Cancer stem cells (CSC) have been implicated in disease recurrence, metastasis and therapeutic resistance, but effective targeting strategies for these cells are still wanting. VS-5584 is a potent and selective dual inhibitor of mTORC1/2 and Class I PI3K kinases. Here we report that VS-5584 is up to 30-fold more potent in inhibiting the proliferation and survival of CSC compared to non-CSC in solid tumor cell populations. VS-5584 preferentially diminished CSC levels in multiple mouse xenograft models of human cancer, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Likewise, VS-5584 treatment ex vivo preferentially reduced CSC in surgically resected breast and ovarian patient tumors. In contrast, chemotherapeutics such as paclitaxel and cisplatin were less effective in targeting CSC than bulk tumor cells. Mechanistic investigations revealed that preferential targeting of CSC required inhibition of multiple components of the PI3K/mTOR pathway: coordinate RNAi-mediated silencing of PI3K alpha, PI3K beta and mTOR phenocopied the effect of VS-5584, exhibiting the strongest preferential targeting of CSC, while silencing of individual PI3K isoforms or mTOR failed to replicate the effect of VS-5584. Consistent with CSC ablation, VS-5584 delayed tumor regrowth following chemotherapy in xenograft models of small cell lung cancer. Taken together, the preferential targeting of CSC prompts a new paradigm for clinical testing of VS-5584: clinical trials designed with CSC-directed endpoints may facilitate demonstration of the therapeutic benefit of VS-5584. We suggest that combining VS-5584 with classical chemotherapy that debulks tumors may engender a more effective strategy to achieve durable remissions in cancer patients.

Cancer stem cells (CSC) have been implicated in disease recurrence, metastasis, and therapeutic resistance, but effective targeting strategies for these cells are still wanting. VS-5584 is a potent and selective dual inhibitor of mTORC1/2 and class I PI 3-kinases. Here, we report that VS-5584 is up to 30-fold more potent in inhibiting the proliferation and survival of CSC compared with non-CSC in solid tumor cell populations. VS-5584 preferentially diminished CSC levels in multiple mouse xenograft models of human cancer, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Likewise, VS-5584 treatment ex vivo preferentially reduced CSC in surgically resected breast and ovarian patient tumors. In contrast, chemotherapeutics such as paclitaxel and cisplatin were less effective in targeting CSC than bulk tumor cells. Mechanistic investigations revealed that preferential targeting of CSC required inhibition of multiple components of the PI3K–mTOR pathway: coordinate RNAi-mediated silencing of PI3Kα, PI3Kβ, and mTOR phenocopied the effect of VS-5584, exhibiting the strongest preferential targeting of CSC, while silencing of individual PI3K isoforms or mTOR failed to replicate the effect of VS-5584. Consistent with CSC ablation, VS-5584 delayed tumor regrowth following chemotherapy in xenograft models of small-cell lung cancer. Taken together, the preferential targeting of CSC prompts a new paradigm for clinical testing of VS-5584: clinical trials designed with CSC-directed endpoints may facilitate demonstration of the therapeutic benefit of VS-5584. We suggest that combining VS-5584 with classic chemotherapy that debulks tumors may engender a more effective strategy to achieve durable remissions in patients with cancer. Cancer Res; 75(2); 1–10. ©2014 AACR.

Soluble growth factors and cytokines within the tumor microenvironment aid in the induction of the epithelial-to-mesenchymal transition (EMT). Although EMT promotes the development of cancer-initiating cells (CIC), cellular mechanisms by which cancer cells maintain mesenchymal phenotypes remain poorly understood. Work presented here indicates that induction of EMT stimulates non–small cell lung cancer (NSCLC) to secrete soluble factors that function in an autocrine fashion. Using gene expression profiling of all annotated and predicted secreted gene products, we find that NF-κB activity is required to upregulate INHBA/Activin, a morphogen in the TGFβ superfamily. INHBA is capable of inducing and maintaining mesenchymal phenotypes, including the expression of EMT master-switch regulators and self-renewal factors that sustain CIC phenotypes and promote lung metastasis. Our work demonstrates that INHBA mRNA and protein expression are commonly elevated in primary human NSCLC and provide evidence that INHBA is a critical autocrine factor that maintains mesenchymal properties of CICs to promote metastasis in NSCLC. Cancer Res; 75(2); 1–10. ©2014 AACR.

Soluble growth factors and cytokines within the tumor microenvironment aid in the induction of the epithelial-to-mesenchymal transition (EMT). Although EMT promotes the development of cancer-initiating cells (CICs), cellular mechanisms by which cancer cells maintain mesenchymal phenotypes remain poorly understood. Work presented here indicates that induction of EMT stimulates non-small cell lung cancer (NSCLC) to secrete soluble factors that function in an autocrine fashion. Using gene expression profiling of all annotated and predicted secreted gene products, we find that NF-κB activity is required to upregulate INHBA/Activin, a morphogen in the TGF beta superfamily. INHBA is capable of inducing and maintaining mesenchymal phenotypes, including the expression of EMT master-switch regulators and self-renewal factors that sustain CIC phenotypes and promote lung metastasis. Our work demonstrates that INHBA mRNA and protein expression is commonly elevated in primary human NSCLC and provide evidence that INHBA is a critical autocrine factor that maintains mesenchymal properties of CICs to promote metastasis in NSCLC.

Kinase inhibitors such as imatinib have dramatically improved outcomes for patients with gastrointestinal stromal tumor (GIST), but many patients develop resistance to these treatments. Although in some patients this event corresponds with mutations in the GIST driver oncogenic kinase KIT, other patients develop resistance without KIT mutations. In this study, we address this patient subset in reporting a functional dependence of GIST on the FGF receptor FGFR3 and its crosstalk with KIT in GIST cells. Addition of the FGFR3 ligand FGF2 to GIST cells restored KIT phosphorylation during imatinib treatment, allowing sensitive cells to proliferate in the presence of the drug. FGF2 expression was increased in imatinib-resistant GIST cells, the growth of which was blocked by RNAi-mediated silencing of FGFR3. Moreover, combining KIT and FGFR3 inhibitors synergized to block the growth of imatinib-resistant cells. Signaling crosstalk between KIT and FGFR3 activated the MAPK pathway to promote resistance to imatinib. Clinically, an IHC analysis of tumor specimens from imatinib-resistant GIST patients revealed a relative increase in FGF2 levels, with a trend toward increased expression in imatinib-naïve samples consistent with possible involvement in drug resistance. Our findings provide a mechanistic rationale to evaluate existing FGFR inhibitors and multikinase inhibitors that target FGFR3 as promising strategies to improve treatment of patients with GIST with de novo or acquired resistance to imatinib. Cancer Res; 75(5); 1–12. ©2014 AACR.