Combination Therapy Strategies Research Articles

Co istotnego zapamiętamy z nowości kończącego się roku w nadciśnieniu płucnym?

Pulmonary hypertension (PH) is a complex dise- ase with multifactorial etiology. Currently, a new definition and PH recognition is discussed if mean pulmonary artery pressure during right heart catheterisation is above 20 mm Hg (previo- usly > 25 mm Hg).In recent years, the use of so-called targeted therapy — focused on pathomechanisms of pulmonary arterial hypertension (PAH) contributed to improved survival in this group of patients. Further progress was made due to the development of new combined therapy strategies and escalation of treatment — in order to obtain patients’ low risk profile. Contemporary proceedings in PAH, in addition to the prompt diagnosis of the disease and the implementation of specialized treatment, include regular controls with a comprehensive assessment of exercise tole-rance, right ventricular function and the clinical status of patients.

Abstract P6-17-33: A multi-scale mathematical model of combination targeted and cytotoxic therapy to evaluate treatment response in HER2+ breast cancer

Abstract Introduction: We have previously established a tissue scale mathematical model with 5 coupled, ordinary differential equations (ODEs) describing the longitudinal relationship of vasculature, hypoxia, necrosis, immune infiltration, and tumor growth in a preclinical model of human epidermal growth factor receptor 2 positive (HER2+) breast cancer undergoing trastuzumab treatment. The purpose of this study is to expand this model to multiple scales (tissue and cellular) and to explicitly include the effects of combination trastuzumab-paclitaxel therapy as measured by time-resolved microscopy of in vitro HER2+ breast cancer cells. This requires interlacing experimental data and additional ODEs at the cellular scale to incorporate drug dynamics within the tissue scale model for overall tumor growth. An integrated multi-scale mathematical-experimental approachbridging in vivo and in vitro experimental data has potential to elucidate the optimal strategies for combination therapy for HER2+ breast cancer. Experimental: In vitro data was collected to longitudinally evaluate BT474 HER2+ human cancer cells growth when treated with trastuzumab (25-50 ug/ml) and/or paclitaxel (10-250 nmol/L) measured by time-resolved microscopy. Changes in confluence before and after treatment are recorded every 3 hours over the course of 7 days. Ongoing studies are quantifying the change in confluence for these cells with alternating dosing and timing of the two therapies. Modeling: Thein vivo, tissue-scale model parameters were calibrated using mean and 95% confidence intervals of tumor volume from caliper measurements, vasculature and hypoxia from imaging data, and necrosis from histology. The in vitro scale of the model is calibrated with confluence data for controls to define intervals for growth rates and carrying capacities. Experimental results for single drug applications are used to estimate cell death, and data from combinations of both the targeted anti-HER2 therapy and cytotoxic therapies provide estimates of synergistic drug effects. After calibration of parameters, the two scales of the model are coupled via the tumor volume and vasculature components to simulate the effects of combination therapy in vivo. Results and Discussion: The confluence data shows that trastuzumab dosing in combination with paclitaxel results in the greatest reduction in tumor cells than either drug alone (p < 0.05), and there are distinct differences in alternating the order and timing of these drugs. Further, preliminary results show that the model's in vitro scale equations can be calibrated with the available longitudinal data. As dosing of combination therapy in vitro provides the opportunity to quantify the effects of combination therapy that would not be feasible to collect in an in vivo setting, our aim is to generate experimentally testable predictions for improved combination therapy in vivo with our multiscale model. This is an important step for future clinical translation to provide temporal guidance of standard-of-care, combination therapies, potentially leading to significantly improved anticancer response in HER2+ breast cancer. We acknowledge the support of NCI U01CA174706, NCI R01CA186193, CPRIT RR160005, and ACS RSG-18-006-01-CCE. Citation Format: Jarrett AM, Shah A, Bloom MJ, Yankeelov TE, Sorace AG. A multi-scale mathematical model of combination targeted and cytotoxic therapy to evaluate treatment response in HER2+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-33.

FUS/circ_002136/miR-138-5p/SOX13 feedback loop regulates angiogenesis in Glioma

BackgroundAngiogenesis plays a critical role in the progression of glioma. Previous studies have indicated that RNA-binding proteins (RBPs) interact with RNAs and participate in the regulation of the malignant behaviors of tumors. As a type of endogenous non-coding RNAs, circular RNAs (circRNAs) are abnormally expressed in various cancers and are involved in diverse tumorigeneses including angiogenesis.MethodsThe expression levels of FUS, circ_002136, miR-138-5p, SOX13, and SPON2 were determined using quantitative real-time PCR (qRT-PCR) and western blot. Transient cell transfection was performed using the Lipofectamine 3000 reagent. The RNA-binding protein immunoprecipitation (RNA-IP) and the RNA pull-down assays were used to detect the interaction between FUS and circ_002136. The dual-luciferase reporter assay system was performed to detect the binding sites of circ_002136 and miR-138-5p, miR-138-5p and SOX13. The chromatin immunoprecipitation (ChIP) assays were used to examine the interactions between transcription factor SOX13 and its target proteins .ResultsWe demonstrated that down-regulation of FUS or circ_002136 dramatically inhibited the viability, migration and tube formation of U87 glioma-exposed endothelial cells (GECs). MiR-138-5p was down-regulated in GECs and circ_002136 functionally targeted miR-138-5p in an RNA-induced silencing complex (RISC). Inhibition of circ_002136, combined with the restoration of miR-138-5p, robustly reduced the angiogenesis of GECs. As a target gene of miR-138-5p, SOX13 was overexpressed in GECs and was proved to be involved in circ_002136 and miR-138-5p-mediated angiogenesis in gliomas. In addition, we found that SOX13 was directly associated with and activated the SPON2 promoter, thereby up-regulating the expression of SPON2 at the transcriptional level. Knockdown of SPON2 suppressed the angiogenesis in GECs. More important, SOX13 activated the FUS promoter and increased its expression, forming a feedback loop.ConclusionOur data suggests that the feedback loop of FUS/circ_002136/miR-138-5p/SOX13 played a crucial role in the regulation of angiogenesis in glioma. This also provides a potential target and an alternative strategy for combined glioma therapy.

Expression patterns of PD-L1 and other immune checkpoint molecules in gastric cancer.

49 Background: Immune checkpoint inhibitors (ICPIs) have provided clinical benefit for various malignancies. A new attempt was recently made to combine PD-1/PD-L1 inhibitors and other ICPIs. In order to establish a new strategy for ICPI combination therapy, we must first understand the expression patterns of PD-L1 and other ICP molecules. To identify possible candidate agents for application in combination therapy with PD-1/PD-L1, we analyzed the expression patterns of ICP molecules in gastric cancer. Methods: Tumor samples were obtained from 278 patients who underwent gastrectomy for gastric cancer from 2014 to 2016, and a comprehensive gene expression analysis using a DNA microarray. We analyzed the TCGA dataset to identify the candidate ICP molecules in relation to PD-L1. The immune landscape was evaluated according to the expression patterns of known immune cell markers reported by Bindea et al. Results: Seven ICP molecules ( PD-L2, IDO1, CD80, ICOS, CTLA4, LAG3 and TIM3) were coexpressed with PD-L1 in the TCGA dataset. In our cohort, all of these molecules were also coexpressed (R > 0.5, P < 0.0001). Using a cluster analysis based on these gene expression profiles, patients were divided into Group A, characterized by the co-overexpression of PD-L1 and other ICP molecules, and Group B, characterized by the underexpression of PD-L1. In Group A, PD-L2 (R = 0.67) and IDO1 (R = 0.61) were particularly strongly associated with the PD-L1 expression. In Group B, elevated expressions of CTLA4 and ICOS were predominantly observed, and these expressions were strongly correlated with each other (R = 0.90). The tumor stage was significantly more advanced in Group B than in Group A (P = 0.02). Accordingly, Group A exhibited a better survival outcome than Group B. The immune landscape of Group A was particularly enriched for Th1, NK CD56dim and activated dendritic cells. Conclusions: PD-L1 and several immune checkpoint molecules tend to be coexpressed in gastric cancer. The combination of PD-L1/-L2 and IDO1 target agents may be a new strategy for treating gastric cancer with PD-L1 overexpression. In contrast, the underexpression of PD-L1 might be an indication for the use of CTLA4- and/or ICOS-directed therapies.

Risk stratification and medical therapy of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) remains a severe clinical condition despite the availability over the past 15 years of multiple drugs interfering with the endothelin, nitric oxide and prostacyclin pathways. The recent progress observed in medical therapy of PAH is not, therefore, related to the discovery of new pathways, but to the development of new strategies for combination therapy and on escalation of treatments based on systematic assessment of clinical response. The current treatment strategy is based on the severity of the newly diagnosed PAH patient as assessed by a multiparametric risk stratification approach. Clinical, exercise, right ventricular function and haemodynamic parameters are combined to define a low-, intermediate- or high-risk status according to the expected 1-year mortality. The current treatment algorithm provides the most appropriate initial strategy, including monotherapy, or double or triple combination therapy. Further treatment escalation is required in case low-risk status is not achieved in planned follow-up assessments. Lung transplantation may be required in most advanced cases on maximal medical therapy.

Epigenetic Co-Deregulation of EZH2/TET1 is a Senescence-Countering, Actionable Vulnerability in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) cells lack the expression of ER, PR and HER2. Thus, TNBC patients cannot benefit from hormone receptor-targeted therapy as non-TNBC patients, but can only receive chemotherapy as the systemic treatment and have a worse overall outcome. More effective therapeutic targets and combination therapy strategies are urgently needed to improve the treatment effectiveness.Methods: We analyzed the expression levels of EZH2 and TET1 in TCGA and our own breast cancer patient cohort, and tested their correlation with patient survival. We used TNBC and non-TNBC cell lines and mouse xenograft tumor model to unveil novel EZH2 targets and investigated the effect of EZH2 inhibition or TET1 overexpression in cell proliferation and viability of TNBC cells.Results: In TNBC cells, EZH2 decreases TET1 expression by H3K27me3 epigenetic regulation and subsequently suppresses anti-tumor p53 signaling pathway. Patients with high EZH2 and low TET1 presented the poorest survival outcome. Experimentally, targeting EZH2 in TNBC cells with specific inhibitor GSK343 or shRNA genetic approach could induce cell cycle arrest and senescence by elevating TET1 expression and p53 pathway activation. Using mouse xenograft model, we have tested a novel therapy strategy to combine GSK343 and chemotherapy drug Adriamycin and could show drastic and robust inhibition of TNBC tumor growth by synergistic induction of senescence and apoptosis.Conclusions: We postulate that the well-controlled dynamic pathway EZH2-H3K27me3-TET1 is a novel epigenetic co-regulator module and provide evidence regarding how to exploit it as a novel therapeutic target via its pivotal role in senescence and apoptosis control. Of clinical and therapeutic significance, the present study opens a new avenue for TNBC treatment by targeting the EZH2-H3K27me3-TET1 pathway that can modulate the epigenetic landscape.

Salidroside enhances the anti-cancerous effect of imatinib on human acute monocytic leukemia via the induction of autophagy-related apoptosis through AMPK activation.

As the typical tyrosine kinase inhibitor, imatinib has been the first-line antineoplastic agent for both chronic myeloid leukemia and acute lymphoblastic leukemia. However, a large number of patients are still resistant to the benefits of imatinib, and they have a dissatisfactory prognosis. Salidroside, a compound that is extracted from natural plants, has been reported to have an excellent anticancer effect and few side effects. In the present study, we have developed a new combination therapy strategy of salidroside and imatinib for combating the growth of acute lymphoblastic leukemia. As demonstrated by the anti-proliferation assay, salidroside exhibited excellent cytotoxicity against myeloid leukemia cells. Moreover, cells treated by the combination therapy of salidroside and imatinib displayed a clear lower growth rate than cells only treated by imatinib, indicating that salidroside has a positive effect on enhancing the cytotoxicity of imatinib against leukemia cells. Subsequently, the underlying mechanisms were investigated. The results revealed that autophagy marker proteins in leukemia cells, including LC3, p62, and Beclin1, displayed a significant expression change after treating them with salidroside plus imatinib, with the levels of LC3 and Beclin1 dramatically increasing while the expression of p62 was significantly decreased. Moreover, an obvious down-regulation of p-PI3K, p-AKT and p-mTOR expression levels in leukemia cells after treatment with salidroside plus imatinib suggested that the PI3K/mTOR pathway plays an important role in the process of cell apoptosis induced by salidroside or imatinib. Further studies showed that pre-incubating the cells with an autophagy inhibitor dramatically inhibited the ability of imatinib to induce autophagy, but did not inhibit the ability of salidroside. The underlying causes were subsequently explored and the results showed that silencing AMPKα1, the most important regulator of autophagy, dramatically attenuates the ability of salidroside to induce cell apoptosis. These results together indicated that salidroside enhances the cytotoxicity of imatinib on acute monocytic leukemia via the induction of autophagy-related apoptosis through AMPK activation. The unique advantages of combination therapy were further confirmed by in vivo experiments, with the tumor-bearing cells treated with salidroside plus imatinib achieving the best anti-tumor effect.

Supramolecular Hydrogel Based on Chlorambucil and Peptide Drug for Cancer Combination Therapy.

Supramolecular hydrogels of self-assembling peptide-drug conjugates have been considered as effective self-delivery drug systems for cancer therapy in recent years. Here, a novel self-assembling peptide-based supramolecular hydrogel was developed by simultaneously conjugating small-molecule drug chlorambucil (CRB) and peptide drug tyroservatide (YSV) to the self-assembling peptide. The resulting hydrogel with a nanofiber structure showed enhanced stability against proteinase K degradation and an improved cellular uptake performance in comparison with the free molecules. As a consequence, it exhibited enhanced antitumor efficiency both in vitro and in vivo with favorable biocompatibility. This biocompatible self-delivery drug system could not only significantly improve the delivery efficiency of the small-molecule drugs but also adequately synergize the antitumor effect of CRB and YSV, inspiring the design of new strategies of cancer combination therapy.

Conophylline suppresses pancreatic cancer desmoplasia and cancer-promoting cytokines produced by cancer-associated fibroblasts.

Despite recent advances in cancer treatment, pancreatic cancer is a highly malignant tumor type with a dismal prognosis and it is characterized by dense desmoplasia in the cancer tissue. Cancer‐associated fibroblasts (CAF) are responsible for this fibrotic stroma and promote cancer progression. We previously reported that a novel natural compound conophylline (CnP) extracted from the leaves of a tropical plant reduced liver and pancreatic fibrosis by suppression of stellate cells. However, there have been no studies to investigate the effects of CnP on CAF, which is the aim of this work. Here, we showed that CAF stimulated indicators of pancreatic cancer malignancy, such as proliferation, invasiveness, and chemoresistance. We also showed that CnP suppressed CAF activity and proliferation, and inhibited the stimulating effects of CAF on pancreatic cancer cells. Moreover, CnP strongly decreased the various cytokines involved in cancer progression, such as interleukin (IL)‐6, IL‐8, C‐C motif chemokine ligand 2 (CCL2), and C‐X‐C motif chemokine ligand 12 (CXCL12), secreted by CAF. In vivo, CAF promoted tumor proliferation and desmoplastic formation in a mouse xenograft model, CnP reduced desmoplasia of tumors composed of pancreatic cancer cells + CAF, and combination therapy of CnP with gemcitabine remarkably inhibited tumor proliferation. Our findings suggest that CnP is a promising therapeutic strategy of combination therapy with anticancer drugs to overcome refractory pancreatic cancers.

Modulating the Tumor Microenvironment via Oncolytic Viruses and CSF-1R Inhibition Synergistically Enhances Anti-PD-1 Immunotherapy.

Immunotherapy based on the immune checkpoint blockade has emerged as the most promising approach for cancer therapy. However, the proportion of colorectal cancer patients who benefit from immunotherapy is small due to the immunosuppressive tumor microenvironment. Hence, combination immunotherapy is an ideal strategy to overcome this limitation. In this study, we developed a novel combination of CSF-1R (colony-stimulating factor 1 receptor) inhibitor (PLX3397), oncolytic viruses, and anti-PD-1 antibody. Our results demonstrated that the triple treatment synergistically conferred significant tumor control and prolonged the survival of mouse models of colon cancer. Approximately 43% and 82% of mice bearing the CT26 and MC38 tumor, respectively, survived long term following the triple treatment. This combination therapy reprogrammed the immunosuppressive tumor microenvironment toward a CD8+ Tcell-biased anti-tumor immunity by increasing Tcell infiltration in the tumor and augmenting anti-tumor CD8+ Tcell function. Our results provide a robust strategy for clinical combination therapy.

Combination therapy of sorafenib with mesenchymal stem cells as a novel cancer treatment regimen in xenograft models of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common liver malignancy and the second leading cause of cancer-related deaths in the world. Sorafenib is the first-line treatment of HCC. Although sorafenib has positive effects on the survival of patients, novel therapeutic strategies are needed to extend survival and improve the efficacy of sorafenib. This study combines sorafenib with mesenchymal stem cells (MSCs) as a new approach to enhance the efficacy of sorafenib. A subcutaneous xenograft model of HCC, established by human HepG2 cell lines, was implanted into the flank of nude miceand was used to evaluate tumor growth after treatment with sorafenib alone or in combination with MSCs. The aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine levels were measured for safety assessment. Histopathological studies were performed using hematoxylin and eosin staining, and immunohistochemistry tests were performed to evaluate proliferation (Ki67) and angiogenesis (CD34). The TUNEL assay was used to detect apoptosis and measure the expression of major inflammatory cytokines (IL-1a, IL-10, and TNF-α) with real-time polymerase chain reaction. Sorafenib, in combination with MSCs, strongly inhibited tumor growth in the xenograft model. Furthermore, the combination therapy significantly inhibited HCC cell proliferation, decreased tumor angiogenesis, and induced apoptosis and maintained antitumor-associated anti-inflammatory effects of MSCs. This combination therapy strategy could be used as a new therapeutic approach to the treatment of HCC that significantly improves upon the results achieved using sorafenib as monotherapy.

A Novel PAA Derivative with Enhanced Drug Efficacy in Pancreatic Cancer Cell Lines.

Nanoparticles have been shown to be effective drug carriers in cancer therapy. Pancreatic cancer forms dense tumours which are often resistant to drug molecules. In order to overcome such multidrug resistance, new drug entities, novel delivery systems and combination therapy strategies are being explored. In this paper, we report the design and synthesis of a poly(allylamine)-based amphiphile modified with hydrophobic naphthalimido pendant groups. Bisnaphthalimide compounds have been shown to possess anticancer activity. The potential of this polymer to encapsulate, solubilize and enhance drug (5-fluorouricil and bis-(naphthalimidopropyl)-diaminooctane) cytotoxicity in BxPC-3 cells was evaluated. Our studies showed that the insoluble drugs could be formulated up to 4.3 mg mL−1 and 2.4 mg mL−1 inside the amphiphiles, respectively. Additionally, the novel poly(allylamine)-naphthalimide carrier resulted in an amplification of cytotoxic effect with drug treatment after 24 h, and was capable of reduction of 50% cell population at concentrations as low as 3 μg mL−1.

Back Pain Treatment: A Review of the 2018 Recommendations

In 2018, published works that focus on European and American recommendations for the treatment of chronic back pain, where using opioid analgesics, it is recommended to take into account their complexity in multimorbid pathology in patients, since there is no effectiveness of pain reduction due to polypharmacotherapy. The peculiarity of the treatment is that the prevalence of musculoskeletal pain more often reaches its peak in elderly patients and is about 60%, which allows to establish an important direction in therapy - taking into account multimorbid pathology. It is necessary to take into account the accompanying chronic pathology because in patients with back pain hypertension occurs in 58.7%, chronic kidney disease occurs in 29.1% of patients, diabetes mellitus occurs in 23.1% of patients, depressive disorders occur in 15 , 3% of patients. Today it is proved that the common use of NSAIDs is critical for adverse events on the part of their use in patients with multimorbid pathology. In a number of clinical studies, the significant effect of tolperisone has been proven to relieve muscular spasm in acute and chronic back pain compared to tizanidine, where tolperisone is more often used in multimorbid pathology as a relaxant of skeletal muscles. The combination therapy strategy potentially represents a paradigm shift in rehabilitation therapy and can be very important for early intervention in spasticity management processes. In order to expand the scope of potential therapeutic use, the basic requirement for safe treatment is the quantitative basis of evidence of safety and efficacy, which, for example, is now presented in full on tolperisone hydrochloride. The new therapeutic options include a progressive addition of convergent therapy, where it is assumed that the potential for therapeutic purposes will be strengthened in view of the multimorbid pathology in the patient with back pain.

An alternative strategy for combination therapy: Interactions between polymyxin B and non-antibiotics

Antimicrobial resistance is increasing and few new antibiotics are in the development pipeline. Alternative strategies to treat infectious diseases, such as combination therapy, are urgently needed. Polymyxin B is a neglected and disused antibiotic with moderate antibacterial activity. In this study, we aimed to find synergistic interactions between polymyxin B and a wide range of non-antibiotics (non-ABs) to improve its efficacy. Thirty non-AB compounds from various drug classes were screened for synergistic potential with sub-minimum inhibitory concentrations (MICs) of polymyxin B in an agar diffusion assay against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa (3 isolates per species). Potential candidates were further studied in in vitro checkerboard assays, up to 5 isolates per species, using optical density to assess growth. Interactions were assessed with fractional inhibitory concentration index (FICi) analysis and surface response analysis with Loewe, Bliss and Highest Single Agent analysis using the Combenefit program. Twenty non-ABs enhanced polymyxin B activity in the agar diffusion test in one or more species. Of these, three showed a consistent synergistic effect (FICi ≤ 0.5) in the checkerboard assay for at least one species: citalopram, sertraline and spironolactone. Surface response analyses were largely in concordance, and further assessment showed only spironolactone was synergistic with polymyxin B at clinically relevant levels. The screening strategy used showed consistent synergism in vitro between polymyxin B and some non-ABs for A. baumannii, E. coli and K. pneumoniae. The synergistic interactions found merit further exploration as alternative strategies for difficult-to-treat infections.

Doxorubicin-loaded poly(ε-caprolactone)-Pluronic micelle for targeted therapy of esophageal cancer.

There is still lack of effective treatment of esophageal cancer, and it is urgently necessary to develop a new programs to treat this disease. More and more evidence suggests that the combination of 2 or more treatment strategies can enhance the antitumor activity in cancer treatment. We have established a new therapeutic strategy that combines doxorubicin-loaded poly(ε-caprolactone) (PCL)-Pluronic micelles and miR-34a to better combat esophageal cancer. Doxorubicin was loaded into PCL-Pluronic micelle to achieve better uptake. Confocal microscopy was used to assess in vitro cellular uptake of PCL-Pluronic micelle. Finally, the in vivo effect of this new combination therapy strategy was also studied. The results showed that PCL-Plannick micelles significantly enhanced the uptake of doxorubicin in esophageal cancer cells in vitro, thereby improving the accumulation of doxorubicin in the cells. In vitro and in vivo combination of doxorubicin-loaded PCL-Pluronic micelles and miR-34a, achieving a significantly synergistic therapeutic effect over the corresponding single treatment. These results suggested that the combinational therapy based on doxorubicin-loaded PCL-Pluronic micelle and miR-34a may provide a reasonable strategy for improving the outcome of esophageal cancer treatment.

Abstract PR03: Dissecting the playbook of cancer: Genomic analysis of 100,000 human tumors reveals elaborate patterns of activation of the RTK-RAS-MAPK pathway

Abstract Massive tumor sequencing allows us to progress beyond cataloging driver mutations, to discovery of the intricate patterns of genomic alterations that are required to enable a successful tumor. We used patterns of mutation co-occurrence to map activation of the RAS pathway in over 16,000 lung adenocarcinomas. This revealed a network of 40 highly mutually exclusive mutations in 19 genes, 18 of which are known RAS pathway genes (the RTKs EGFR, ERBB2, MET, ALK, RET, ROS1, KIT, FGFR2; NRAS and KRAS, BRAF and CRAF, MEK1/2, CBL, NF1, SOS1 and RIT1), indicating that each mutation is a strong pathway driver, and is redundant with other pathway mutations. Together, these mutations cover 71% of all lung adenocarcinomas, and include several previously uncharacterized mutations. Analysis of the remaining tumors uncovers additional patterns of RAS pathway activation. Some have additional mutations in RAS pathway genes that have the opposite pattern—they are co-occurent with each other, either in an additive manner where several mutations are enriched with each other, such as rare RAF and RAS family mutations, or synergistic, where a particular pair of mutations is highly co-occurrent, often in the same gene. These pairs are exclusive of other pathway mutations, suggesting that they are also strong pathway activators. We also see partially exclusive mutations, including amplifications of several genes and loss of function RAF family mutations that are likely to be weak activators of the RAS pathway in additional tumors. Finally, this approach also identifies recurrent mutations that are independent of other pathway mutations, giving clinical evidence that their function in human tumors is distinct from the common function of other RAS pathway mutations. We validated these patterns of exclusivity, additivity, and synergy in 80,000 genomic profiles from 400 other tumor types, and identified additional exclusive RAS pathway alterations in several other tissues. Across all cancers, 30% of all tumors had exclusive mutations indicating that they were RAS pathway driven. The individual mutations varied greatly between tissues, suggesting that there exist extensive tissue-specific enhancers and inhibitors of each oncogenic mutation, which may be of great relevance to drug and biomarker development. This initial foray into the future of massive tumor genomic datasets shows that we can transform genomic data from individual mutations into patterns of pathway activation, and distinguish pathway-driving mutations from other hotspots, providing a better understanding of the oncogenic strategies used by each human tumor. The degree of mutational exclusivity seen can predict the degree of oncogene addiction, and patterns of co-mutation can suggest strategies for combination therapies, as well as allow reverse translation to enable better matching of preclinical and basic research models to oncogenic strategies that are most relevant to human tumors. This abstract is also being presented as Poster B37. Citation Format: Gerard Manning, Nicholas Lounsbury, Ryan Hartmaier, Sally Trabucco, Ethan Sokol, Jacob Rinaldi, Jinfeng Liu, Florian Gnad. Dissecting the playbook of cancer: Genomic analysis of 100,000 human tumors reveals elaborate patterns of activation of the RTK-RAS-MAPK pathway [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR03.

Present and future of cancer immunotherapy: A tumor microenvironmental perspective.

Modulation of the tumor microenvironment is becoming an increasingly popular research topic in the field of immunotherapy, and studies regarding immune checkpoint blockades and cancer immunotherapy have pushed cancer immunotherapy to a climax. Simultaneously, the manipulation of the immune regulatory pathway can create an effective immunotherapy strategy; however, the tumor microenvironment serves an important role in suppressing the antitumor immunity by its significant heterogeneity. A number of patients with cancer do not have a good response to monotherapy approaches; therefore, combination strategies are required to achieve optimal therapeutic benefits. Targeting the tumor microenvironment may provide a novel strategy for immunotherapy, break down the resistance of conventional cancer therapy and produce the foundation for personalized precision medicine. The present review summarized the research regarding cancer immunotherapy from the perspective of how the tumor microenvironment affects the immune response, with the aim of proposing a novel strategy for cancer immunotherapy and combination therapy.

Abstract 4630: Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy

Abstract Rhabdomyosarcoma is the most common soft tissue sarcoma in children. The aggressive alveolar subtype (aRMS) is characterized by chromosomal translocations, most often a t(2;13) resulting in the expression of the oncogenic fusion protein PAX3-FOXO1 which is critical for tumorigenesis and cell survival. Our aim here was to identify a pharmacological combination therapy approach interfering with PAX3-FOXO1 biology at different levels. Since loss of the fusion protein results in cell death, we first aimed to pharmacologically enhance this effect. To this end, we screened aRMS tumor cells with a library of 208 drugs while simultaneously silencing PAX3-FOXO1 by shRNA. This identified the BH3-mimetic ABT-263 to sensitize aRMS cells to cell death after PAX3-FOXO1 depletion. To further characterize the cell death mechanisms we used combined shRNA and CRISPR approaches to perform a BH3 protein profiling. In accordance with identification of ABT-263 we demonstrate that aRMS cells undergo intrinsic apoptosis in a NOXA-dependent manner upon depletion of PAX3-FOXO1. In a parallel approach, and to identify drugs altering PAX3-FOXO1 protein stability, we screened the same drug library directly measuring fusion protein levels as read-out. This revealed that inhibition of Aurora kinase A negatively affects PAX3-FOXO1 protein levels. Finally, using both aRMS cell lines and patient-derived xenografts we demonstrate that combination treatment of Aurora kinase A inhibitors together with ABT-263 synergistically induces cell death and greatly slows tumor growth in vitro and in vivo. Taken together, these data show a novel functional interaction of Aurora kinase A and PAX3-FOXO1 and suggest new opportunities for targeted combination treatment of aRMS. Citation Format: Johannes Ommer, Marco Wachtel, Beat W. Schaefer. Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy [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 4630.

Abstract 1843: Combination therapy targeting Rb/Wee1 kinase pathways for rhabdomyosarcoma treatment

Abstract Introduction Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. New therapeutic strategies are needed particularly for high-risk patients where the survival rate is 20-40%. The FDA approved CDK4/6 inhibitor palbociclib (PD), and the WEE1 kinase inhibitor MK-1775 have anticancer effects when used as single agents. PD causes cell arrest at G1 phase (requiring intact Rb), while MK-1775 promotes cell death by abrogating DNA damage during the G2/M checkpoint phase by forcing premature mitosis entry. As the two drugs target tumor cells at different phases of the cell cycle, cells that are not in the targeted phase may escape single therapy. We hypothesized that sequential combination therapy will increase therapeutic efficacy. The use of PD first, will induce the cells to go under G1 arrest. Upon recovery, more cells will synchronize in the S/G2 phase, increasing their susceptibility to MK1775, resulting in synergistic cell killing. Methods Rb+ RMS (Rh-30) and Rb- osteosarcoma (OS) cell lines (CCH-D, LM7, and SASO2) were treated with increasing doses of PD for 6 days, then stained with propidium iodide (PI) and analyzed for cell cycle status to determine whether PD induced G1 arrest. To determine the optimal recovery time for Rb+ cells to be synchronized in S/G2 phase, cells were treated with PD (IC50-IC75) for 6 days and then were harvested either immediately or after 3-24 h recovery in drug free medium. Cells were stained with PI for cell cycle analysis. A 12-day long combination therapy assay was performed with or without recovery time between the two treatments. Combination index (CI) was calculated by using the statistical program calcusyn to determine whether the combinatorial drug treatment had acted in an antagonistic, additive or synergistic manner. Results Six days of treatment with PD resulted in reversible G1 arrest only in Rb+ cell lines, confirming that Rb, the downstream target of CDK4, must be intact to induce G1 arrest by palbociclib. Six days of treatment with PD followed by 9-12 h recovery in fresh medium (the optimal recovery time prior to treatment with MK-1775), led to re-entry of the Rb+ Rh-30 cells into the cell cycle and successful synchronization at S/G2 phase. By contrast, Rb- cells failed to arrest in G1 phase following treatment with PD and were not synchronized at S/G2 phase. Since cells synchronized in S/G2 phase are more vulnerable to MK1775, combination therapy resulted in a synergistic effect in the Rb+ cells but an antagonistic effect in the Rb- cells. Conclusion: Our results suggest that the combination therapy strategy of PD preceding MK1775 may be a novel therapeutic approach for patients with Rb+ RMS. This strategy will be investigated in xenograft and PDX RMS model. Citation Format: Simin Kiany, Gangxiong Huang, Melanie Justice, Khandan Keyomarsi, Eugenie Kleinerman. Combination therapy targeting Rb/Wee1 kinase pathways for rhabdomyosarcoma treatment [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 1843.

Abstract 336: Anti-tumor activity of the Chk1 inhibitor prexasertib (LY2606368) as a single agent in triple negative breast cancer models

Abstract Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, is diagnosed in approximately 15% of all breast cancer patients and characterized by high level of genomic instability, defects in DNA damage response (DDR) and increased replication stress (RS). Currently the treatment options for TNBC are limited, and new approaches are needed. Checkpoint kinase 1 (Chk1) is a key kinase that regulates the cell cycle, DDR and RS through the modulation of cell cycle checkpoints and replication fork licensing; and has emerged as an attractive target for anti-cancer therapy. Prexasertib (LY2606368), an ATP-competitive inhibitor of Chk1 has demonstrated single agent activity in vitro and in vivo. Prexasertib is being evaluated in an ongoing TNBC Phase 2 trial sponsored by NCI; and preliminary results suggest modest single-agent activity in sporadic TNBC patients (https://academic.oup.com/annonc/article/27/suppl_6/231PD/2798970). To understand the biology and mechanisms underlying prexasertib's activity and/or identify the potential biomarkers that may predict response or identify novel combinations that could improve prexasertib's efficacy, we evaluated the anti-tumor activity of prexasertib as a single agent in TNBC cancer cell lines in vitro and in vivo, including xenograft, orthotopic and patient-derived xenograft (PDX) tumor models. Prexasertib inhibited cell proliferation in 12 TNBC cell lines with IC50 values ranging from 0.32 nM to 117.3 nM; and demonstrated inhibition of Chk1 auto-phosphorylation and activation of γH2AX, RPA32 and DNAPKc in TNBC cell lines including HCC1187, HCC1806, MX-1 and MDA-MB-231. Treatment with prexasertib inhibited tumor growth by 83.8%, 85.5% and 94.2% in HCC1187, MX-1 and HCC1806 xenograft models, respectively. Prexasertib also inhibited primary tumor growth by 74.4% and lung metastasis by 97.48% in a MDA-MB-231 mammary fat pad orthotopic model. The anti-tumor effect of prexasertib was further assessed in 40 TNBC PDX models. Prexasertib treatment resulted in 10% (4/40) of models achieving complete tumor regression, 40% (16/40) achieving partial tumor regression/stasis and 50% (20/40) not deriving benefit. Potential biomarkers of prexasertib were investigated. TNBC cells with higher phosphorylation levels of DNAPKc and RPA32 demonstrated higher sensitivity to prexasertib treatment. TNBC PDX tumors with higher RNA message levels of CyclinE1, Cyclin D1 and c-Myc showed better prexasertib response. Together these findings will inform subsets of TNBC and/or individual patient characteristics that should be considered for future clinical assessments and development of potential combination therapy strategies. Citation Format: Wenjuan Wu, Constance King, Gregory Donoho, Philip Iversen, Andrew Capen, Mark Castanares, Jennifer Stephens, Yan Ding, Susan Pratt, Ricardo Martinez, Sean Buchanan, Christoph Reinhard, Richard Beckmann, Aimee Lin. Anti-tumor activity of the Chk1 inhibitor prexasertib (LY2606368) as a single agent in triple negative breast cancer models [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 336.