Synergistic Therapeutic Strategy Research Articles

Design Strategy to Access siRNA-Encapsulating DNA "Nanosuitcases" That Can Conditionally Release Their Cargo.

DNA nanotechnology enables the design and assembly of DNA nanostructures with unprecedented control over their size and shape. Additionally, the programmable base-pairing alphabet of DNA allows the incorporation of responsive units within these DNA nanostructures. Here, we describe a general design strategy to construct responsive DNA prisms that can encapsulate and selectively release an encapsulated siRNA upon recognition of an oligonucleotide trigger. This prismatic DNA scaffold design is adaptable and can encapsulate oligonucleotides of any length and type. Moreover, the prism can be made to respond to an oligonucleotide trigger of interest like a messenger RNA (mRNA) or a microRNA (miRNA), thus enabling dual or synergistic therapeutic strategies. We present an overview of the design strategy used to access these DNA nanostructures, followed by the steps involved in DNA sequence generation, assembly, and validation of this construct.

Synergistic Therapeutic Strategy of Dual Drug-loaded Lipid Polymer Hybrid Nanoparticles for Breast Cancer Treatment

In this investigation, a smart nanocarrier-loaded docetaxel, a microtubules disrupting agent and vorinostat, a histone deacetylase inhibitor was developed to achieve a synergistic anticancer effect. Dual drug-loaded lipid polymer hybrid nanoparticles were prepared, with easy fabrication and favorable properties including small size, narrow distribution and a high loading efficacy. The in vitro drug release conducted in phosphate-buffered saline, pH 7.4 and acetate-buffered saline, pH 5.5 media demonstrated the sustained, pH-dependent release profile. The nanoparticles were effectively taken up by cells, which ensured greater suppression of cell growth. The co-delivery of both drugs exhibited a synergistic effect on the induction of cancer cell apoptosis, resulting in greater inhibition of SCC-7, MCF-7, and MDA-MB-231 cancer cells by the drug-loaded carrier. These promising results may lead to clinical applications with enhanced docetaxel activity.

CARM1 Inhibition: Evaluation of Response and Efficacy in Acute Myeloid Leukemia

Small molecule protein arginine methyltransferase inhibitors (PRMTi) are being actively pursued for the treatment of a variety of cancers; however, the mechanisms of response to PRMTi remain poorly understood. CARM1, also known as PRMT4, is significantly overexpressed in AML, as well as many solid tumors, and regulates myeloid differentiation. We have shown the dependency of AML cells, but not normal blood cells, on CARM1 activity, based on CARM1 knockout, CARM1 knockdown, and chemical inhibition (Greenblatt et al. Cancer Cell 2018). These experiments showed that CARM1 regulates essential processes in leukemia cells, and is critical for leukemic transformation. Although several small molecule inhibitors of CARM1 have been reported recently, many display a lack of selectivity for CARM1 or fail to produce a biological response. The recent discovery of potent and selective CARM1 inhibitors (Drew et al., 2017), has made it possible to investigate the implications of pharmacological inhibition of CARM1 in vitro and in vivo.In vitro, a selective CARM1 inhibitor, EPZ025654, reduced the methylation of a CARM1 substrate, BAF155, in a time and concentration-dependent manner, while the specific histone targets of CARM1 remained unchanged. Translocation (8;21) AML samples in the Eastern Cooperative Oncology Group cohort, have significantly higher CARM1 expression compared to normal CD34+ controls. This led us to hypothesize that CARM1 is a direct target of the AML1-ETO fusion protein. Therefore, we assessed whether EPZ025654 could inhibit AML1-ETO driven gene expression. AML1-ETO specific target genes showed significant changes in expression following EPZ025654 treatment. AML1-ETO positive patient samples also displayed decreased colony formation in methylcellulose and increased myeloid differentiation in response to CARM1 inhibition.We next evaluated EZM2302, a compound structurally related to EPZ025654, that is highly orally bioavailable and is well tolerated in mice (Drew et al., 2017). We generated AE9a-GFP primary transplantation mice and treated them with 100 mg/kg of EZM2302 or vehicle twice-daily (BID). The inhibitor treated mice showed significantly improved survival as well as fewer GFP+ cells in the peripheral blood over time. GFP+ AE9a bone marrow cells also showed decreased colony formation in vitro and induced macrophage differentiation in methylcellulose. GFP+ cells were isolated by FACS and submitted for RNA-sequencing. Flow cytometry analysis post-treatment revealed a significant downregulation of c-Kit and increased differentiation of hematopoietic stem and progenitor cells.Resistance to epigenetic targeted therapeutics has been observed, often through the induction of kinase signaling pathways. Therefore, we explored synergistic combinations with CARM1 inhibition using RNA-sequencing and proteomics analysis in leukemia cell lines. We used L1000 profiling (Subramanian et al., 2017) to simultaneously profile the transcriptional response of 18 AML cell line and CD34+ cells after 6 days of treatment. The AML1-ETO positive cell lines exhibited an IC50 in the 0.4-3 μM range, while CD34+ cells and several AML cell lines appeared to be resistant to CARM1 inhibition. While gene expression changes resulting from alterations in RNA stability were observed, the most significant differences between sensitive and resistant cell lines were genes associated with the regulation of cell cycle progression. Gene expression changes were evaluated over time in an AML1-ETO positive cell line, SKNO-1. SKNO-1 cell lines showed an upregulation of a gene expression signature associated with PI3K/AKT/mTOR signaling, with the most significant gene expression changes occurring 7-14 days post treatment. We simultaneously profiled these cells using multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in response to CARM1 inhibition over time. A comparison of this response to chemical perturbation signatures in the L1000 database, identified several chemical inhibitors of the PI3K/AKT/mTOR axis that could reverse the gene expression changes induced by CARM1 inhibition. This finding elucidated a response mechanism for CARM inhibition and a synergistic therapeutic strategy that has the potential to improve CARM1 directed therapy. DisclosuresNo relevant conflicts of interest to declare.

Combination Therapy with BTK Inhibitor Plus Anti-PD-1 Antibody Results in a Hyperprogressor Phenotype in a Mouse Model of CLL

Chronic lymphocytic leukemia (CLL) is often driven by aberrant activation of the B-cell receptor signaling pathway. We and others have shown in preclinical animal models as well as human clinical trials that the BTK inhibitor, ibrutinib, is effective in reducing CLL tumor burden and improving survival rates in both humans and mice. Preclinical studies also suggest that BTK inhibitors likely act not only through their direct effect on CLL tumor cells but also by alleviating immunosuppression in the tumor microenvironment through BTK inhibition in myeloid-derived suppressor cells. Additionally, we have previously demonstrated that CLL cells are susceptible to cytotoxic killing by T cells targeting the aberrantly expressed TCL1 oncoprotein (Weng et al. Blood 2012). Therefore, we hypothesized that the combination of BTK-pathway inhibition in conjunction with activation of antigen-specific T-cells by immune checkpoint blockade will be a synergistic therapeutic strategy. Here, we examined the effect of acalabrutinib (previously known as ACP-196), a selective BTK inhibitor with limited effect on other kinases, alone or in combination with immune checkpoint blockade in a mouse model of CLL.Eµ-TCL1 mice, which overexpress TCL1 in B-cells, were used as a model of CLL. We generated cohorts of Eµ-TCL1 mice and treated them with anti-PD-1 antibody (n = 18), acalabrutinib (n = 20), and acalabrutinib+anti-PD-1 antibody (n= 19). Treatment cohorts had a significant reduction in CD5+CD19+ tumor cells in peripheral blood and spleens as compared to vehicle-treated mice (n =16) (p <0.05). However, the most pronounced anti-tumor effects were observed in mice treated with acalabrutinib alone. In fact, MRI imaging of splenic volume and flow cytometry of CD5+CD19+ cells during the treatment phase revealed that acalabrutinib monotherapy was superior to the combinatorial therapy. Even more surprising was the fact that while anti-PD-1 and acalabrutinib monotherapies provided an improvement in survival compared to vehicle treatment (p = 0.05 and p < 0.0001, respectively), the combination of these two agents actually diminished overall survival (p = 0.77). Shockingly, histopathological analyses of tumors from these acalabrutinib+anti-PD-1 treated mice revealed a significant increase in lymph node involvement and tumors with a high mitotic index. Consistent with this, we observed that treatment of CD5+CD19+ tumor cells from Eµ-TCL1 transgenic mice in vitro with anti-PD-1 antibody plus acalabrutinib markedly increased the proliferative index as measured by EdU incorporation assay compared to acalabrutinib treated group.To understand the molecular events responsible for the observed acalabrutinib+anti-PD-1 hyperproliferative phenotypes, we are examining complementary pathways that may be aberrantly activated/repressed by this combination. Currently, we have identified expression changes in both the NFkb and PKC signaling pathways when these agents are used in combination, and are working to understand how this combination may impact tumor progression. We are also assessing the possibility that anti-PD-1 interferes with the anti-tumorigenic effect of acalabrutinib by enhancing the BCR signaling pathway in CLL tumor cells.In summary, our results demonstrate that the selective BTK inhibitor, acalabrutinib greatly enhances the survival of Eµ-TCL1 mice compared to either vehicle or ibrutinib alone. Surprisingly, we found that the combination of anti-PD-1 antibody + acalabrutinib therapy is actually detrimental in this CLL model and results in a hyperprogressor phenotype. Therefore, results from ongoing clinical trials evaluating combination strategies of anti-PD-1 antibody therapy plus BTK inhibitor need to be analyzed carefully to ensure that this combination is not leading to a similar hyperprogressor phenotype in patients. The results provided here offer some initial insights into the potential mechanisms of the “hyperprogressive” phenotypes following anti-PD-1 treatment and may highlight pathways that could be useful in blocking these deleterious effects. DisclosuresNo relevant conflicts of interest to declare.

IDH2 Mutations Induce Altered STAT Signaling and Cytokine Responses Which Are Restored By Enasidenib

Acute myeloid leukemia (AML) is a heterogeneous myeloid malignancy characterized by mutational and clonal heterogeneity. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are common, occurring in approximately 15-20% of patients, and actionable, with recently approved inhibitors for both mutations. These inhibitors lead to leukemia cell differentiation in vitro, in vivo and in patients. Healthy myeloid differentiation is governed by precise regulation of intracellular signaling, but this regulation is disrupted in AML. Given that signal transducer and activator of transcription (STAT) proteins are involved in both leukemogenesis and myeloid differentiation, we sought to determine the role of phosphorylated STATs and other signaling proteins in mIDH AML and inhibitor-induced differentiation of mIDH2 AML.To simultaneously dissect single cell signaling, differentiation and epigenetic changes, we employed fluorescence flow cytometry and mass cytometry to study an in vitro model of mIDH2R140Q leukemia, consisting of a parental TF-1 leukemia cell line and a CRISPR/Cas9 gene-edited mIDH2R140Q-mutated cell line. In order to generate provoked signaling profiles, we stimulated both the wild-type IDH2 and mutant IDH2 cells with nine cytokines. Additionally, we treated both cell lines and primary mIDH2 AML samples ex vivo with enasidenib and measured changes in high-dimensional single cell phenotype and phospho-protein expression via mass cytometry.mIDH2 leukemia cells displayed elevated basal pSTAT1, pSTAT3 and pNFkB-s529 expression, and concomitant low basal IkBa levels when compared to parental cells. Further, mIDH2 cells had increased PMA induced pS6 and IL1β induced pNFkB-s529 than the parental cell line, while wtIDH2 cells had higher levels of cytokine-induced pSTAT1, pSTAT3 and pSTAT5.After prolonged enasidenib treatment (28 days), model mIDH2 cells expressed lower basal pSTAT1, pSTAT3 and pNFkB-s529 than vehicle-treated mIDH2 cells. Further, when stimulated with GM-CSF, enasidenib-treated mIDH2 leukemia cells showed increased response at pSTAT3 and pSTAT5 as compared to vehicle-treated leukemia cells. We dissected enasidenib-induced differentiation using 18 cell surface markers and visualized results using t-distributed stochastic neighbor embedding (tSNE). Presence of mIDH2 leads to baseline expression differences including higher CD90 and CD71. Following treatment with enasidenib, mIDH2 leukemia cells had increased CD45 and CD11b expression as compared to vehicle-treated controls. Additionally, prolonged treatment with enasidenib increased proliferation as shown by increased Ki67 and decreased histone hypermethylation at suppressive histone marks, H3K27 and H3K9, while 7 days of enasidenib did not result in changes to these marks.We further explored the impact of mIDH2 in primary leukemia samples. First, we developed an ex vivo stromal co-culture system that allowed for treatment and expansion of four primary mIDH2 AML samples for 16 days. While the basal levels of signaling markers were sample dependent, consistently across all samples enasidenib-treated primary AML was more sensitive to IL-6 and GM-CSF-induced pSTAT1, pSTAT3 and pSTAT5 signaling. The enasidenib-treated AML also showed increased expression of mature myeloid markers, including CD33 and CD11c.Here we have shown the presence of mIDH2 mutations leads to decreased STAT signaling in response to cytokine stimulation as compared to wtIDH2 AML cells. We have also demonstrated increased response of pNFkB-s529 and pS6 to cytokine stimulation in mIDH2 AML cells as compared to wtIDH2 AML cells. Moreover, in both mIDH2 AML cell line and primary samples, IDH2 inhibitor-induced differentiation restored sensitivity to cytokine responses and reduced histone hypermethylation. Future work exploring these aberrant signaling events may reveal precise connections between mutated IDH, associated epigenetic changes, and intracellular signaling, potentially uncovering synergistic therapeutic strategies for mIDH AML. DisclosuresFerrell:Incyte: Research Funding.

Nanocatalyst Complex Can Dephosphorylate Key Proteins in MAPK Pathway for Cancer Therapy

Controlling phosphorylation processes of proteins is a facile way for manipulating cell fates. Herein, a synergistic therapeutic strategy utilizing a near-infrared (NIR)-responsive nanocatalyst (NC) complex is presented, which is comprised of photoactive NC and protein phosphatase 2A (PP2A), to synergistically inhibit hyperphosphorylation of mitogen-activated protein kinase (MAPK) pathway for cancer therapy, as an example of many biological processes this approach can apply to. NIR-triggered release of PP2A specially dephosphorylates and inactivates mitogen-activated protein kinase kinase (MAP2K, also known as MEK) and extracellular regulated protein kinases (ERK) in the MAPK pathway, meanwhile, the NIR-triggered activation of NC decreases the level of intracellular adenosine triphosphate to attenuate protein phosphorylation of MEK and ERK. The synergistic therapeutics effectively suppress melanoma progression by inhibiting hyperphosphorylation of the MAPK pathway. In addition, the nanocatalyst complex reduces the risk of drug-resistance through inhibiting a rebound of RAS-GTP. The NIR-responsive nanocatalyst complex paves a novel way for cancer therapeutics.

Abstract 3716: Tumor multicomponent targeting nano-micelles with synergistic combination to overcome drug resistance and reprogramming of macrophages in renal cell carcinoma

Abstract Renal Cell Carcinoma (RCC) contributes &amp;gt;90% of the most common form of kidney tumor and remains one of the ten leading causes of cancer death in the US. Tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors have increased therapeutic possibilities for treating RCC. Although the impact on disease progression is encouraging, a substantial proportion of patients do not respond adequately, and therapy resistance almost inevitably occurs. Eventually, new strategies have emerged that include immunotherapy such as PD-1 inhibitor (Nivolumab) and the combination of chemo-immune therapy. Possibly, combination treatment aimed at different, non-related pathways may be advantageous. In this regard, we would like to come up with synergistic therapeutic strategies for nonresponsive, highly aggressive tumor types to tackle the current clinical challenges. We have worked on different RCCs tumor models that in general have a poor prognosis with conventional therapies. We also pursued different combination regimens, including drugs that work on the mTOR inhibition (everolimus), inhibit RTK-inhibitor or VEGFR (cabozantinib or sorafenib). Also, there is a critical need to develop safe and effective delivery vehicles that can carry the payload to the right target tissue and cell. Thus, different types of nanoparticles have been designed to deliver a variety of therapeutic agents to target hypoxic tumor microenvironment by using carbonic anhydrase-9 (CA9); FRα for targeting cancer epithelial cells; and FRβ for targeting tumor-associated macrophages (TAM) and combination delivery of RTK-inhibitor Cabozantinib (CB) and sorafenib with our own apoptosis inducer/CARP-1 protein activator CFM-4.16 (C4.16) for overcoming drug resistance and reprograming TAM for RCC therapy. The current work also focused on multimodal approaches, including (a) Optimization of hypoxia marker conjugated targeted polymer-lipid nano-formulation (PLNP) using copper-free click chemistry; (b) In vitro and in vivo pre-clinical testing of targeted -PLNP loaded with polypharmacy in inhibiting RCCs using mice bearing resistant RCCs and patient-derived xenografts (PDX). The results of efficacy and biodistribution of targeted PLNPs in animals bearing RCC xenografts and PDX model showed higher accumulation of drugs at tumor sites with higher tumor growth inhibition. Also, the targeted formulation showed high binding affinity and specific tumor uptake, faster normal tissue clearance, and less non-target organ uptake. These findings portent promising therapeutic potential of hypoxia-targeted -PLNPs for treating RCCs. Citation Format: Hashem O. Alsaab, Samaresh Sau, Vino T. Cheriyan, Rami Alzhrani, Ulka Vaishampayan, Arun K. Rishi, Arun K. Iyer. Tumor multicomponent targeting nano-micelles with synergistic combination to overcome drug resistance and reprogramming of macrophages in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3716.

High-Performance Poly(lactic-co-glycolic acid)-Magnetic Microspheres Prepared by Rotating Membrane Emulsification for Transcatheter Arterial Embolization and Magnetic Ablation in VX2 Liver Tumors.

Interventional embolization is a popular minimally invasive vascular therapeutic technique and has been widely applied for hepatocellular carcinoma (HCC) therapy. However, harmful effects caused by transcatheter arterial chemoembolization (TACE) and radioembolization, such as the toxicity of chemotherapy or excessive radiation damage, are serious disadvantages and significantly reduce the therapeutic efficacy. Here, a synergistic therapeutic strategy combined transcatheter arterial embolization and magnetic ablation (TAEMA) by using poly(lactic-co-glycolic acid) (PLGA)-magnetic microspheres (MMs) has been successfully applied to orthotopic VX2 liver tumors of rabbits. These MMs fabricated by novel rotating membrane emulsification system with well-controlled sizes (100-1000 μm) exhibited extremely low hemolysis ratio and excellent biocompatibility with HepG2 cells and L02 cells. Moreover, experimental results demonstrated that, while exposed to alternating magnetic field (AMF) after TAE, the tumor edge could be heated up by more than 15 °C both in vivo and in vitro, whereas only a negligible increase of temperature was observed in the normal hepatic parenchyma (NHP) nearby. Sufficient temperature increase induces apoptosis of tumor cells. This can further inhibit the tumor angiogenesis and results in necrosis compared to the rabbits only treated with TAE. In stark contrast, tumors rapidly grow and subtotal metastasis occurs in the lungs or kidneys, causing severe complications for rabbits only irradiated under AMF. Importantly, the results from the biochemical examination and the gene expression of relative HCC markers further confirmed that the treatment protocol using PLGA-MMs could achieve good biosafety and excellent therapeutic efficacy, which are promising for liver cancer therapy.

684P - Targeting c-met and DNA double-strand break (DSB) repair pathways for BRCA-mutated gastric carcinomas

Background: In the present study, we underline the importance of combining c-Met Inhibitor (Inh.) and DSB repair Inhibitor (PARP Inh.) with the goal of accomplishing a synergistic therapeutic strategy in BRCA-mutated gastric carcinomas. Methods: Firstly, to verify whether c-Met affects tumor response to PARP Inh., AGS (low c-Met expression) and Hs746T (high c-Met expression) cell lines, were subjected to knockdown c-Met expression, in the presence of PARP Inh. (NU1025). Subsequently, we examined the correlation between BRCA1/2 and c-Met. We knocked down BRCA1/2 expression in AGS and Hs746T cells and treated them with PARP Inh. Next, we examined the effects of combining c-Met Inh. (SU11274) and PARP Inh. (NU1025), measured by MTT, clonogenic cell survival and Annexin, assays. We also evaluated the effect of combining PARP Inh. and c-Met Inh. in AGS/Hs746T xenograft tumor models. AGS/Hs746T -pcDNA3 and AGS/Hs746T -siBRCA cells were injected into SCID mice. Tumor sizes were measured every 3 days. DNA damage was assessed by γ-H2AX staining. Results: Silence of Met expression promoted Hs746T cells more sensitive to PARP Inh. to similar levels as the AGS cells, as indicated by decreased cell viability. Silence of BRCA1/2 expression sensitized only AGS cells, signifying that increased expression of c-Met renders cells resistant to PARP Inh. in the context of inactivating BRCA1/2. Combining c-Met Inh. and PARP Inh. suppressed cell growth and clonogenicity and enhanced apoptosis in both AGS and Hs746T cells. The dual inhibition was demonstrated to be even more successful when cells were knockdown for BRCA1/2. Also, co-inhibition treatment substantially reduced tumor growth to AGS/Hs746T -pcDNA3 and more even effectively to AGS/Hs746T –siBRCA1/2 xenograft models, compared to either Inh. alone. DNA damage was higher in AGS/Hs746T –siBRCA1/2 compare to AGS/Hs746T -pcDNA3 xenograft models. Conclusions: BRCA deficiency renders gastric tumor cells sensitive to PARP inhibition. In addition, treatment with c-Met Inh. enhanced Hs746T sensitivity to the PARP Inh. Our data demonstrate that dual Met/PARP inhibition is synergistic providing an effective therapeutic strategy in BRCA-mutated gastric carcinomas. Legal entity responsible for the study: M.V. Karamouzis Funding: None Disclosure: All authors have declared no conflicts of interest.

Cholangiocarcinoma stem-like subset shapes tumor-initiating niche by educating associated macrophages

A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium. CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect. CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease. Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.

Abstract 1935: Gene expression analysis reveals distinct pathways of resistance to antiangiogenic therapy in ovarian cancer

Abstract Purpose: The rapid development of resistance after an initial response phase is a major challenge of antiangiogenic therapies. The molecular mechanisms underlying clinical resistance to antiangiogenic therapies are not yet well understood. We aimed to identify potential mediators of resistance to antiangiogenic therapy, with a view toward eventually developing therapeutic approaches to improve antiangiogenic therapies. Methods: We developed preclinical in vivo phenotypic resistance models of antiangiogenic therapy using human ovarian carcinoma cells. We used two representative anti-VEGF agents to block VEGF (bevacizumab, 10 mg/kg BIW, IP) or its receptor (sorafenib, 30 mg/kg daily, PO). The treatment was given until tumors showed a long-term trend toward continued progression after an initial response (resistant tumor). Whereas treatment-sensitive tumors were defined by a long-term trend toward stable disease or regression. We conducted next generation sequencing to compare changes in the gene expression in resistant and sensitive tumors, distinguishing their origin (tumor vs stroma cells). To get a more detailed perspective of the overall changes in gene expression, Gene Set Enrichment Analysis (GSEA) was performed by pair-wise comparison according to treatment outcome (resistant vs sensitive) with respect to bevacizumab and sorafenib, of which the entire MSigDB collections were compared to. Further, significantly enriched pathways were validated with clinical gene expression profiling studies of various tumor types deposited in GEO and ArrayExpress. Results: Our GSEA analysis revealed significant differences (FDR&amp;lt;0.25) in enrichment of various pathways between sensitive and resistant tumors. We observed ERK, P38 MAPK, ATP dependent helicase activity, and phosphatidylinositide 3-kinases pathways are differentially enriched between sensitive and resistant phenotypes of bevacizumab-treated group. In sorafenib-resistant group, six pathways were enriched including E2F1, KRAS and PDGF regulated pathways. Conclusions: Here, we report distinct pathways of response to bevacizumab and sorafenib in preclinical ovarian cancer models, which may provide critical insights into ovarian tumor's adaption during anti-VEGF blockade. These pathways may also provide important clues for identifying synergistic therapeutic strategies to overcome or delay resistance to antiangiogenic agents. Citation Format: Bharat Kumar Devapatla, Pharavee Jaiprasart, Sukyung Woo. Gene expression analysis reveals distinct pathways of resistance to antiangiogenic therapy in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1935. doi:10.1158/1538-7445.AM2015-1935

Potential synergy between tau aggregation inhibitors and tau chaperone modulators

Tau is a soluble, microtubule-associated protein known to aberrantly form amyloid-positive aggregates. This pathology is characteristic for more than 15 neuropathies, the most common of which is Alzheimer’s disease. Finding therapeutics to reverse or remove this non-native tau state is of great interest; however, at this time only one drug is entering phase III clinical trials for treating tauopathies. Generally, tau manipulation by therapeutics can either directly or indirectly alter tau aggregation and stability. Drugs that bind and change the conformation of tau itself are largely classified as aggregation inhibitors, while drugs that alter the activity of a tau-effector protein fall into several categories, such as kinase inhibitors, microtubule stabilizers, or chaperone modulators. Chaperone inhibitors that have proven effective in tau models include heat shock protein 90 inhibitors, heat shock protein 70 inhibitors and activators, as well as inducers of heat shock proteins. While many of these compounds can alter tau levels and/or aggregation states, it is possible that combining these approaches may produce the most optimal outcome. However, because many of these compounds have multiple off-target effects or poor blood–brain barrier permeability, the development of this synergistic therapeutic strategy presents significant challenges. This review will summarize many of the drugs that have been identified to alter tau biology, with special focus on therapeutics that prevent tau aggregation and regulate chaperone-mediated clearance of tau.

Treatment of acute high-risk pulmonary embolism

At present, high-risk pulmonary embolism represents a cardiovascular emergency burdened with high in-hospital mortality and characterized by acute right ventricular dysfunction and hemodynamic impairment. In addition to circulatory support and anticoagulation, thrombolytic therapy has become the cornerstone of the treatment in patients presenting with this condition. Despite the recommendations, a consistent proportion of patients does not currently receive thrombolytic therapy. Although performed in a limited number of patients, transcatheter and surgical embolectomy procedures are an alternative or synergistic therapeutic strategy to thrombolysis, enabling a prompt resolution of right ventricular volume overload. In this review, data from the literature are discussed with the aim of defining an algorithm for the treatment of high-risk patients.

Combinations of the FLT3 Inhibitor CEP-701 and Chemotherapy Synergistically Kill Infant and Childhood MLL-Rearranged ALL Cells in a Sequence-Dependent Manner.

Rearrangement of the MLL gene at chromosome 11q23, which occurs in 80% of infants and 5% of older children with acute lymphoblastic leukemia (ALL), confers a dismal prognosis despite maximally intensified chemotherapy and allogeneic transplantation. Novel therapies are clearly needed for these patients. The FLT3 tyrosine kinase represents an attractive potential target in these leukemias, due to the marked overexpression of FLT3 in cases of MLL-rearranged ALL. We have previously shown that MLL-rearranged infant and childhood ALL leukemic cells express high levels of constitutively activated FLT3 receptor protein, and that these cells are selectively killed by exposure to the FLT3 inhibitor CEP-701. However, FLT3 inhibition is unlikely to represent curative therapy for these patients if used as monotherapy. Thus, we examined various combinations of CEP-701 with chemotherapy to look for potentially synergistic therapeutic strategies. We performed MTT cytotoxicity and annexin V binding (AVB) apoptosis assays on MLL-rearranged ALL cell lines (SEM-K2 and HB-1119) and three primary patient samples (all of which we show to express high levels of constitutively activated FLT3) after exposure to multiple dose combinations of CEP-701 and six chemotherapy agents (vincristine, l-asparaginase, dexamethasone, daunorubicin, etoposide and cytarabine). Since we have previously demonstrated that the nature of the interactions between CEP-701 and chemotherapy agents (i.e., synergistic, additive or antagonistic) are sequence-dependent in AML cells, we performed the MTT and AVB assays in three sequences designed to mimic the potential clinical uses of the combinations (Seq1: chemotherapy followed by CEP-701; Seq2: simultaneous exposure to both; and Seq3: CEP-701 followed by chemotherapy). The nature of the interaction between CEP-701 and each chemotherapy agent was determined using the median effect method of Chou and Talalay, which calculates a combination index (CI) for each combination (CI < 0.9 - synergistic; CI 0.9-1.1 - additive; CI > 1.1 antagonistic). A striking pattern of sequence-dependent synergy was observed: Seq1 was markedly synergistic (mean CI +/− SD = 0.59 +/− 0.18), Seq2 was additive (CI = 0.99 +/− 0.29) and Seq3 was antagonistic (CI = 1.23 +/− 0.40). The sequence-dependence is attributable to the G1 cell cycle arresting effects of CEP-701 in these cells. An MLL-rearranged cell line that expresses low levels of minimally activated FLT3 (RS4-11) demonstrates minimal cell cycle arrest when treated with CEP-701, and mere additivity in all 3 CEP-701/chemotherapy combination sequences (Seq1: CI = 0.95 +/− 0.10; Seq2: CI = 1.01 +/− 0.09; Seq3: CI = 1.06 +/− 0.10), confirming that the observed pattern of synergy is mediated through the effects of CEP-701 on FLT3 signaling. These results can help guide the combination of CEP-701 with existing chemotherapy regimens for infants and children with MLL-rearranged ALL, with the goal of improving the dismal outcome for these patients.