demonstrate the existence of novel cross-talk between thrombin, FGF, and Notch signaling pathways, which play important roles in vascular formation and remodeling.

Secretion without Golgi.

A growing number of proteins devoid of signal peptides have been demonstrated to be released through the non-classical pathways independent of endoplasmic reticulum and Golgi. Among them are two potent proangiogenic cytokines FGF1 and IL1alpha. Stress-induced transmembrane translocation of these proteins requires the assembly of copper-dependent multiprotein release complexes. It involves the interaction of exported proteins with the acidic phospholipids of the inner leaflet of the cell membrane and membrane destabilization. Not only stress, but also thrombin treatment and inhibition of Notch signaling stimulate the export of FGF1. Non-classical release of FGF1 and IL1alpha presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders.

Sphingosine kinase 1 is a critical component of the copper-dependent FGF1 export pathway.

Sphingosine kinase 1 catalyzes the formation of sphingosine-1-phosphate, a lipid mediator involved in the regulation of angiogenesis. Sphingosine kinase 1 is constitutively released from cells, even though it lacks a classical signal peptide sequence. Because copper-dependent non-classical stress-induced release of FGF1 also regulates angiogenesis, we questioned whether sphingosine kinase 1 is involved in the FGF1 release pathway. We report that (i) the coexpression of sphingosine kinase 1 with FGF1 inhibited the release of sphingosine kinase 1 at 37 degrees C; (ii) sphingosine kinase 1 was released at 42 degrees C in complex with FGF1; (iii) sphingosine kinase 1 null cells failed to release FGF1 at stress; (iv) sphingosine kinase 1 is a high affinity copper-binding protein which formed a complex with FGF1 in a cell-free system, and (v) sphingosine kinase 1 over expression rescued the release of FGF1 from inhibition by the copper chelator, tetrathiomolybdate. We propose that sphingosine kinase 1 is a component of the copper-dependent FGF1 release pathway.

Thrombin induces rapid PAR1-mediated non-classical FGF1 release.

Thrombin induces cell proliferation and migration during vascular injury. We report that thrombin rapidly stimulated expression and release of the pro-angiogenic polypeptide fibroblast growth factor 1 (FGF1). Thrombin failed to induce FGF1 release from protease-activated receptor 1 (PAR1) null fibroblasts, indicating that this effect was dependent on PAR1. Similarly to thrombin, FGF1 expression and release were induced by TRAP, a specific oligopeptide agonist of PAR1. These results identify a novel aspect of the crosstalk between FGF and thrombin signaling pathways which both play important roles in tissue repair and angiogenesis.

Release of FGF1 and p40 synaptotagmin 1 correlates with their membrane destabilizing ability.

Fibroblast growth factor (FGF)1 is released from cells as a constituent of a complex that contains the small calcium binding protein S100A13, and the p40 kDa form of synaptotagmin (Syt)1, through an ER-Golgi-independent stress-induced pathway. FGF1 and the other components of its secretory complex are signal peptide-less proteins. We examined their capability to interact with lipid bilayers by studying protein-induced carboxyfluorescein release from liposomes of different phospholipid (pL) compositions. FGF1, p40 Syt1, and S100A13 induced destabilization of liposomes composed of acidic but not of zwitterionic pL. We produced mutants of FGF1 and p40 Syt1, in which specific basic amino acid residues in the regions that bind acidic pL were substituted. The ability of these mutants to induce liposomes destabilization was strongly attenuated, and they exhibited drastically diminished spontaneous and stress-induced release. Apparently, the non-classical release of FGF1 and p40 Syt1 involves destabilization of membranes containing acidic pL.

Molecular mechanism of inhibition of nonclassical FGF-1 export.

Fibroblast growth factor (FGF-1) lacks a signal sequence and is exported by an unconventional release mechanism. The nonclassical export of FGF-1 has been shown to be inhibited by an anti-allergic and anti-inflammatory drug, amlexanox (AMX). We investigate the molecular mechanism(s) underlying the inhibitory action of AMX on the release of FGF-1, using a variety of biophysical techniques including multidimensional NMR spectroscopy. AMX binds to FGF-1 and enhances its conformational stability. AMX binds to locations close to Cys30 and sterically blocks Cu(2+)-induced oxidation, leading to the formation of the homodimer of FGF-1. AMX-induced inhibition of the formation of the FGF-1 homodimer is observed both under cell-free conditions and in living cells. Results of this study suggest a novel approach for the design of drugs against FGF-1-mediated disorders.

The intracellular domain of Notch ligand Delta1 induces cell growth arrest.

Notch signaling involves proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands, Delta or Jagged; and the resultant soluble intracellular domain of Notch stimulates a cascade of transcriptional events. The Delta1 ligand also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. We demonstrate that the expression of the intracellular domain of Delta1 results in a non-proliferating senescent-like cell phenotype which is dependent on the expression of the cell cycle inhibitor, p21, and is abolished by co-expression of constitutively active Notch1. These data suggest a new intracellular role for Delta1.

The non-transmembrane form of Delta1, but not of Jagged1, induces normal migratory behavior accompanied by fibroblast growth factor receptor 1-dependent transformation.

The interactions between Notch (N) receptors and their transmembrane ligands, Jagged1 (JI) and Delta1 (Dl1), mediate signaling events between neighboring cells that are crucial during embryonal development and in adults. Since the non-transmembrane extracellular form of J1 acts as an antagonist of N activation in NIH 3T3 mouse fibroblast cells and induces fibroblast growth factor 1 (FGF1)-dependent transformation (Small, D., Kovalenko, D., Soldi, R., Mandinova, A., Kolev, V., Trifonova, R., Bagala, C., Kacer, D., Battelli, C., Liaw, L., Prudovsky, I., and Maciag, T. (2003) J. Biol. Chem. 278, 16405-16413), we examined the potential redundant functions of the two subfamilies of Notch ligands and report that while the soluble (s) forms of both Dl1 and J1 act as N signaling antagonists in NIH 3T3 cells, they do display disparate functions. While sJ1 induced an attenuation of cell motility which is accompanied by a decrease in actin stress fibers and an increase in adherence junctions, sDl1 does not. However, sJ1, like sDl1, induces a NIH 3T3 cell tranformed phenotype mediated by FGF signaling. Because the inhibition of N signaling by sJ1 and sDl1 is rescued by dominant-negative Src expression, we suggest that there may be cooperation between the Notch and Src signaling pathways.

The non-classical export routes: FGF1 and IL-1alpha point the way.

Non-classical protein release independent of the ER-Golgi pathway has been reported for an increasing number of proteins lacking an N-terminal signal sequence. The export of FGF1 and IL-1alpha, two pro-angiogenic polypeptides, provides two such examples. In both cases, export is based on the Cu2+-dependent formation of multiprotein complexes containing the S100A13 protein and might involve translocation of the protein across the membrane as a 'molten globule'. FGF1 and IL-1alpha are involved in pathological processes such as restenosis and tumor formation. Inhibition of their export by Cu2+ chelators is thus an effective strategy for treatment of several diseases.