Regenerating islet-derived family member, 4 modulates multiple receptor tyrosine kinases and mediators of drug resistance in cancer.

Regenerating islet-derived family member, 4 (Reg IV) is a secreted protein and member of the C-type lectin superfamily. Expression analyses have characterized Reg IV as a prognostic marker for certain cancers; however, the functional role of Reg IV in cancer, including downstream signaling, has only begun to be elucidated. To investigate the biological role of Reg IV in cancer, phosphorylation events were studied in cancer cell lines in the context of either Reg IV stimulation (HCT116 cells) or knockdown of endogenous Reg IV (PC3 and KM12 cells). In addition to the previously observed impact on epidermal growth factor receptor and Akt phosphorylation, we observed modulation in the phosphorylation of multiple additional receptor tyrosine kinases (RTKs), including insulin receptor, insulin-like growth factor receptor as well as their downstream effectors, mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways. Furthermore, knockdown of Reg IV impacted the ability of insulin and EGF to stimulate downstream tyrosine phosphorylation. Knockdown of Reg IV in cancer cell lines inhibited anchorage-dependent and anchorage-independent (both soft-agar and spheroid assays) cell growth and induced cell cycle arrest. This was accompanied by upregulation of p21 and p27. Transiently silencing Reg IV in cancer cells induced apoptosis and downregulated Bcl-2. Conversely, stimulation of HCT116 cells with recombinant Reg IV induced Bcl-2. Hsp27, a molecule implicated in drug resistance, was similarly modulated by Reg IV. Consistent with our observations with Reg IV siRNA-mediated knockdown, monoclonal antibodies directed against Reg IV inhibited PC3 and KM12 cell growth. Collectively, Reg IV plays an important role in cancer by modulation of key signaling molecules including Hsp27, Bcl-2 and multiple RTKs.

A Phase Ib Open-Label, Multicenter Study of Inhaled DV281, a TLR9 Agonist, in Combination with Nivolumab in Patients with Advanced or Metastatic Non-small Cell Lung Cancer.

PURPOSE: Although PD-(L)1 inhibitors have shown efficacy in advanced/metastatic non-small cell lung cancer (NSCLC), many patients do not respond to this treatment and more effective combinations with acceptable toxicities are needed. To assess the potential benefit of combining localized innate immune stimulation with checkpoint blockade, the TLR9 agonist DV281 was combined with nivolumab in a phase Ib study. PATIENTS AND METHODS: Patients after one or two prior lines of systemic therapy were enrolled in a dose-escalation study with a 3+3 design. DV281 was administered via inhalation in five dose cohorts at 1 to 25 mg; nivolumab 240 mg was administered intravenously every 2 weeks. Safety, tolerability, pharmacodynamics, and response to treatment were assessed. RESULTS: Twenty-six patients with advanced NSCLC enrolled. Baseline programmed death ligand 1 (PD-L1) expression was present in 16 patients (61.5%); 21 (80.7%) had received previous anti-PD-1/PD-L1. Thirteen patients (50%) had stable disease, nine (34.6%) had progressive disease, and four (15.4%) were not evaluable. Median duration of disease control was 124 days. Adverse events were seen in 16 patients (61.5%), mostly grade 1/2 chills, fatigue, flu-like symptoms, diarrhea, and rash; there was only one grade 3 adverse event (dyspnea). Pharmacodynamic assessment, measured by IFN- inducible gene expression, showed target engagement in all dose cohorts. Systemic pharmacodynamic responses plateaued in the 2 highest dose cohorts. CONCLUSIONS: DV281 with nivolumab was well tolerated with target engagement observed at every dose. Pharmacodynamic advantages at doses above 10 mg were unclear. The long duration of disease control in 50% of patients suggests clinically relevant activity in this population of heavily pretreated patients.

Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-alpha and survivin.

INTRODUCTION: Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer. METHODS: To investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-alpha (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER). RESULTS: AGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown. CONCLUSIONS: Transiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.

Low-dose metronomic cyclophosphamide complements the actions of an intratumoral C-class CpG TLR9 agonist to potentiate innate immunity and drive potent T cell-mediated anti-tumor responses.

The synthetic oligonucleotide SD-101 is a potent and specific agonist for toll-like receptor 9. Intratumoral injection of SD-101 induces significant anti-tumor immunity in preclinical and clinical studies, especially when combined with PD-1 blockade. To build upon this strategy, we studied the enhancement of SD-101 activities by combination with low-dose cyclophosphamide, a well-characterized agent with potentially complementary activities. In multiple mouse tumor models, we demonstrate substantial anti-tumor activity of the combination, compared to each single agent. Combination therapy generated CD8+ T cell dependent immunity leading to rejection of both non-injected and injected tumors and long-term survival, even in very large tumors. Mechanistic studies encompassing global gene expression changes and characterization of immune cell infiltrates show the rapid, sequential induction of innate and adaptive responses and identify discrete contributions of SD-101 and cyclophosphamide. Importantly, these changes were prominent in tumors not injected directly with SD-101. Combination treatment resulted in creation of a permissive environment for a systemic anti-tumor immune response, including a reduction of intratumoral regulatory T cells (Tregs) and an increase in "M1" versus "M2" tumor-associated macrophage (TAM) phenotypes. Additionally, we observed increased immunogenic cell death as well as antigen processing in response to combination treatment.

Discovery and Optimization of HKT288, a Cadherin-6-Targeting ADC for the Treatment of Ovarian and Renal Cancers.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Microarray analysis of primary endothelial cells challenged with different inflammatory and immune cytokines.

To investigate the potential molecular mediators of tissue-specific recruitment, we explored the influence of different cytokine challenges on gene expression regulation in five primary endothelial cells (ECs), representing two different phenotypes: iliac artery and aortic (macrovascular); lung, colon and dermal (microvascular). We challenged ECs with cytokines that elicit different patterns of inflammatory and immune responses in immune cells: tumor necrosis factor (TNF-alpha), interferon-gamma (IFN-gamma) or interleukin-4 (IL-4), and used microarrays containing approximately 40,000 unique cDNAs, to assess changes in differential gene expression relative to untreated cells. Five hundred and sixty three sequences changed by at least 2.5 fold in one or more of the 15 possible EC /cytokine combinations. The list included highly regulated adhesion molecules, chemokines, cytokines, metalloproteases, and IFN-gamma-induced genes. Overall, IFN-gamma caused the largest number of gene expression changes and its profile was least correlated with IL-4. In addition to clusters that were predominantly EC/cytokine specific, we also observed several clusters that were regulated by more than one cytokine across several ECs. Furthermore, we identified genes that were reciprocally expressed in response to different cytokines that could serve as markers of inflammatory and immune expression. These results confirm the importance of microenvironment in primary ECs that could have important applications in developing targeted therapies for vascular diseases.