Imaging guided trials of the angiogenesis inhibitor sunitinib in mouse models predict efficacy in pancreatic neuroendocrine but not ductal carcinoma

Preclinical trials in mice represent a critical step in the evaluation of experimental therapeutics. Genetically engineered mouse models (GEMMs) represent a promising platform for the evaluation of drugs, particularly those targeting the tumor microenvironment. We evaluated sunitinib, an angiogenesis inhibitor that targets VEGF and PDGF receptor signaling, in two GEMMs of pancreatic cancer. Sunitinib did not reduce tumor burden in pancreatic ductal adenocarcinoma (PDAC), whereas tumor burden was reduced in the pancreatic neuroendocrine tumor (PNET) model, the latter results confirming and extending previous studies. To explore the basis for the lack of pathologic response in PDAC, we used noninvasive microbubble contrast-enhanced ultrasound imaging, which revealed that sunitinib reduced blood flow both in PDAC and in PNET, concomitant with a reduction in vessel density; nevertheless, PDAC tumors continued to grow, whereas PNET were growth impaired. These results parallel the response in humans, where sunitinib recently garnered FDA and European approval in PNET, whereas two antiangiogenic drugs failed to demonstrate efficacy in PDAC clinical trials. The demonstration of on-target activity but with discordant benefit in the PDAC and PNET GEMMs illustrates the potential value of linked preclinical and clinical trials

Endoglin requirement for BMP9 signaling in endothelial cells reveals new mechanism of action for selective anti-endoglin antibodies.

Endoglin (ENG), a co-receptor for several TGFβ-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFβ and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFβ1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFβ only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFβ1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials

Cancer cell-intrinsic resistance to BiTE therapy is mediated by loss of CD58 costimulation and modulation of the extrinsic apoptotic pathway

Background Bispecific T-cell engager (BiTE) molecules induce redirected lysis of cancer cells by T cells and are an emerging modality for solid tumor immunotherapy. While signs of clinical activity have been demonstrated, efficacy of T-cell engagers (TCEs) in solid tumors settings, molecular determinants of response, and underlying mechanisms of resistance to BiTE therapy require more investigation.Methods To uncover cancer cell-intrinsic genetic modifiers of TCE-mediated cytotoxicity, we performed genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loss-of-function and CRISPRa (CRISPR activation) gain-of-function screens using TCEs against two distinct tumor-associated antigens (TAAs). By using in vitro T-cell cytotoxicity assays and in vivo efficacy studies, we validated the roles of two common pathways identified in our screen, T-cell costimulation pathway and apoptosis pathway, as key modifiers of BiTE activity.Results Our genetic screens uncovered TAAs-independent cancer cell-intrinsic genes with functions in autophagy, T-cell costimulation, the apoptosis pathway, chromatin remodeling, and cytokine signaling that altered responsiveness to BiTE-mediated killing. Notably, loss of CD58 (the ligand of the CD2 T-cell costimulatory receptor), a gene frequently altered in cancer, led to decreased TCE-mediated cytotoxicity, T-cell activation and antitumor efficacy in vitro and in vivo. Moreover, the effects of CD58 loss were synergistically compounded by concurrent loss of CD80/CD86 (ligands for the CD28 T-cell costimulatory receptor), whereas joint CD2 and CD28 costimulation additively enhanced TCE-mediated killing, indicating non-redundant costimulatory mechanisms between the two pathways. Additionally, loss of CFLAR (Caspase-8 and FADD Like Apoptosis Regulator), BCL2L1, and BID (BH3 Interacting Domain Death Agonist) induced profound changes in sensitivity to TCEs, indicating that key regulators of apoptosis, which are frequently altered in cancer, impact tumor responsiveness to BiTE therapy.Conclusions This study demonstrates that genetic alterations central to carcinogenesis and commonly detected in cancer samples lead to significant modulation of BiTE antitumor activity in vitro and in vivo, findings with relevance for a better understanding of patient responses to BiTE therapy and novel combinations that enhance TCE efficacy

GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the deregulation of the hedgehog signaling pathway. The Sonic Hedgehog ligand (Shh), absent in the normal pancreas, is highly expressed in pancreatic tumors and is sufficient to induce neoplastic precursor lesions in mouse models. We investigated the mechanism of Shh signaling in PDAC carcinogenesis by genetically ablating the canonical bottleneck of hedgehog signaling, the transmembrane protein Smoothened (Smo), in the pancreatic epithelium of PDAC-susceptible mice. We report that multistage development of PDAC tumors is not affected by the deletion of Smo in the pancreas, demonstrating that autocrine Shh–Ptch–Smo signaling is not required in pancreatic ductal cells for PDAC progression. However, the expression of Gli target genes is maintained in Smo-negative ducts, implicating alternative means of regulating Gli transcription in the neoplastic ductal epithelium. In PDAC tumor cells, we find that Gli transcription is decoupled from upstream Shh–Ptch–Smo signaling and is regulated by TGF-β and KRAS, and we show that Gli1 is required both for survival and for the KRAS-mediated transformed phenotype of cultured PDAC cancer cells

Model of ENG-mediated signaling and BMP9 signaling inhibition by anti-ENG antibodies.

<p>A) In primary human ECs, parallel BMP9/ALK1/pSMAD1/5/8 and TFGβ/ALK5/pSMAD2/3 signaling cascades coexist and both pathways demonstrate a requirement for Endoglin (*). Anti-ENG antibodies TRC105 and M999 inhibit BMP9 binding to ENG and BMP9/pSMAD1/5/8 signaling (B) Model of ENG protein with amino acid (AA) numbers depicting: the orphan domain, the ZIP domain (ZP), the trans-membrane domain (TM) and the cytoplasmic domain (CD) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050920#pone.0050920-Alt1" target="_blank">[52]</a>. Distinct regions of the orphan domain associate with the following monoclonal antibodies SN6h (AA119-231), TRC105 (AA231-277) or SN6 (AA277-340) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050920#pone.0050920-She1" target="_blank">[53]</a>. M999, a novel anti-ENG monoclonal antibody competes with TRC105 for ENG binding, indicating that their epitopes overlap. TRC105 and M999 inhibit the interaction between recombinant BMP9 and recombinant ENG.</p

M999 competes with TRC105 for ENG binding and both antibodies block BMP9/ENG interaction.

<p>A) Sequence of protein layering in the Bio Layer Interferometry antibody competition assay: 1) a strepatavidin-coated biosensor is loaded to saturation with 2) biotinylated recombinant human Endoglin (10 μg/ml), then with 3) a negative control IgG1k or the M999 anti-Endoglin rat IgG2 antibodies (10 μg/ml) prior to the addition of 4) M999, TRC105, SN6, or SN6h (10 μg/ml). (B) Wavelength shifts (WS) triggered by the addition of the individual anti-ENG antibodies to ENG in the presence of a ctrl antibody (blue bars), or in the presence of the anti-ENG antibody M999 (red bars). Competition with M999, calculated as (WS^CTRL-WS^M999)/WS^CTRL*100 is indicated at the bottom of the graph. (C) Sequence of protein layering in the Bio Layer Interferometry BMP9 binding assay: 1) a strepatavidin-coated biosensor is loaded to saturation with 2) biotinylated recombinant human Endoglin (10 μg/ml), then with 3) a negative control IgG1k or the tested anti-ENG antibodies (M999, TRC105, SN6, SN6h) (10 μg/ml) prior to the addition of 4) BMP9 (10 μg/ml). (B) Wavelength shifts (WS) triggered by the addition of BMP9 in addition to the individual anti-ENG antibodies. BMP9 binding inhibition, calculated as [1-(WS^ENGAB/WS^CTRLAB)]*100, is indicated at the bottom of the graph. Results are the mean +/− standard deviation of technical triplicates.</p

ENG-mediated BMP9 signaling is required for VEGF and bFGF-induced HUVEC tube formation.

<p>A–B) HUVECs were dispensed in a 96 well plate (3000 cells/well) and grown in 0.1% FBS and incubated with the following antibodies at various concentrations for 72 hours, with a daily change of media: (A) mIgG2b (mouse IgG2b isotype control) and Ab3209 (mouse IgG2b anti-BMP9); (B) rIgG2a (rat IgG2a isotype control), M999 (rat IgG2a anti-human ENG), hIgG2 (human IgG2 isotype control) and cSN6j (human IgG2 anti-human ENG). After 72 hours, cell growth in each well was assessed using Cell Titer-Glo®. (C–D) GFP-labeled HUVECs admixed with human dermal fibroblasts dispensed in 96 well plates were treated on day 0 with angiogenic factors: VEGF (1 ng/ml) or bFGF (2 ng/ml). Negative controls wells (No AF) contained no angiogenic factors. Measurement of Branch Points were performed at day 13.5 following treatment with PBS (no Ab), Suramin (20 μM – positive control that inhibits RTK-mediated tube formation), or the following antibodies: hIgG2 (isotype control), cSN6j (hIgG2 anti-human ENG), rIgG2a (isotype control), M999 (rIgG2a anti-human ENG), mIgG2 (isotype control) or Ab3209 (mIgG2 anti-human BMP9). Antibody concentrations used in (C): 10 μg/ml; in (D): 20 μg/ml. (E) Representative image of individual wells from the HUVEC tube formation assay following treatment with bFGF (2 ng/ml) and the indicated antibody (20 μg/ml). (F) HUVECs were dispensed in each well of a 96-well plate (2500 cells/well) in serum free media. Cells were incubated with antibodies at various concentrations for 1 hour and stimulated with BMP9 (0.1 ng/ml) (+) or PBS (−) for 30 min prior to lysis. Levels of pSMAD1 normalized for levels of total SMAD1 were monitored using a SMAD1/pSMAD1 MSD assay. The following antibodies were used: mIgG1k (mouse IgG1k isotype control), SN6h (mouse IgG1k anti-human ENG), hIgG1 (human IgG1 isotype control), TRC105 (human IgG1 anti-human ENG). (G) GFP-labeled HUVECs admixed with human dermal fibroblasts dispensed in 96-well plates were treated on day 0 with bFGF (2 ng/ml); No Angiogenic Factor (No AF) wells did not receive bFGF. Measurement of Branch Points were performed at day 13.5 following treatment with the following antibodies: mIgG1k (mouse IgG1k isotype control), SN6h (mouse IgG1k anti-human ENG), hIgG1 (human IgG1 isotype control), TRC105 (human IgG1 anti-human ENG). Antibody concentration used in (G): 10 μg/ml. Results are the mean +/− standard deviation of technical triplicates. Results are the mean +/− standard deviation of technical triplicates (Δ =  P value >0.05; *  =  P value <0.05; **  =  P value <0.005; *** P  =  value <0.001).</p

The SMAD1/5/8 pathway in primary endothelial cells is activated by BMP9, not TGFβ1.

<p>A–C) HUVECs were serum-starved for 3 hours and stimulated with increasing amounts of TGFβ1 or BMP9 for 30 minutes. Levels of SMAD3, pSMAD3, pSMAD1/5/8 and β-Actin in the total cell extracts were assessed by western blotting. (D) Total RNA extract from HUVECs was subjected to reverse transcription and quantitative PCR assessment of the SMAD1, 2, 3, 5 and 8 transcripts relative to the levels of the β-Actin transcript. (E) HUVECs were serum-starved for 3 hours and treated with increasing amounts of BMP9 and levels of phospho-SMAD1 normalized for levels of total SMAD1 were monitored using a SMAD1/pSMAD1 MSD assay. (F-G) Primary human ECs (HUVEC, HMVECd, HAEC, HPAEC –2500 cells/well) and immortalized mouse EC lines (MS1, EOMA, bEnd.3, SVR, C166–1250 cells/well) were serum-starved for 3 hours and monitored for pSMAD1 induction with a SMAD1/pSMAD1 MSD assay upon treatment with BMP9 or increasing amounts of TGFβ1 for 30 minutes. Results are the mean +/− standard deviation of technical triplicates.</p