Search

Interstitial macrophages (IMs) reside in the lung tissue surrounding key structures including airways, vessels, and alveoli. Recent work has described IM heterogeneity during homeostasis, however, there are limited data on IMs during inflammation. We sought to characterize IM origin, subsets, and transcriptomic profiles during homeostasis and lipopolysaccharide (LPS) induced acute lung inflammation. During homeostasis, we used three complementary methods, spectral flow cytometry, single-cell RNA-sequencing, and gene regulatory network enrichment, to demonstrate that IMs can be divided into two core subsets distinguished by surface and transcriptional expression of folate receptor β ( Folr2/FRβ). These subsets inhabited distinct niches within the lung interstitium. Within FRβ+ IMs we identified a subpopulation marked by coexpression of LYVE1. During acute LPS-induced inflammation, lung IM numbers expand. Lineage tracing revealed IM expansion was due to recruitment of monocyte-derived IMs. At the peak of inflammation, recruited IMs were comprised two unique subsets defined by expression of genes associated with interferon signaling and glycolytic pathways. As recruited IMs matured, they adopted the overall transcriptional state of FRβ− resident IMs but retained expression in several origin-specific genes, such as IL-1β. FRβ+ IMs were of near-pure resident origin. Taken together our data show that during LPS-induced inflammation, there are distinct populations of IMs that likely have unique functions. FRΒ+ IMs comprise a stable, resident population, whereas FRβ− ΙΜs represent a mixed population of resident and recruited IMs.

Supplementary Figure S1:Effect of CB-5083 on cell growth and survival in multiple myeloma and solid tumor cell lines; Supplementary Figure S2:CB-5083 activates UPR;Supplementary Figure S3:CB-5083 transcriptional response is unique in comparison to proteasome inhibitors;Supplementary Figure S4:CB-5083 enhances the antitumor activity of proteasome inhibitors; Supplementary Figure S5: Nrf1 upregulation induced by bortezomib is inhibited by CB-5083; Supplementary Figure S6:CB-5083 demonstrates a broad activity in multiple myeloma relevant in vivo models; Supplementary Table S1: List of antibodies used in the manuscript.

Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375–86. ©2017 AACR.

Objectives The purpose of this study was to assess serum concentrations of gabapentin in cats with chronic kidney disease (CKD) vs clinically healthy cats. Methods Five healthy cats were enrolled in a pharmacokinetic study. A single 20 mg/kg dose of gabapentin was administered orally and blood was obtained at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 8, 12, 24 and 36 h via a jugular catheter. Serum gabapentin concentrations were measured using liquid chromatography coupled to tandem mass spectrometry. Non-compartmental pharmacokinetic analysis was performed. The same five healthy cats plus 25 cats with stable International Renal Interest Society stage 2 (n = 14) and 3 (n = 11) CKD were enrolled in a limited sampling study. Cats in both groups received a single 10 mg/kg dose of gabapentin, and serum gabapentin concentrations and compliance scores were obtained 3 and 8 h post-administration. Results Cats with CKD had significantly higher dose-normalized serum gabapentin concentrations than normal cats at 3 h ( P = 0.0012 CKD vs normal 10 mg/kg; P = 0.008 CKD vs normal 20 mg/kg) and 8 h ( P Conclusions and relevance Cats with CKD that received 10 mg/kg of gabapentin had significantly higher dose-normalized serum concentrations than normal cats that received 20 mg/kg, supporting the need to dose-reduce in this patient population.

Wild-type YAP does not enhance metastasis in zebrafish embryos.

Movie over 12 hours of a whole zebrafish embryo injected with YAP-AA over-expressing A375 cells

Movie of A375 cells over-expressing YAP-AA in the inter-segmental vessels (ISVs) over the course of ~3 hours.

The oncogene YAP has been shown previously to promote tumor growth and metastasis. However, how YAP influences the behavior of tumor cells traveling within the circulatory system has not been as well explored. Given that rate-limiting steps of metastasis are known to occur while tumor cells enter, travel through, or exit circulation, we sought to study how YAP influences tumor cell behavior within the circulatory system. Intravital imaging in live zebrafish embryos revealed that YAP influenced the distribution of tumor cells within the animal following intravenous injection. Control cells became lodged in the first capillary bed encountered in the tail, whereas cells overexpressing constitutively active YAP were able to travel through this capillary plexus, reenter systemic circulation, and seed in the brain. YAP controlled transit through these capillaries by promoting active migration within the vasculature. These results were corroborated in a mouse model following intravenous injection, where active YAP increased the number of circulating tumor cells over time. Our results suggest a possible mechanism whereby tumor cells can spread to organs beyond the first capillary bed downstream from the primary tumor. These results also show that a specific gene can affect the distribution of tumor cells within an animal, thereby influencing the global pattern of metastasis in that animal.Significance:These findings demonstrate that YAP endows tumor cells with the ability to move through capillaries, allowing them to return to and persist in circulation, thereby increasing their metastatic spread.See related commentary by Davidson, p. 3797

Movie of A375 control cells in the tail over the course of 12 hours following injection.

Movie showing blood flow through the Duct of Cuvier of a 2-day-old zebrafish embryo.

Movie over 12 hours of a whole zebrafish embryo injected with EV control A375 cells

Supplementary materials and methods

Movie of A375 EV control cells in the inter-segmental vessels (ISVs) over the course of ~3 hours.

Movie over 12 hours of the head of a zebrafish embryo injected with EV control A375 cells.

Legends for the supplemental figures and videos.

Movie over 12 hours of the head of a zebrafish embryo injected with YAP-AA-over-expressing A375 cells.

Movie of A375 cells over-expressing YAP-AA in the tail over the course of 12 hours following injection.

Pulmonary macrophages are heterogeneous. Distinct populations of resident tissue macrophages exist in the lung airspace and tissue compartments during homeostasis. During inflammation, these are joined by monocyte-derived recruited macrophages. Flow cytometry can be used to identify and purify lung macrophage subsets. Here, we describe methods for identifying and isolating macrophages from bronchoalveolar lavage and digested lung tissues from mouse and human. We also describe basic staining for flow cytometry analysis of different macrophage subsets.

Adeno-associated virus (AAV) vectors are a promising vehicle for noninvasive gene delivery to the central nervous system via intravenous infusion. However, naturally occurring serotypes have a limited ability to transduce the brain, and translating engineered capsids from mice to nonhuman primates has proved challenging.In this study, we use an mRNA-based directed-evolution strategy in multiple strains of mice as well as a de novo selection in cynomolgus macaques to identify families of engineered vectors with increased potency in the brain and decreased tropism for the liver.We compare the transgene expression capabilities of several engineered vectors and show that while some of our novel macaque-derived variants significantly outperform AAV9 in transducing the macaque brain following systemic administration, mouse-derived variants-both those identified in this study and those reported by other groups-universally do not.Together, the results of this work introduce a class of primate-derived engineered AAV capsids with increased therapeutic potential and highlight the critical need for using appropriate animal models to both identify and evaluate novel AAVs intended for delivery to the human central nervous system.This work was funded primarily through an anonymous philanthropic gift to the P.C.S. lab at the Broad Institute of MIT and Harvard and by a grant from the Howard Hughes Medical Institute to P.C.S.

Despite encouraging clinical results with immune checkpoint inhibitors and other types of immunotherapies, the rate of failure is still very high. The development of proper animal models which could be applied to the screening of effective preclinical antitumor drugs targeting human tumor antigens, such as mesothelin, is a great need. Mesothelin (MSLN) is a 40 kDa cell-surface glycoprotein which is highly expressed in a variety of human cancers, and has great value as a target for antibody-based therapies. The present study reports the establishment of an immunocompetent transgenic mouse expressing human MSLN (hMSLN) only in thyroid gland by utilizing an expression vector containing a thyroid peroxidase (TPO) promoter. These mice do not reject genetically modified tumor cells expressing hMSLN on the cell membrane, and tolerate high doses of hMSLN-targeted immunotoxin. Employing this TPO-MSLN mouse model, we find that combination treatment of LMB-100 and anti-CTLA-4 induces complete tumor regression in 91% of the mice burdened with 66C14-M tumor cells. The combination therapy provides a significant survival benefit compared with both LMB-100 and anti-CTLA-4 monotherapy. In addition, The cured mice reject tumor cells when rechallenged, indicating the development of long-term antitumor immunity. This novel TPO-MSLN mouse model can serve as an important animal tool to better predict tumor responses to any immunomodulatory therapies that target MSLN.

Existing pre-clinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates using an autochthonous small cell lung cancer model, we extrapolated half-life times in the circulation of 50-100 seconds and intravasation rates between 4,000 and 27,000 CTCs/hour – an average daily shedding rate equivalent to ∼0.07% of the total number of primary tumor cells in the lung. Additionally, transfer of 1-2% of daily-shed CTCs from late-stage tumor-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis.

Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.

Significance Despite the usefulness of genetically engineered mouse models in cancer research, their small total blood volume and the rarity of circulating tumor cells (CTCs) preclude the use of existing liquid biopsy techniques for longitudinal CTC studies in mice. We have devised a method for collecting CTCs from an unanesthetized mouse longitudinally, spanning multiple days or weeks, to study acute perturbations (e.g., drug treatment) or potentially long-term phenotypes (e.g., tumor progression) within the same mouse. Here, we show that our optofluidic-based approach eliminates confounding biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice., Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.

LMB-100 is a recombinant immunotoxin being developed for cancer treatment that is composed of a Fab that binds to mesothelin and a portion of Pseudomonas exotoxin A. LMB-100 is in clinical trials for the treatment of mesothelioma and pancreatic cancer. To determine if check point modulating antibodies enhance the formation of anti-drug antibodies (ADA) against LMB-100, we treated mice with LMB-100 and four different immune modulating monoclonal antibodies that have different mechanisms of action; anti-CTLA4, anti-OX40, anti-PD-1 and anti-PDL-1. We found that anti-PD-1 and anti PDL-1 do not increase the formation of ADA, but anti-CTLA-4 and anti-OX-40 do increase the onset of ADA. These results indicate that combining anti-CTLA-4 and anti-OX-40 with antibodies and other protein-based therapeutics may enhance ADA formation in humans.

Recombinant immunotoxins (RITs) are genetically engineered proteins being developed to treat cancer. They are composed of an Fv that targets a cancer antigen and a fragment of a bacterial toxin that kills tumor cells. Because the toxin is a foreign protein, it is immunogenic. The clinical success of RITs in patients with a normal immune system is limited by their immunogenicity. In this review, we discuss our progress in therapeutic protein deimmunization and the balancing act between immunogenicity and therapeutic potency. One approach is to prevent the activation of B cells by mapping and elimination of B-cell epitopes. A second approach is to prevent helper T-cell activation by interfering with major histocompatibility complex II presentation or T-cell recognition. Immunizing mice with RITs that were deimmunized by elimination of the murine B- or T-cell epitopes showed that both approaches are effective. Another approach to control immunogenicity is to modify the host immune system. Nanoparticles containing synthetic vaccine particles encapsulating rapamycin can induce immune tolerance and prevent anti-drug antibody formation. This treatment restores RIT anti-tumor activity that is otherwise neutralized because of immunogenicity.

Recombinant immunotoxins (RITs) are chimeric proteins being developed for cancer treatment. They are composed of an Ab fragment that targets a cancer Ag and a cytotoxic portion of Pseudomonas exotoxin A. They are effective for patients with hematologic malignancies with defective immunity, but their efficacy against solid tumors is limited by anti-drug Ab (ADA) responses in immune-competent patients. Pre-existing Abs or immune memory owing to previous toxin exposure represent additional hurdles because they induce rapid and strong ADA responses. Here, we evaluated the efficacy of methotrexate (MTX) to prevent ADA formation against the mesothelin-targeting RIT LMB-100 in naive mice and in mice with pre-existing Abs. We found that low-dose MTX combined with LMB-100 completely suppressed the formation of ADAs in a dose- and frequency-dependent manner. Suppression of the immune response restored blood levels of LMB-100 and prevented its neutralization. Furthermore, combination of MTX with LMB-100 did not compromise the immune response against a second Ag given after stopping MTX, indicating specific immune tolerance. Adoptive transfer of splenocytes suppressed Ab responses to LMB-100 in recipient mice, indicating a durable immune tolerance. We conclude that combination of MTX and LMB-100 is effective at preventing immune responses in a durable, Ag-specific manner. We propose combining low-dose MTX in immune-competent cancer patients receiving RIT therapy to prevent immunogenicity. This approach could be applied to other immunogenic therapeutic agents and to proteins for which there is pre-existing immunity.

The oncogene YAP has been shown previously to promote tumor growth and metastasis. However, how YAP influences the behavior of tumor cells traveling within the circulatory system has not been as well explored. Given that rate-limiting steps of metastasis are known to occur while tumor cells enter, travel through, or exit circulation, we sought to study how YAP influences tumor cell behavior within the circulatory system. Intravital imaging in live zebrafish embryos revealed that YAP influenced the distribution of tumor cells within the animal following intravenous injection. Control cells became lodged in the first capillary bed encountered in the tail, whereas cells overexpressing constitutively active YAP were able to travel through this capillary plexus, reenter systemic circulation, and seed in the brain. YAP controlled transit through these capillaries by promoting active migration within the vasculature. These results were corroborated in a mouse model following intravenous injection, where active YAP increased the number of circulating tumor cells over time. Our results suggest a possible mechanism whereby tumor cells can spread to organs beyond the first capillary bed downstream from the primary tumor. These results also show that a specific gene can affect the distribution of tumor cells within an animal, thereby influencing the global pattern of metastasis in that animal. Significance: These findings demonstrate that YAP endows tumor cells with the ability to move through capillaries, allowing them to return to and persist in circulation, thereby increasing their metastatic spread. See related commentary by Davidson, p. 3797

SS1P is an anti-mesothelin immunotoxin composed of a targeting antibody fragment genetically fused to a truncated fragment of Pseudomonas exotoxin A. Delayed responses reported in mesothelioma patients receiving SS1P suggest that anti-tumor immunity is induced. The goal of this study is to evaluate if SS1P therapy renders mesothelioma tumors more sensitive to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) immune checkpoint blockade. We evaluated the ability of SS1P to induce adenosine triphosphate (ATP) secretion and calreticulin expression on the surface of AE17M mouse mesothelioma cells. Both properties are associated with immunogenic cell death. Furthermore, we treated these tumors with intra-tumoral SS1P and systemic CTLA-4. We found that SS1P increased the release of ATP from AE17M cells in a dose and time-dependent manner. In addition, SS1P induced calreticulin expression on the surface of AE17M cells. These results suggest that SS1P promotes immunogenic cell death and could sensitize tumors to anti-CTLA-4 based therapy. In mouse studies, we found that the combination of anti-CTLA-4 with intra-tumoral SS1P induced complete regressions in most mice and provided a statistically significant survival benefit compared to monotherapy. The surviving mice were protected from tumor re-challenge, indicating the development of anti-tumor immunity. These findings support the use of intra-tumoral SS1P in combination with anti-CTLA-4.

Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naive and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.

Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375–86. ©2017 AACR.

SummaryRecognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMOD477G, confer resistance to these inhibitors. Here, we report that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMOD477G medulloblastoma.

In response to oral glucose, glucagon-like peptide-1 receptor (Glp1r) knockout (Glp1r−/−) mice become hyperglycaemic due to impaired insulin secretion. Exercise also induces hyperglycaemia in Glp1r−/− mice. In contrast to oral glucose, exercise decreases insulin secretion. This implies that exercise-induced hyperglycaemia in Glp1r−/− mice results from the loss of a non-insulinotropic effect mediated by the Glp1r. Muscle glucose uptake (MGU) is normal in exercising Glp1r−/− mice. Thus, we hypothesize that exercise-induced hyperglycaemia in Glp1r−/− mice is due to excessive hepatic glucose production (HGP). Wild-type (Glp1r+/+) and Glp1r−/− mice implanted with venous and arterial catheters underwent treadmill exercise or remained sedentary for 30 min. [3-3H]glucose was used to estimate rates of glucose appearance (Ra), an index of HGP, and disappearance (Rd). 2[14C]deoxyglucose was used to assess MGU. Glp1r−/− mice displayed exercise-induced hyperglycaemia due to an excessive increase in Ra but normal Rd and MGU. Exercise-induced glucagon levels were ∼2-fold higher in Glp1r−/− mice, resulting in a ∼2-fold higher glucagon:insulin ratio. Since inhibition of the central Glp1r stimulates HGP, we tested whether intracerebroventricular (ICV) infusion of the Glp1r antagonist exendin(9–39) (Ex9) in Glp1r+/+ mice would result in exercise-induced hyperglycaemia. ICV Ex9 did not enhance glucose levels or HGP during exercise, suggesting that glucoregulatory effects of Glp1 during exercise are mediated via the pancreatic Glp1r. In conclusion, functional disruption of the Glp1r results in exercise-induced hyperglycaemia associated with an excessive increase in glucagon secretion and HGP. These results suggest an essential role for basal Glp1r signalling in the suppression of alpha cell secretion during exercise.

Recombinant immunotoxins (rITs) are composed of a tumor antigen-targeting antibody fragment fused to a portion of Pseudomonas exotoxin A. rITs have been effective in clinical trials for patients with hematologic malignancies. The CD22 targeting rIT Moxetumomab Pasudotox has achieved overall response rates of 86% and complete remission rates of 46% in patients with relapsed-refractory Hairy Cell Leukemia. However, the therapeutic efficacy of rITs against solid tumors is limited by their immunogenicity in immune-competent patients. In clinical trials to treat mesothelioma patients with SS1P, a rIT targeting mesothelin, 90% of patients developed neutralizing antibodies against SS1P after one cycle of treatment. When immunosuppressive chemotherapy and SS1P were combined, more cycles of rIT could be given and several patients with advanced chemo-refractory mesothelioma had striking tumor regressions. This implicates high therapeutic potential for rITs against solid tumors once immunogenicity is surmounted. Methotrexate (MTX) is a folate antagonist which interferes with purine biosynthesis, and is used to treat osteosarcomas and other cancers. MTX also interferes with T cell responses and is used to treat autoimmune diseases. Based on its immunosuppressive properties, Joly et al. demonstrated that low-dose MTX prevented the formation of ADAs against recombinant human alglucosidase alfa in mice in an antigen-specific manner. We hypothesized that MTX would similarly prevent the formation of ADAs against rITs in an antigen-specific manner. To test our hypotheses, mice were treated with the mesothelin-targeting rIT RG7787 with or without MTX given 0, 24, and 48 hours after RG7787 treatment. Serum was collected and anti-RG7787 ADAs were measured by direct ELISA. We found that six doses of RG7787 combined with low dose MTX (1 mg/kg) inhibited the formation of ADAs against RG7787. This inhibition was sustained through six challenges with RG7787 without additional MTX. Further, we found that immunization with RG7787 plus MTX induced RG7787-specific tolerance, and had no effect on the ADA response against a second protein, ovalbumin. We conclude that combination of MTX and RG7787 is effective at preventing primary immune responses in a durable, antigen-specific manner. We propose to combine this agent in immune-competent cancer patients receiving rIT therapy to prevent rIT immunogenicity. Citation Format: Emily M. King, Ronit Mazor, Ira Pastan. Methotrexate prevents primary immune responses against recombinant immunotoxin in murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2017-68

Objective: Leptin alleviates hyperglycemia in rodent models of Type 1 diabetes by activating leptin receptors within the central nervous system. Here we delineate whether non-canonical leptin signaling through the Creb-regulated transcriptional coactivator 1 (Crtc1) contributes to leptin-dependent improvements in diabetic glucose metabolism. Methods: We employed mice with a targeted genetic disruption of Crtc1, tracer dilution techniques and neuroanatomical studies to interrogate whether Crtc1 enables leptin to improve glucose metabolism in streptozotocin-induced (STZ) diabetes. Results: Here we show that leptin improves diabetic glucose metabolism through Crtc1-dependent and independent mechanisms. We find that leptin reduces diabetic hyperglycemia, hepatic gluconeogenic gene expression and selectively increases glucose disposal to brown adipose tissue and heart, in STZ-diabetic Crtc1WT mice but not Crtc1+/− mice. By contrast, leptin decreases circulating glucagon levels in both STZ-diabetic Crtc1WT and Crtc1+/− mice. We also demonstrate that leptin promotes Crtc1 nuclear translocation in pro-opiomelanocortin (Pomc) and non-Pomc neurons within the hypothalamic arcuate nucleus (ARC). Accordingly, leptin's ability to induce Pomc gene expression in the ARC is blunted in STZ-diabetic Crtc1+/− mice. Conclusions: Our study reveals that Crtc1 functions as a conduit for leptin's glucoregulatory actions in insulin-dependent diabetes. This study also highlights a new role for Crtc1 in modulating peripheral glucose metabolism.

Long-living Ames dwarf mice (df/df) characterized by growth hormone (GH) deficiency are widely used in aging research because of their 40–60 % lifespan extension compared to normal (N) littermates. Importantly, these mice not only live longer but are also protected from age-related diseases including insulin resistance. Several studies demonstrate that df/df mice have enhanced insulin signaling in different insulin-sensitive tissues and suggest that this is a mechanism for extended lifespan. However, it is unknown whether the enhanced insulin signaling in df/df mice translates to improved insulin action on hepatic glucose production and tissue glucose uptake. We performed hyperinsulinemic-euglycemic clamps to assess tissue-specific insulin action in vivo for the first time in these small long-living dwarfs. Our results demonstrate that the glucose infusion rate required to maintain euglycemia was ∼2-fold higher in df/df mice compared to N controls. Insulin-mediated glucose production was completely suppressed in dwarf mice, and stimulation of gastrocnemius and vastus muscle and adipose tissue glucose uptake was also enhanced in df/df mice (100, 86, and 65 %, respectively). These findings show that improved insulin signaling in df/df mice is associated with enhanced tissue-specific insulin action in vivo. This improved functionality of insulin action and glucose homeostasis may play a key role in promoting healthy aging and longer lifespan in df/df mice.

The functional role of individual kinases in multiple myeloma (MM) cells has been primarily interrogated using RNAi approaches, which are mechanistically dissimilar to the pharmacological action of small molecule inhibitors used in the clinic; and in stroma-free cultures, which don’t account for stroma-induced signaling events. To address these dissimilarities, we performed functional mapping of the kinome in 16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure) targeting a total of 43 known primary oncogenic targets. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors (consistent with high proliferative rates of MM cell lines in vitro); modest to minimal for selective PDK1, PI3K (excluding those that also inhibit mTOR), and Akt inhibitors; virtually none for selective inhibitors of c-met, ALK, EGFR, HER2, c-kit, PDGFR, VEGFR, Flt3, FAK, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases). We also observed modest activity for FGFR3 inhibitors against FGFR3expressing cell lines; and none for BRAF inhibitors in our BRAF wild-type cell lines (some of which had increased proliferation upon treatment; consistent with known adaptive activation of signaling through C-RAF). Testing 8 different MEK1/2 inhibitors identified lines with low, intermediate vs. high response; the latter being KRAS-mutant and BRAF-wild-type cells with high baseline p-ERK levels (e.g. due to MEK2-activating mutation in one cell line). Importantly, comparison of results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our studies demonstrate the feasibility of functional annotations of the kinome in MM using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of cell lines. Our results provide insight into the relative role of different classes of kinases in MM, and how the genetic (e.g. MEK2 or BRAF mutation status) and microenvironmental context can influence the role of these kinases and possible future individualized administration of inhibitors against these kinases in MM. BP-032

Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors, most recently culminating in the FDA-approval of vismodegib (Genentech/Roche) for the treatment of locally advanced or metastatic basal cell carcinoma. Vismodegib and other hedgehog pathway antagonists currently being evaluated in the clinical setting are small molecules that act by binding to a common site within Smoothened (SMO), a critical pathway component. Acquired mutations in SMO that map to these overlapping binding pockets compromise the potency and clinical efficacy of vismodegib and other clinically relevant agents in this space through an apparent class effect. As a consequence there are currently no visible therapeutic options for maintaining profound on-target efficacies in patients that present with recurrent disease harboring these point mutations. We report here that itraconazole and arsenic trioxide, two FDA-approved agents recently reported to inhibit Hedgehog signaling through mechanisms distinct from that of current SMO antagonists, maintain potent inhibition of hedgehog signaling in the face of acquired SMO mutations, both as single agents and when used in combination. Using a preclinical mouse model of medulloblastoma that mimics clinical mechanisms of vismodegib-associated acquired resistance, we demonstrate that itraconazole and arsenic trioxide inhibit pathway activation, proliferation, and tumor growth of medulloblastoma harboring SMO-D477G mutation, both in vitro and in vivo. These drugs act as effectively in syngeneic models of medulloblastoma demonstrating constitutive signaling through wild-type SMO. Combination of itraconazole and arsenic trioxide further suppress tumor growth and improves survival in an orthotopic model of vismodegib-resistant medulloblastoma compared to either single agent alone. Furthermore, itraconazole and arsenic trioxide retain activity in all reported drug-resistant SMO mutants, including those reported in relation to NVP-LDE225 (Novartis). Taken together, these results support clinical evaluation of itraconazole and arsenic trioxide for the treatment of Hh-dependent tumors, most notably those with acquired resistance to cyclopamine-mimics. These readily available FDA-approved agents have well defined pharmacokinetic and toxicity profiles. Our data support the rapid entry of itraconazole and arsenic trioxide into clinical testing to address patient populations for which vismodegib may not be available or is no longer efficacious. Citation Format: Blake T. Aftab, James Kim, Jean Y. Tang, Emily M. King, Daniel Kim, Jynho Kim, Ervin H. Epstein, Philip A. Beachy, Charles M. Rudin. Itraconazole and arsenic trioxide inhibit hedgehog pathway activation and tumor growth associated with acquired resistance to vismodegib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5644. doi:10.1158/1538-7445.AM2013-5644