Rapid detection of EGFR mutation in CTCs based on a double spiral microfluidic chip and the real-time RPA method.

Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumors and spread into the peripheral bloodstream. Mutation detection in CTCs can reveal vital genetic information about the tumors and can be used for "liquid biopsy" to indicate cancer treatment and targeted medication. However, current methods to measure the mutations in CTCs are based on PCR or DNA sequencing which are cumbersome and time-consuming and require sophisticated equipment. These largely limited their applications especially in areas with poor healthcare infrastructure. Here we report a simple, convenient, and rapid method for mutation detection in CTCs, including an example of a deletion at exon 19 (Del19) of the epidermal growth factor receptor (EGFR). CTCs in the peripheral blood of NSCLC patients were first sorted by a double spiral microfluidic chip with high sorting efficiency and purity. The sorted cells were then lysed by proteinase K, and the E19del mutation was detected via real-time recombinase polymerase amplification (RPA). Combining the advantages of microfluidic sorting and real-time RPA, an accurate mutation determination was realized within 2 h without professional operation or complex data interpretation. The method detected as few as 3 cells and 1% target variants under a strongly interfering background, thus, indicating its great potential in the non-invasive diagnosis of E19del mutation for NSCLC patients. The method can be further extended by redesigning the primers and probes to detect other deletion mutations, insertion mutations, and fusion genes. It is expected to be a universal molecular diagnostic tool for real-time assessment of relevant mutations and precise adjustments in the care of oncology patients.

Discovery of vimseltinib (DCC-3014), a highly selective CSF1R switch-control kinase inhibitor, in clinical development for the treatment of Tenosynovial Giant Cell Tumor (TGCT).

Based on knowledge of kinase switch-control inhibition and using a combination of structure-based drug design and standard medicinal chemistry principles, we identified a novel series of dihydropyrimidone-based CSF1R kinase inhibitors displaying exquisite selectivity for CSF1R versus a large panel of kinases and non-kinase protein targets. Starting with lead compound 3, an SAR optimization campaign led to the discovery of vimseltinib (DCC-3014; compound 20) currently undergoing clinical evaluation for the treatment of Tenosynovial Giant Cell Tumor (TGCT), a locally aggressive benign tumor associated with substantial morbidity. 2021 Elsevier ltd. All rights reserved.

Discovery of acyl ureas as highly selective small molecule CSF1R kinase inhibitors.

Based on the structure of an early lead identified in Deciphera's proprietary compound collection of switch control kinase inhibitors and using a combination of medicinal chemistry guided structure activity relationships and structure-based drug design, a novel series of potent acyl urea-based CSF1R inhibitors was identified displaying high selectivity for CSF1R versus the other members of the Type III receptor tyrosine kinase (RTK) family members (KIT, PDGFR-α, PDGFR-ß, and FLT3), VEGFR2 and MET. Based on in vitro biology, in vitro ADME and in vivo PK/PD studies, compound 10 was selected as an advanced lead for Deciphera's CSF1R research program.

Enhanced Separation Efficiency and Purity of Circulating Tumor Cells Based on the Combined Effects of Double Sheath Fluids and Inertial Focusing.

Circulating tumor cells (CTCs) play a crucial role in solid tumor metastasis, but obtaining high purity and viability CTCs is a challenging task due to their rarity. Although various works using spiral microchannels to isolate CTCs have been reported, the sorting purity of CTCs has not been significantly improved. Herein, we developed a novel double spiral microchannel for efficient separation and enrichment of intact and high-purity CTCs based on the combined effects of two-stage inertial focusing and particle deflection. Particle deflection relies on the second sheath to produce a deflection of the focused sample flow segment at the end of the first-stage microchannel, allowing larger particles to remain focused and entered the second-stage microchannel while smaller particles moved into the first waste channel. The deflection of the focused sample flow segment was visualized. Testing by a binary mixture of 10.4 and 16.5 µm fluorescent microspheres, it showed 16.5 µm with separation efficiency of 98% and purity of 90% under the second sheath flow rate of 700 µl min-1. In biological experiments, the average purity of spiked CTCs was 74% at a high throughput of 1.5 × 108 cells min-1, and the recovery was more than 91%. Compared to the control group, the viability of separated cells was 99%. Finally, we validated the performance of the double spiral microchannel using clinical cancer blood samples. CTCs with a concentration of 2-28 counts ml-1 were separated from all 12 patients' peripheral blood. Thus, our device could be a robust and label-free liquid biopsy platform in inertial microfluidics for successful application in clinical trials.

Vimseltinib: A Precision CSF1R Therapy for Tenosynovial Giant Cell Tumors and Diseases Promoted by Macrophages.

Macrophages can be co-opted to contribute to neoplastic, neurologic, and inflammatory diseases. Colony-stimulating factor 1 receptor (CSF1R)-dependent macrophages and other inflammatory cells can suppress the adaptive immune system in cancer and contribute to angiogenesis, tumor growth, and metastasis. CSF1R-expressing osteoclasts mediate bone degradation in osteolytic cancers and cancers that metastasize to bone. In the rare disease tenosynovial giant cell tumor (TGCT), aberrant CSF1 expression and production driven by a gene translocation leads to the recruitment and growth of tumors formed by CSF1R-dependent inflammatory cells. Small molecules and antibodies targeting the CSF1/CSF1R axis have shown promise in the treatment of TGCT and cancer, with pexidartinib recently receiving FDA approval for treatment of TGCT. Many small-molecule kinase inhibitors of CSF1R also inhibit the closely related kinases KIT, PDGFRA, PDGFRB, and FLT3, thus CSF1R suppression may be limited by off-target activity and associated adverse events. Vimseltinib (DCC-3014) is an oral, switch control tyrosine kinase inhibitor specifically designed to selectively and potently inhibit CSF1R by exploiting unique features of the switch control region that regulates kinase conformational activation. In preclinical studies, vimseltinib durably suppressed CSF1R activity in vitro and in vivo, depleted macrophages and other CSF1R-dependent cells, and resulted in inhibition of tumor growth and bone degradation in mouse cancer models. Translationally, in a phase I clinical study, vimseltinib treatment led to modulation of biomarkers of CSF1R inhibition and reduction in tumor burden in TGCT patients.

Ripretinib (DCC-2618) Is a Switch Control Kinase Inhibitor of a Broad Spectrum of Oncogenic and Drug-Resistant KIT and PDGFRA Variants.

Ripretinib (DCC-2618) was designed to inhibit the full spectrum of mutant KIT and PDGFRA kinases found in cancers and myeloproliferative neoplasms, particularly in gastrointestinal stromal tumors (GISTs), in which the heterogeneity of drug-resistant KIT mutations is a major challenge. Ripretinib is a "switch-control" kinase inhibitor that forces the activation loop (or activation "switch") into an inactive conformation. Ripretinib inhibits all tested KIT and PDGFRA mutants, and notably is a type II kinase inhibitor demonstrated to broadly inhibit activation loop mutations in KIT and PDGFRA, previously thought only achievable with type I inhibitors. Ripretinib shows efficacy in preclinical cancer models, and preliminary clinical data provide proof-of-concept that ripretinib inhibits a wide range of KIT mutants in patients with drug-resistant GISTs.

Carnosic acid protects mice from high-fat diet-induced NAFLD by regulating MARCKS.

Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of liver damage characterized by abnormal hepatic fat accumulation and inflammatory response. Although the molecular mechanisms responsible for the disease are not yet fully understood, the pathogenesis of NAFLD likely involves multiple signals. The identification of effective therapeutic strategies to target these signals is of utmost importance. Carnosic acid (CA), as a phenolic diterpene with anticancer, anti-bacterial, anti-diabetic and neuroprotective properties, is produced by many species of the Lamiaceae family. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. In the present study, wild-type C57BL/6 and MARCKS-deficient mice were randomly divided into the normal chow- or high-fat (HF) diet-fed groups. The HF diet increased the fasting glucose and insulin levels, and promoted glucose intolerance in the wild-type mice. MARCKS deficiency further upregulated intolerance, fasting glucose and insulin. The HF diet also promoted hepatic steatosis, serum alanine transaminase (ALT) and aspartate transaminase (AST) activity, inflammation and lipid accumulation in the wild-type mice. These responses were accelerated in the MARCKS-deficient mice. Importantly, increased inflammation and lipid accumulation were associated with phosphoinositide 3-kinase (PI3K)/AKT, NLR family pyrin domain containing 3 (NLRP3)/nuclear factor-κB (NF-κB) and sterol regulatory element binding protein-1c (SREBP-1c) signaling pathway activation. The mice treated with CA exhibited a significantly improved glucose and insulin tolerance. The production of pro-inflammatory cytokines and lipid accumulation were suppressed by CA. Significantly, MARCKS was reduced in mice fed the HF diet. CA treatment upregulated MARCKS expression compared to the HF group. Furthermore, the activation of the PI3K/AKT, NLRP3/NF-κB and SREBP-1c signaling pathways was inhibited by CA. Taken together, our data suggest that CA suppresses inflammation and lipogenesis in mice fed a HF diet through MARCKS regulation. Thus, CA may be prove to be a useful anti-NAFLD agent.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors.

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486-501. ©2017 AACR.

Thyroid nodule sizes influence the diagnostic performance of TIRADS and ultrasound patterns of 2015 ATA guidelines: a multicenter retrospective study.

To evaluate the impact of thyroid nodule sizes on the diagnostic performance of thyroid imaging reporting and data system (TIRADS) and ultrasound patterns of 2015 American Thyroid Association (ATA) guidelines. Total 734 patients with 962 thyroid nodules were recruited in this retrospective study. All nodules were divided into three groups according to the maximal diameter (d < 10 mm, d = 10-20 mm and d > 20 mm). The ultrasound images were categorized based on TIRADS and ATA ultrasound patterns, respectively. A total of 931 (96.8%) and 906 (94.2%) patterns met the criteria for TIRADS and ATA ultrasound patterns. The AUC (0.849) and sensitivity (85.3%) of TIRADS were highest in d = 10-20 mm group. However, ATA had highest AUC (0.839) and specificity (89.8%) in d > 20 mm group. ATA ultrasound patterns had higher specificity (P = 0.04), while TI-RADS had higher sensitivity (P = 0.02). In nodules d > 20 mm, the specificity of ATA patterns was higher than TIRADS (P = 0.003). Our results indicated that nodule sizes may influence the diagnostic performance of TIRADS and ATA ultrasound patterns. The ATA patterns may yield higher specificity than TIRADS, especially in nodules larger than 20 mm.

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers.

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated Drug Resistance via Balanced Inhibition of MET, TIE2, and VEGFR2.

Altiratinib (DCC-2701) was designed based on the rationale of engineering a single therapeutic agent able to address multiple hallmarks of cancer (1). Specifically, altiratinib inhibits not only mechanisms of tumor initiation and progression, but also drug resistance mechanisms in the tumor and microenvironment through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 (KDR) kinases. This profile was achieved by optimizing binding into the switch control pocket of all three kinases, inducing type II inactive conformations. Altiratinib durably inhibits MET, both wild-type and mutated forms, in vitro and in vivo. Through its balanced inhibitory potency versus MET, TIE2, and VEGFR2, altiratinib provides an agent that inhibits three major evasive (re)vascularization and resistance pathways (HGF, ANG, and VEGF) and blocks tumor invasion and metastasis. Altiratinib exhibits properties amenable to oral administration and exhibits substantial blood-brain barrier penetration, an attribute of significance for eventual treatment of brain cancers and brain metastases.

Discovery of 1-(3,3-dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea (LY3009120) as a pan-RAF inhibitor with minimal paradoxical activation and activity against BRAF or RAS mutant tumor cells.

The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036.

Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.

Ski, a modulator of wound healing and scar formation in the rat skin and rabbit ear.

We recently demonstrated that Ski is a novel wound healing-related factor that promotes fibroblast proliferation and inhibits collagen secretion. Here, we show that increasing local Ski expression by gene transfer not only significantly accelerated wound healing by relieving inflammation, accelerating re-epithelialization and increasing formation of granulation tissue, but also reduced scar formation by decreasing collagen production in rat dermal wounds. Similarly, ski gene transfer accelerated wound healing, reduced the protuberant height and volume of scars and increased collagen maturity in a hypertrophic scar model in the rabbit ear. Conversely, reducing Ski expression in the wound by RNA interference resulted in significantly slower wound healing and increased scar area in rat dermal wounds. We demonstrated that these effects of Ski are associated with transforming growth factor-ß-mediated signalling pathways through both Smad2/3-dependent and Smad-independent pathways. Together, our results define a dual role for Ski in promoting wound healing and alleviating scar formation, identifying a new target for therapeutic approaches to preventing scar hyperplasia and accelerating wound healing.

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

[Extramedullary infiltration of acute monocytic leukemia/monoblastic sarcoma: a clinicopathologic and immunophenotype analysis of 5 cases].

OBJECTIVE: To study the clinicopathologic features, diagnosis and differential diagnosis of extramedullary infiltration of acute monocytic leukemia/monoblastic sarcoma. METHODS: Five cases of extramedullary infiltration of acute monocytic leukemia/monoblastic sarcoma were selected from 102 cases of myeloid sarcoma diagnosed during the period from 1990 to 2006. The clinicopathologic findings and followup data were retrospectively analyzed. Immunohistochemical study was also carried out with SP method. RESULTS: Among the 5 cases studied, 3 were males and 2 were females, including 2 children and 3 adults. Generalized lymphadenopathy was found in 4 patients and skin lesions were observed in 2 patients. The tumor cells in all cases were positive for CD68 (KP1), CD68 (PGM1), lysozyme and CD45. They were negative for MPO, CD15, CD163, TdT, CD117, T and B cell markers. The Ki-67 index ranged from 40% to 80%. Follow-up data were available in all the 5 patients. Four of the 5 patients died of the disease, with the average survival time being 6.25 months. CONCLUSIONS: Monoblastic sarcoma is a rare disease with poor prognosis. It is almost impossible to distinguish monoblastic sarcoma from granulocytic sarcoma and other types of small round cell tumors on the basis of morphologic examination alone. Immunohistochemistry is mandatory for a correct diagnosis.