Evaluation of EDB Fibronectin in Plasma, Patient-Derived Xenograft Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tumor Tissues Using Immunoaffinity LC-MS/MS.

The fibronectin (FN) isoform including the extradomain B (EDB) segment (EDB + FN) is a promising tumor target and is highly expressed in some tumor types, such as breast, head, and neck cancer. To date, mostly immunohistochemistry (IHC) and Western blot have been used for the analysis of EDB + FN. However, complete quantitative measurements of EDB + FN expression in a tumor and circulation are important for the development of anti-EDB therapeutics. To this end, a method using protein enrichment followed by online antipeptide antibody enrichment coupled with a nanoflow LC-MS/MS was developed to quantify EDB + FN in human and cynomolgus plasma, patient-derived xenograft (PDX) tumors, and PDX formalin-fixed paraffin-embedded (FFPE) samples. Mouse plasma EDB + FN was analyzed using a protein immunoaffinity method followed by nanoflow LC-MS/MS. EDB + FN concentrations were 63.1 pmol/g in PDX breast cancer tumor and 49.6 pmol/g in PDX head and neck tumor. Mean plasma concentration was 1.1 nM (pmol/mL, 47.4 ng/mL) in normal healthy humans and 0.35 nM (15.1 ng/mL) in naive cynomolgus. The assay sensitivity was 0.018 nM based on calibration with recombinant human EDB + FN (rhEDB + FN).

Anti-Extra Domain B Splice Variant of Fibronectin Antibody-Drug Conjugate Eliminates Tumors with Enhanced Efficacy When Combined with Checkpoint Blockade.

Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.

Oligomycin-induced bioenergetic adaptation in cancer cells with heterogeneous bioenergetic organization.

Cancer cells constantly adapt to oxidative phosphorylation (OXPHOS) suppression resulting from hypoxia or mitochondria defects. Under the OXPHOS suppression, AMP-activated protein kinase (AMPK) regulates global metabolism adjustments, but its activation has been found to be transient. Whether cells can maintain cellular ATP homeostasis and survive beyond the transient AMPK activation is not known. Here, we study the bioenergetic adaptation to the OXPHOS inhibitor oligomycin in a group of cancer cells. We found that oligomycin at 100 ng/ml completely inhibits OXPHOS activity in 1 h and induces various levels of glycolysis gains by 6 h, from which we calculate the bioenergetic organizations of cancer cells. In glycolysis-dominant cells, oligomycin does not induce much energy stress as measured by glycolysis acceleration, ATP imbalance, AMPK activation, AMPK substrate acetyl-CoA carboxylase phosphorylation at Ser(79), and cell growth inhibition. In OXPHOS-dependent LKB1 wild type cells, oligomycin induces 5-8% ATP drops and transient AMPK activation during the initial 1-2 h. After AMPK activation is completed, oligomycin-induced increase of acetyl-CoA carboxylase phosphorylation at Ser(79) is still detected, and cellular ATP is back at preoligomycin treatment levels by sustained elevation of glycolysis. Cell growth, however, is inhibited without an increase in cell death and alteration in cell cycle distribution. In OXPHOS-dependent LKB1-null cells, no AMPK activation by oligomycin is detected, yet cells still show a similar adaptation. We also demonstrate that the adaptation to oligomycin does not invoke activation of hypoxia-induced factor. Our data suggest that cancer cells may grow and survive persistent OXPHOS suppression through an as yet unidentified regulatory mechanism.

Discovery of the catechol structural moiety as a Stat3 SH2 domain inhibitor by virtual screening.

The Stat3 SH2 domain is essential for its activation, and development of a potent SH2 inhibitor will be therapeutically valuable in treating cancers with constant Stat3 activation. We report here the identification of the catechol (1,2-dihydroxybenzene) structural moiety by virtual screening as a Stat3 SH2 inhibitor. The catechol compound docked to the Stat3 SH2 domain in computer modeling forms hydrogen bonds with the conserved pTyr-interacting amino acids. In the biochemical assay, a catechol-containing compound, but not the hydroxyl group-acetalized analogue, was able to inhibit Stat3 DNA-binding activity. Furthermore, the catechol compound was demonstrated to compete with pTyr peptides in binding to the Stat3 SH2 domain, suggesting that the catechol moiety is a pTyr bioisostere and may potentially be used for designing cell-permeable SH2 inhibitors. In our preliminary effort, we also demonstrated that the potency of catechol compound as Stat3 SH2 inhibitors could be improved by modifying the non-catechol part of the compound structure.

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in vitamin D receptor transactivation during keratinocyte differentiation.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)2D3)], controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the vitamin D receptor (VDR)-interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the VDR. However, the relationship between these two classes of coactivators is not clear. Using glutathione-S-transferase-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP and SRC proteins were expressed in keratinocytes. DRIP205 expression decreased during differentiation, although SRC-3 levels increased. Both DRIP205 and SRC-3 potentiated vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are sequentially involved in vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in VDR transactivation during keratinocyte differentiation.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)), controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the Vitamin D receptor (VDR) interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the Vitamin D receptor. However, the relationship between these two classes of coactivators is not clear. Using GST-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP205 and SRC-3 potentiated Vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are differentially involved in Vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Inhibition of the signal transducer and activator of transcription-3 (STAT3) signaling pathway by 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid esters.

The JAK-STAT3 pathway regulates genes that are important in cell proliferation and thus is a promising target for cancer therapy. A high-throughput screening (HTS) campaign using an Apo-ONE Homogenous Caspase 3/7 assay in U266 cells identified 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid ethyl ester 4 as a potential STAT3 pathway inhibitor. Optimization of this HTS hit led to the identification of the 7-cyano analogue 8, which inhibited STAT3-Y705 phosphorylation with an EC 50 of 170 nM. Compound 8 also inhibited cytokine induced JAK activation but did not inhibit BCR-ABL activated STAT5 phosphorylation in K562 cells.

Reciprocal recruitment of DRIP/mediator and p160 coactivator complexes in vivo by estrogen receptor.

Two functionally distinct classes of coactivators are recruited by liganded estrogen receptor, the DRIP/Mediator complex and p160 proteins, although the relative dynamics of recruitment is unclear. Previously, we have shown a direct, estradiol-dependent interaction between the DRIP205 subunit of the DRIP complex and the estrogen receptor (ER) AF2 domain. Here we demonstrate the in vivo recruitment of other endogenous DRIP subunits to ER in response to estradiol treatment in MCF-7 cells. To explore the relationship between DRIP and p160 coactivators, we examined the kinetics of coactivator recruitment to the ER target promoter, pS2, by chromatin immunoprecipitation. We observed a cyclic association and dissociation of coactivators with the promoter, with recruitment of p160s and DRIPs occurring in opposite phases, suggesting an exchange between these coactivator complexes at the target promoter.