Bosutinib inhibits migration and invasion via ACK1 in KRAS mutant non-small cell lung cancer.

The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in "normal" tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches.

Circulating Biomarkers for Prediction of Immunotherapy Response in NSCLC.

Non-small cell lung cancer (NSCLC) constitutes the majority of the lung cancer population and the prognosis is poor. In recent years, immunotherapy has become the standard of care for advanced NSCLC patients as numerous trials demonstrated that immune checkpoint inhibitors (ICI) are more efficacious than conventional chemotherapy. However, only a minority of NSCLC patients benefit from this treatment. Therefore, there is an unmet need for biomarkers that could accurately predict response to immunotherapy. Liquid biopsy allows repeated sampling of blood-based biomarkers in a non-invasive manner for the dynamic monitoring of treatment response. In this review, we summarize the efforts and progress made in the identification of circulating biomarkers that predict immunotherapy benefit for NSCLC patients. We also discuss the challenges with future implementation of circulating biomarkers into clinical practice.

Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK.

Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules, we have identified SB203580, a specific p38 MAP kinase inhibitor, as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations <10 microM, induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers, so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly, transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly, cardiomyogenesis was strongly inhibited at SB203580 concentrations > or =15 microM. Thus, modulation of the p38MAP kinase pathway, in combination with factors released by END2 cells, plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process.

Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells.

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.

Somatic mutation in the ACK1 ubiquitin association domain enhances oncogenic signaling through EGFR regulation in renal cancer derived cells.

Activated Cdc42-associated Kinase, ACK1, is a non-receptor tyrosine kinase with numerous interacting partners, including Cdc42 and EGFR. Gene amplification and overexpression of ACK1 were found in many cancer types such as those of the lung and prostate. Previously, we identified both somatic- and germ line missense mutations in the ACK1 coding sequence, by surveying 261 cancer cell lines and 15 control tissues. Here, we verified and characterized the non-synonymous mutation, ACK-S985 N, located in the ubiquitin association domain of the protein. Both overexpression and silencing experiments in MCF7 and A498 cells, respectively, demonstrated a role of the ACK1 S985 N mutation in enhancing cell proliferation, migration and anchorage-independent growth as well as the epithelial-mesenchymal transition. Further, we showed that the ACK1 S985 N mutant is unable to bind ubiquitin, unlike the wild type kinase. This contributed to ACK1 protein stability and stabilized EGFR after EGF stimulation, thereby prolonging mitogenic signaling in cancer cells. In addition, the ACK1 S985 N-EGFR interaction is enhanced, but not the ubiquitination of the receptor. Intriguingly, silencing of ACK1 in A498 cells sensitized the renal carcinoma cells to gefitinib, against which they are otherwise resistant. The work demonstrates that other than gene amplification, a single somatic mutation in ACK1 can result in extended protein stability enabling the oncoprotein to exert its oncogenic function in tumor progression. It also provides a rationale to target ACK1 in combination with other chemotherapeutic drugs, such as EGFR inhibitors, to potentiate therapeutic action against resistant tumors.