RNA Expression-based Analysis to Predict Response in Patients with Metastatic Mismatch Repair Proficient Colorectal Cancer Treated with Regorafenib and Nivolumab.

Introduction We previously conducted a phase I/Ib study (NCT03712943) with regorafenib and nivolumab in patients with refractory metastatic mismatch repair proficient (pMMR) colorectal cancer (CRC). This study aimed to investigate the role of Xerna™ TME Panel in predicting the treatment response. Methods 22 archival pretreatment tumor samples were subjected to the Xerna™ TME Panel, a machine learning-based RNA-sequencing biomarker assay. The Xerna TME subtypes were evaluated for correlation with overall survival (OS), progression free survival (PFS), disease control rate (DCR), and other biomarkers including KRAS, PD-L1, CD8 expression, and Treg cells in tumor microenvironment. Results Based on Xerna™ TME Panel, four patients with immune active (IA) subtype and six patients with immune suppressed (IS) subtype were classified as biomarker-positive, and five with angiogenic (A) subtype and seven with immune desert (ID) subtype were biomarker-negative. While not reaching statistical significance, Xerna TME biomarker-positive patients seemed to have longer median PFS (7.9 vs. 4.1 months, P=0.254), median OS (15.75 vs. 11.9 months, P=0.378), and higher DCR (70% vs. 58%, P=0.675). The IA subtype in our cohort had higher levels of CD4+ FOXP3+ Treg cells, whereas the A subtype showed lower levels of Treg cells. Conclusion Xerna™ TME Panel analysis in patients with refractory metastatic pMMR CRC who were treated with regorafenib plus nivolumab might be of value for predictive clinical benefit. Further studies are needed to evaluate the predictive role of Xerna™ TME Panel analysis in patients with refractory metastatic pMMR CRC.

Xerna™ TME Panel is a machine learning-based transcriptomic biomarker designed to predict therapeutic response in multiple cancers.

Introduction: Most predictive biomarkers approved for clinical use measure single analytes such as genetic alteration or protein overexpression. We developed and validated a novel biomarker with the aim of achieving broad clinical utility. The Xerna™ TME Panel is a pan-tumor, RNA expression-based classifier, designed to predict response to multiple tumor microenvironment (TME)-targeted therapies, including immunotherapies and anti-angiogenic agents. Methods: The Panel algorithm is an artificial neural network (ANN) trained with an input signature of 124 genes that was optimized across various solid tumors. From the 298-patient training data, the model learned to discriminate four TME subtypes: Angiogenic (A), Immune Active (IA), Immune Desert (ID), and Immune Suppressed (IS). The final classifier was evaluated in four independent clinical cohorts to test whether TME subtype could predict response to anti-angiogenic agents and immunotherapies across gastric, ovarian, and melanoma datasets. Results: The TME subtypes represent stromal phenotypes defined by angiogenesis and immune biological axes. The model yields clear boundaries between biomarker-positive and -negative and showed 1.6-to-7-fold enrichment of clinical benefit for multiple therapeutic hypotheses. The Panel performed better across all criteria compared to a null model for gastric and ovarian anti-angiogenic datasets. It also outperformed PD-L1 combined positive score (>1) in accuracy, specificity, and positive predictive value (PPV), and microsatellite-instability high (MSI-H) in sensitivity and negative predictive value (NPV) for the gastric immunotherapy cohort. Discussion: The TME Panel's strong performance on diverse datasets suggests it may be amenable for use as a clinical diagnostic for varied cancer types and therapeutic modalities.

RhoB loss prevents streptozotocin-induced diabetes and ameliorates diabetic complications in mice.

RhoB is an early-response gene whose expression is elevated by multiple cellular stresses; this gene plays an important role in cancer, macrophage motility, and apoptosis. These factors are essential for the onset of type 1 diabetes mellitus and related complications. This study explores the role of RhoB in ß-cell depletion and hyperglycemia-associated complications and tests whether the pleiotropic effect of statins on glycemic control is RhoB dependent. We induced ß-cell depletion in RhoB(+/+), RhoB(+/-), and RhoB(-/-) mice with streptozotocin (STZ). Diabetic status was assessed by glucose tolerance and pancreatic islet loss. RhoB(-/-) mice showed a significant reduction in the severity of STZ-induced diabetes; only 13% of the STZ-treated RhoB-null animals became hyperglycemic, as opposed to 61% of the wild-type controls. Diabetes-related complications, such as wound healing rate and onset of nephropathy, were also assessed. Hyperglycemic RhoB(-/-) mice had fewer signs of nephropathy and showed faster wound healing than RhoB(+/+) animals. After assessing the diabetic status of mice treated simultaneously with STZ and simvastatin, we conclude that the effect of statins in improving glycemic control is RhoB independent. We propose that RhoB is a modifier of diabetes, important for the induction of ß-cell loss. Suppression of RhoB expression may have potential application in the treatment of diabetes and associated complications.

VEGF expands erythropoiesis via hypoxia-independent induction of erythropoietin in noncanonical perivascular stromal cells.

Insufficient erythropoiesis due to increased demand is usually met by hypoxia-driven up-regulation of erythropoietin (Epo). Here, we uncovered vascular endothelial growth factor (VEGF) as a novel inducer of Epo capable of increasing circulating Epo under normoxic, nonanemic conditions in a previously unrecognized reservoir of Epo-producing cells (EPCs), leading to expansion of the erythroid progenitor pool and robust splenic erythropoiesis. Epo induction by VEGF occurs in kidney, liver, and spleen in a population of Gli1+SMA+PDGFRß+ cells, a signature shared with vascular smooth muscle cells (VSMCs) derived from mesenchymal stem cell-like progenitors. Surprisingly, inhibition of PDGFRß signaling, but not VEGF signaling, abrogated VEGF-induced Epo synthesis. We thus introduce VEGF as a new player in Epo induction and perivascular Gli1+SMA+PDGFRß+ cells as a previously unrecognized EPC reservoir that could be harnessed for augmenting Epo synthesis in circumstances such as chronic kidney disease where production by canonical EPCs is compromised.

Olaratumab Exerts Antitumor Activity in Preclinical Models of Pediatric Bone and Soft Tissue Tumors through Inhibition of Platelet-Derived Growth Factor Receptor α.

Purpose: Platelet-derived growth factor receptor α (PDGFRα) is implicated in several adult and pediatric malignancies, where activated signaling in tumor cells and/or cells within the microenvironment drive tumorigenesis and disease progression. Olaratumab (LY3012207/IMC-3G3) is a human mAb that exclusively binds to PDGFRα and recently received accelerated FDA approval and conditional EMA approval for treatment of advanced adult sarcoma patients in combination with doxorubicin. In this study, we investigated olaratumab in preclinical models of pediatric bone and soft tissue tumors.Experimental Design: PDGFRα expression was evaluated by qPCR and Western blot analysis. Olaratumab was investigated in in vitro cell proliferation and invasion assays using pediatric osteosarcoma and rhabdoid tumor cell lines. In vivo activity of olaratumab was assessed in preclinical mouse models of pediatric osteosarcoma and malignant rhabdoid tumor.Results:In vitro olaratumab treatment of osteosarcoma and rhabdoid tumor cell lines reduced proliferation and inhibited invasion driven by individual platelet-derived growth factors (PDGFs) or serum. Furthermore, olaratumab delayed primary tumor growth in mouse models of pediatric osteosarcoma and malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin.Conclusions: Overall, these data indicate that olaratumab, alone and in combination with standard of care, blocks the growth of some preclinical PDGFRα-expressing pediatric bone and soft tissue tumor models. Clin Cancer Res; 24(4); 847-57. ©2017 AACR.

Stromal-Based Signatures for the Classification of Gastric Cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

Akt promotes endocardial-mesenchyme transition.

Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identified; however, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells.

Effect of the degree of phosphate substitution in aluminum hydroxide adjuvant on the adsorption of phosphorylated proteins.

Aluminum hydroxide adjuvant was pretreated with six concentrations of potassium dihydrogen phosphate to produce a series of adjuvants with various degrees of phosphate substitution for surface hydroxyl. The adsorption of three phosphorylated proteins (alpha casein, dephosphorylated alpha casein, and ovalbumin) by the phosphate-treated aluminum hydroxide adjuvants was studied. The phosphorylated proteins were adsorbed by ligand exchange of phosphate for hydroxyl even when an electrostatic repulsive force was present. However, the extent (adsorptive capacity) and strength (adsorptive coefficient) of adsorption was inversely related to the degree of phosphate substitution of the aluminum hydroxide adjuvant. Exposure of vaccines containing aluminum hydroxide adjuvant and phosphorylated antigens to phosphate ion in the formulation or during manufacture should be minimized to produce maximum adsorption of the antigen.

Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production.

The effect of the degree of adsorption after exposure to interstitial fluid on the immune response in mice to model vaccines containing ovalbumin, alpha casein or dephosphorylated alpha casein adsorbed to aluminum hydroxide adjuvant was studied. Ovalbumin and dephosphorylated alpha casein were adsorbed in the vaccine but were completely eluted when exposed to interstitial fluid for 4 h. The presence of aluminum hydroxide adjuvant in the vaccine produced immunopotentiation compared to a solution of the protein even though the protein desorbed rapidly upon subcutaneous administration. In contrast, alpha casein was completely adsorbed to aluminum hydroxide adjuvant in both the vaccine and upon exposure to interstitial fluid. Immunopotentiation by aluminum hydroxide adjuvant was also observed in this model vaccine compared to a solution of alpha casein. The results indicated that antigen presenting cells can take up desorbed antigen from interstitial fluid as well as antigen adsorbed to aluminum-containing adjuvants.

Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant.

Hepatitis B surface antigen (HBsAg) differs from many antigens because of its associated lipid bilayer that is largely composed of phospholipids. In general, phosphate groups adsorb strongly to hydroxylated mineral surfaces by ligand exchange. The purpose of this study was to investigate the mechanism of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant with emphasis on the role of phospholipids in this adsorption. The adsorption of HBsAg by aluminum hydroxide adjuvant exhibits a high affinity adsorption isotherm. The Langmuir equation was used to calculate the adsorptive capacity (1.7 microg/microg Al), which is the amount of HBsAg adsorbed at monolayer coverage and the adsorptive coefficient (6.0 ml/microg), which is a measure of the strength of the adsorption force. The relatively high value of the adsorptive coefficient indicates that adsorption is due to a strong attractive force. Ligand exchange between a phosphate of the antigen and a surface hydroxyl of the adjuvant provides the strongest adsorption mechanism. The adsorption capacity of HBsAg was not affected by increased ionic strength indicating that electrostatic attraction is not the predominant adsorption force. Adsorption was also not affected by the addition of ethylene glycol indicating that hydrophobic interactions were not the predominant adsorption force. The strength of the adsorption force was indicated by the resistance of HBsAg to elution when exposed to interstitial fluid. Less than 5% of the HBsAg adsorbed to aluminum hydroxide adjuvant in a model vaccine was eluted during a 12 h in vitro exposure to interstitial fluid at 37 degrees C. Less than 1% of the adsorbed HBsAg in two commercial vaccines was eluted by in vitro exposure to interstitial fluid for 48 h at 37 degrees C. Thus, it was concluded that adsorption of HBsAg by aluminum hydroxide adjuvant is predominantly due to ligand exchange between the phospholipids in HBsAg and surface hydroxyls in aluminum hydroxide adjuvant.

VE-cadherin-p120 interaction is required for maintenance of endothelial barrier function.

Interaction of p120 with juxtamembrane domain (JMD) of VE-cadherin has been implicated in regulation of endothelial cell-cell adhesion. We used a number of approaches to alter the level of p120 available for binding to VE-cadherin as a means to investigate the role of p120-VE-cadherin interaction in regulation of barrier function in confluent endothelial monolayers. Expression of an epitope-tagged fragment corresponding to JMD of VE-cadherin resulted in a decrease in endothelial barrier function as assessed by changes in albumin clearance and electrical resistance. Binding of JMD-Flag to p120 resulted in a decreased level of p120. In addition to decreasing p120 level, expression of JMD also decreased level of VE-cadherin. Expression of JMD also caused an increase in MLC phosphorylation and rearrangement of actin cytoskeleton, which, coupled with decreased cadherin, can contribute to loss of barrier function. Reducing p120 by siRNA resulted in a decrease in VE-cadherin, whereas increasing the level of p120 increased the level of VE-cadherin, demonstrating that p120 regulates the level of VE-cadherin. Overexpression of p120 was, however, associated with decreased barrier function and rearrangement of the actin cytoskeleton. Interestingly, expression of p120 was able to inhibit thrombin-induced increases in MLC phosphorylation, suggesting that p120 inhibits activation of Rho/Rho kinase pathway in endothelial cells. Excess p120 also prevented JMD-induced increases in MLC phosphorylation, correlating this phosphorylation with Rho/Rho kinase pathway. These findings show p120 plays a major role in regulating endothelial barrier function, as either a decrease or increase of p120 resulted in disruption of permeability across cell monolayers.