SummaryCXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.

Resistance to targeted EGFR inhibitors is likely to develop in EGFR mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR TKIs including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002 or AZD9291. Compared to parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumours in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumours. Further, these findings suggest that NRAS modifications in tumour samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition.

αvβ6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvβ6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvβ6, and inhibits binding to all ligands including the latency-associated peptide of TGF-β. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvβ8, but not the integrins α5β1, αvβ3, αvβ5 and α4β1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvβ6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-β-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-β by NCI-H358 tumour cells in a tumour cell-fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvβ6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvβ6 integrin, with some activity against αvβ8 integrin, that reduces both tumour growth and metastasis.

Fibroblasts in the tumour stroma (cancer-associated fibroblasts) influence tumour progression and response to therapeutics; little is known about the mechanisms through which the tumour cell co-opts a normal fibroblast. To study the activation of fibroblasts by tumour cells, a panel of non-small cell lung cancer (NSCLC) cell lines and normal human dermal fibroblasts were co-cultured. A subset of the NSCLC cells induced an activated cancer-associated fibroblast-like fibroblast phenotype defined by induction of fibroblast α-smooth muscle actin expression. Tumour cells that activated fibroblasts were associated with E-Cadherin and EpCAM expression and expression of integrin αvβ6. Co-culture of activating tumour cells with fibroblasts resulted in induction of transcripts associated with tumour cell invasion and growth, TGFβ1 and TGFBR1, SERPINE-1, BMP6, SPHK1 and MMP9. Fibroblast activation was inhibited by an αvβ6/8 integrin blocking antibody (264RAD) and a small molecule inhibitor of the TGF-beta type I receptor activin-like kinase (ALK5) (SB431542), demonstrating that transactivation of the TGFβ pathway initiates fibroblast activation. Both integrin and ALK5 antagonists inhibited initiation. Only ALK5 was effective when added after 3 days of co-culture. This suggests that although activation is αvβ6-dependent, once fibroblasts are activated alternative TGFβ pathway regulators maintain an activation loop. In co-culture activating cells had reduced sensitivity to selumetinib, AZD8931 and afatinib compared with mono-culture. In contrast, non-activating cells were insensitive to selumetinib and AZD8931 in both mono-culture and co-culture. In conclusion NSCLC cell lines, positive for E-Cadherin, EpCAM and αvβ6 expression, activate normal fibroblasts through avβ6/TGFβ signalling in vitro, and influence both gene expression and response to therapeutic agents.

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kβ isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signaling; however, efficacy is often limited by suboptimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signaling in PTEN-null tumors, the PI3Kβ inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kβ and mTOR gave the most effective antiproliferative effects across a panel of PTEN-null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective controlling growth of PTEN-null tumor models of TNBC, prostate, and renal cancers., the combination resulted in increased suppression of pNDRG1, p4EBP1, as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, whilecombination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kβ inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN-null tumors. .