Combination Therapy Strategies Research Articles

Melanoma Cell Intrinsic GABAA Receptor Enhancement Potentiates Radiation and Immune Checkpoint Inhibitor Response by Promoting Direct and T Cell-Mediated Antitumor Activity

Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical. We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABAAR activity. Melanoma cell viability studies were conducted to test whether enhancing GABAAR mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry. Genes coding for subunits of GABAARs express functional GABAARs in melanoma cells. By enhancing GABAAR-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. Invivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8+ T cells. Treated tumors show expression of cytokine-cytokine receptor interactions and overrepresentation of p53 signaling. This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABAARs in melanoma using benzodiazepine.

Pharmacoeconomic savings associated with alternate dosing strategy of nivolumab and ipilimumab combination therapy.

79 Background: Nivolumab (NIVO) plus ipilimumab (IPI) is a commonly used therapy for advanced melanoma. Original weight-based dosing of NIVO 1 mg/kg plus IPI 3 mg/kg (NIVO1+IPI3) was approved in 2017. Recently, the CHECKMATE 511 trial demonstrated improved tolerability of NIVO 3 mg/kg plus IPI 1 mg/kg (NIVO3 + IPI1) compared to the original dosing regimen without observed differences in efficacy.Objective: To determine the pharmacoeconomic implications of NIVO1+IPI3 and NIVO3+IPI1 with a dose banding strategy applied. Methods: Patients with advanced melanoma (N = 21) who received IPI and NIVO in combination between 4/2019 – 7/2019 were evaluated via a single-center retrospective chart review. A total of 118 checkpoint inhibitor doses (59 NIVO, 59 IPI) were analyzed.Weapplied a dose banding strategy to both NIVO1+IPI3 and NIVO3+IPI1 regimens to examine pharmaceutical expenditures using the two dosing regimens, whichincluded our organization 10% dose-vial rounding policy. Pharmaceutical expenditure using average wholesale price (AWP), which was $32.42 per mg of NIVO and $180.03 per mg of IPI, was calculated for each dosing strategy (NIVO1+IPI3 and NIVO3+IPI1). Results: The anticipated cost savings of patients receiving NIVO3+IPI1 combination therapy compared to NIVO1+IPI3, (both with dose rounding strategy applied), was $1,459,473 or >$70,000 per patient, representing a 47.2% savings from the original NIVO1 +IPI3 regimen (Table). Conclusions: In addition to improved tolerability and comparable efficacy for NIVO3+IPI1 vs NIVO1+IPI3, our study shows that adoption of the NIVO3+IPI1 regimen results in significant savings in drug costs. Considerations should be made in future combination trials to compare NIVO3+IPI1 as a treatment arm to NIVO1+IPI3 to see if comparable clinical results can be obtained with less toxicity and decreased pharmaceutical spending. [Table: see text]

Targeting tumor cell-derived CCL2 as a strategy to overcome Bevacizumab resistance in ETV5+ colorectal cancer

In our previous study, ETV5 mediated-angiogenesis was demonstrated to be dependent upon the PDGF-BB/PDGFR-β/Src/STAT3/VEGFA pathway in colorectal cancer (CRC). However, the ability of ETV5 to affect the efficacy of anti-angiogenic therapy in CRC requires further investigation. Gene set enrichment analysis (GSEA) and a series of experiments were performed to identify the critical candidate gene involved in Bevacizumab resistance. Furthermore, the ability of treatment targeting the candidate gene to enhance Bevacizumab sensitivity in vitro and in vivo was investigated. Our results revealed that ETV5 directly bound to the VEGFA promoter to promote translation of VEGFA. However, according to in vitro and in vivo experiments, ETV5 unexpectedly accelerated antiVEGF therapy (Bevacizumab) resistance. GSEA and additional assays confirmed that ETV5 could promote angiogenesis by inducing the secretion of another tumor angiogenesis factor (CCL2) in CRC cells to facilitate Bevacizumab resistance. Mechanistically, ETV5 upregulated CCL2 by activating STAT3 to facilitate binding with the CCL2 promoter. ETV5 induced-VEGFA translation and CCL2 secretion were mutually independent mechanisms, that induced angiogenesis by activating the PI3K/AKT and p38/MAPK signaling pathways in human umbilical vein endothelial cells (HUVECs). In CRC tissues, ETV5 protein levels were positively associated with CD31, CCL2, and VEGFA protein expression. CRC patients possessing high expression of ETV5/VEGFA or ETV5/CCL2 exhibited a poorer prognosis compared to that of other patients. Combined antiCCL2 and antiVEGFA (Bevacizumab) treatment could inhibit tumor angiogenesis and growth more effectively than single treatments in CRCs with high expression of ETV5 (ETV5+ CRCs). In conclusion, our results not only revealed ETV5 as a novel biomarker for anti-angiogenic therapy, but also indicated a potential combined therapy strategy that involved in targeting of both CCL2 and VEGFA in ETV5+ CRC.

Naringin Combined with NF-κB Inhibition and Endoplasmic Reticulum Stress Induces Apoptotic Cell Death via Oxidative Stress and the PERK/eIF2α/ATF4/CHOP Axis in HT29 Colon Cancer Cells.

Currently, combination therapy is considered the most effective solution for a selective chemotherapeutic effect in the treatment of colon cancer. This study investigated the death of both colon cancer HT29 cells and healthy vascular smooth muscle TG-Ha-VSMC cells (VSMCs) induced by naringin combined with endoplasmic reticulum (ER) stress and NF-κB inhibition. Naringin combined with tunicamycin and BAY 11-7082 suppressed the proliferation of HT29 cells in a dose-dependent manner and induced particularly apoptotic death without significantly affecting healthy VSMCs according to Annexin V/PI staining and AO/EB staining analyses. Insufficient antioxidant defense and heat shock response as well as excessive ROS generation were observed in HT29 cells following combination therapy. Quantitative real-time PCR and western blot analysis demonstrated that drug combination-induced mitochondrial apoptosis was activated through the ROS-mediated PERK/eIF2α/ATF4/CHOP pathway. Additionally, naringin combination significantly reduced the sXBP expression induced by tunicamycin+BAY 11-7082 in a dose-dependent manner. In conclusion, this study found that naringin combined with tunicamycin+BAY 11-7082 efficiently induced apoptotic cell death in HT29 colon cancer cells via oxidative stress and the PERK/eIF2α/ATF4/CHOP pathway, suggesting that naringin combined with tunicamycin plus BAY 11-7082 could be a new combination therapy strategy for effective colon cancer treatment with minimal side effects on healthy cells.

Pharmacologic therapy for pulmonary artery hypertension.

Pulmonary arterial hypertension (PAH) is a disease that carries a significant mortality left untreated. This article aims to review pharmacotherapeutics for PAH. PAH-specific therapies have evolved over the last three decades and have expanded from one therapy in the 1990s to 14 FDA-approved medications. Current therapies are directed at restoring the imbalance of vasoactive mediators that include nitric oxide, endothelin and prostacyclin. Although these agents are effective as monotherapy, recent trials have promulgated the strategy of upfront combination therapy. The availability of oral prostacyclin agonists has also allowed for expanded treatment options. Risk assessment is vital in guiding therapy for PAH patients. There is ongoing focus on targeting pathological mechanisms of the disease via novel therapies and repurposing existing drugs. There is an array of medications available for the treatment of PAH. Prudent combination of therapies to maximize treatment effect can improve morbidity and mortality. This article reviews the data supporting these therapies and attempts to outline an approach to patient management.

Salinomycin-Loaded Small-Molecule Nanoprodrugs Enhance Anticancer Activity in Hepatocellular Carcinoma.

BackgroundThere is currently no effective treatment for advanced hepatocellular carcinoma (HCC), and chemotherapy has little effect on long-term survival of HCC patients, largely due to the cancer stem cell (CSC) chemoresistance of HCC.MethodsWe constructed a small-molecule nanometer-sized prodrug (nanoprodrug) loaded with salinomycin (SAL) for the treatment of HCC. SAL was encapsulated by the prodrug LA-SN38 (linoleic acid modified 7-ethyl-10-hydroxycamptothecin) to construct a self-assembled nanoprodrug further PEGylated with DSPE-PEG2000. We characterized this codelivered nanoprodrug and its antitumor activity both in vitro in human HCC cell lines and in vivo in mice.ResultsDelivery of the SAL- and LA-SN38-based nanoprodrugs effectively promoted apoptosis of HCC cells, exerted inhibition of HCC tumor-sphere formation as well as HCC cell motility and invasion, and reduced the proportion of CD133+ HCC-CSC cells. In nude mice, the nanoprodrug suppressed growth of tumor xenografts derived from human cell lines and patient.ConclusionOur results show that SAL-based nanoprodrugs are a promising platform for treating patients with HCC and a novel strategy for combination therapy of cancers.

Erlotinib enhanced chemoradiotherapy sensitivity via inhibiting DNA damage repair in nasopharyngeal carcinoma CNE2 cells.

Nasopharyngeal carcinoma (NPC), arising from nasopharynx epithelium, is a rare type of malignant carcinoma that has a specific geographical distribution and a high risk of distant metastases. For most of the diagnosed NPC patients, the total survival rate decreased significantly due to the high local recurrence rate and metastasis rate. Concurrent chemoradiotherapy (CCRT), as routine therapy strategy of NPC, usually accompanies with high-dosage cytotoxic agents and serious toxic side reaction. Therefore, there is an urgent need for update the existing therapy strategies. In this study, we sought to investigate the effects of a combined therapy strategy, erlotinib combined with cisplatin and radiotherapy, on biological characteristics of NPC CNE2 cells and the potential reasons. CNE2 cells in logarithmic phase seeding in 96-well plates received concentration gradients of erlotinib (at 0, 10, 20, 40, 80, 160, 320 mmol/L) or cisplatin (at 0, 0.25, 0.5, 1, 2, 4, 8 mg/L), in order to obtain the optimal working concentration of erlotinib and cisplatin via 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Then, cells were divided into control group and four treatment groups (Group I-IV). All treatment groups received irradiation of 4 Gy, moreover, Group II-IV respectively received erlotinib, cisplatin and erlotinib plus cisplatin at optimal working concentration. After 24 and 48 h of irradiation, growth inhibition rate was determined; invasion ability and migration ability was respectively detected by Boyden's chamber assay and cell scratch test; flow cytometry was performed for determining apoptosis rate and cell cycle distribution; the expression levels of epidermal growth factor receptor (EGFR) signal pathway proteins were semi-quantitatively analyzed by Western-blot. Compared with Group I, all the other treatment groups showed better inhibition effect on cell viability, invasion and migration ability and higher apoptosis rate, while Group IV showed the strongest growth inhibition effect and highest apoptosis rate. In addition, EGFR signal pathway proteins of Group IV showed the lowest expression level. In combined therapy with radiotherapy/chemotherapy, erlotinib could enhance radiotherapy/chemotherapy sensitivity, probably because it could suppress DNA damage repair after radiotherapy/chemotherapy, thus weakening radiotherapy/chemotherapy resistance of tumor cells.

The clinical application of camrelizumab on advanced hepatocellular carcinoma

ABSTRACT Introduction Camrelizumab (also known as SHR-1210), a humanized monoclonal antibody against PD-1, has been shown to block the binding of PD-1 to PD-L1 and consequently inhibit the immune escape of tumor cells. Recently, camrelizumab was approved as a second-line drug for previously treated advanced hepatocellular carcinoma in China. Areas covered In this paper, the chemical properties, mechanism of action, pharmacokinetics, clinical efficacy, safety, and tolerability of camrelizumab for the treatment of advanced hepatocellular carcinoma are introduced in detail. The strategy for combination therapy and the potential application of camrelizumab in other solid tumors are briefly described. We performed a systematic review of the literature in PubMed and the following keywords were used: ‘SHR-1210,’ ‘Camrelizumab,’ and ‘hepatocellular carcinoma.’ Expert opinion Camrelizumab is a selective, humanized, high-affinity IgG4 kappa mAb against PD-1. Camrelizumab showed promising antitumor activity and manageable toxicities and offers a new second-line drug option for patients with advanced hepatocellular carcinoma. Reactive cutaneous capillary endothelial proliferation is a novel but prevalent immune-related dermatologic toxicity of camrelizumab, which is mild, reversible, and predictable. More clinical trials of camrelizumab are ongoing to develop combination therapy strategies and new indications for malignancies.

Abstract 1221: Novel strategies to target autophagy to improve cancer therapy

Abstract In cancer research, autophagy is thought to be a double-edged sword because it can either prevent malignant transformation during cancer initiation or sustain tumor metabolism, growth and survival in cancer establishment and progression. For established tumors, especially the advanced or metastatic types, the harsh intercellular conditions activate autophagy so that tumor cells can adapt to the stressful conditions and efficiently recycle their nutrients, while also allowing them to respond to anti-tumor therapeutics. Therefore, interventions that deliberately inhibit autophagy in cancer therapy have been used to reduce cancer cell survival, overcome drug resistance and improve clinical outcomes. The vast majority of autophagy inhibitors used in these therapies are lysosomotopic alkaline agents, such as chloroquine (CQ) and hydroxychloroquine (HCQ). However, their efficacy is considered to be imperfect. New strategy to target autophagy for cancer therapy is needed. Through introducing nanotechnology into initial new drug entity design, we developed a series of new chemical entities, BAQ12-18, and all of them are potential building blocks to self-assemble into the single new-chemical-entity nanomedicines (BAQ SNNs). The selected BAQ12 NP and BAQ13 NP not only improve drug delivery and tumor-targeted biodistribution but also endow unique proton sponge effect to dramatically enhance the existing functions of lysosomal disruption and autophagy blockade, and thus generate significant therapeutic efficacy both in vitro and in vivo. The more striking finding is that BAQ13 NP can potently provide both a uniform drug delivery platform and synergistic effect to the combination drug, indicating a new-generation strategy of combination therapy. Considering their capacity to encapsulate different drugs and broad synergistic effect with various treatments, BAQ SNNs may make a tremendous contribution to the outcome of cancer treatment. To the best of our knowledge, this is the first attempt to extend nanotechnology into the design stage of new small-molecule drug entity. The seamless connection between new drug discovery and nanomedicine-based drug delivery will absolutely inspire researchers to develop increasingly advanced nanomedicines with a wide range of practical values and market for improved the therapy against challenging diseases. Citation Format: Yuanpei Li, Zhao Ma, Dailn Zhang, Mythili Ramachandran, Tzu-yin Lin. Novel strategies to target autophagy to improve cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1221.

Prospective clinical study of two different treatment regimens of combined laser photocoagulation and intravitreal bevacizumab for retinopathy of prematurity: the Indian Twin Cities ROP Study (ITCROPS) database report number 9

To evaluate, in a combined treatment strategy for treatment-warranted retinopathy of prematurity (ROP), which of the two is a better treatment sequence, peripheral laser photocoagulation (LPC-IVB) first or intravitreal bevacizumab (IVB-LPC) first. Twenty-two babies (44 eyes) with ROP were recruited from 1 July 2014 to 30 March 2016. All the right eyes received LPC on day one followed by IVB on day four (LPC-IVB group). In all left eyes, IVB was injected on day one followed by LPC on day four (IVB-LPC group). The primary outcome measure was the proportion of eyes that had complete ROP regression with no additional treatment within 2weeks of the onset of therapy. Retinal photography and drawings were used at each visit to document disease course. In LPC-IVB group, 72.7% (16/22) eyes had complete ROP regression with no additional treatment within 2weeks of the onset of therapy. In the IVB-LPC group, 95.5% (21/22 eyes) had complete regression within 2weeks. Additional laser had complete regression in all eyes in each group. One baby (two eyes, one from each group) had late recurrence at 5months. The combined therapy strategy was successful for ROP management. Administration of anti-vascular endothelial growth factor injection before the peripheral laser was better than the reverse strategy of laser first. Late recurrences and adverse events were low.

Immunostimulatory and anti-tumor metronomic cyclophosphamide regimens assessed in primary orthotopic and metastatic murine breast cancer

The impressive successes of immune checkpoint blockade antibodies to treat various types of cancer are limited to minor subsets of patients. Combination therapy strategies, including with chemotherapy, are being explored to possibly improve the efficacy of immunotherapies. Here we report results regarding the use of an immunostimulatory regimen of metronomic cyclophosphamide (CTX). We show that in orthotopic models of syngeneic murine triple-negative breast cancer (EMT6), CTX administered at 140 mg/kg every 6 days (CTX140 1q6d) is superior at inhibiting primary tumor growth when compared to maximum tolerated dose or daily oral (continuous) low-dose CTX. In SCID or SCID beige mice, anti-tumor effects of CTX140 1q6d are reduced, reinforcing the therapeutic contribution of the adaptive and innate immune systems. In a second breast cancer model (SP1-AC2M2), CTX140 1q6d again showed clear superiority in anti-tumor effects, causing complete tumor regressions; however, these mice were not protected from subsequent tumor re-challenge, suggesting absence of immune memory. We also show that in an aggressive and metastatic cisplatin-resistant variant (EMT6-CDDP), CTX140 1q6d is superior and invokes an influx of intra-tumoral CD4+ and CD8+ T cells. CTX increases expression of tumor cell PD-L1; however, when combined with concomitant PD-L1 antibody therapy none of the CTX regimens showed increased benefit. This work sheds light on the potential use of metronomic CTX for the treatment of breast cancer, in particular using the quasi-weekly regimen, but also underscores the complexity of the anti-tumor mechanisms and potential to improve immune checkpoint therapy efficacy.

Targeting Immunometabolism Mediated by CD73 Pathway in EGFR-Mutated Non-small Cell Lung Cancer: A New Hope for Overcoming Immune Resistance.

Despite the relevant antitumor efficacy of immunotherapy in advanced non-small cell lung cancer (NSCLC), the results in patients whose cancer harbors activating epidermal growth factor receptor (EGFR) mutations are disappointing. The biological mechanisms underlying immune escape and both unresponsiveness and resistance to immunotherapy in EGFR-mutant NSCLC patients have been partially investigated. To this regard, lung cancer immune escape largely involves high amounts of adenosine within the tumor milieu with broad immunosuppressive effects. Indeed, besides immune checkpoint receptors and their ligands, other mechanisms inducing immunosuppression and including adenosine produced by ecto-nucleotidases CD39 and CD73 contribute to lung tumorigenesis and progression. Here, we review the clinical results of immune checkpoint inhibitors in EGFR-mutant NSCLC, focusing on the dynamic immune composition of EGFR-mutant tumor microenvironment. The adenosine pathway-mediated dysregulation of energy metabolism in tumor microenvironment is suggested as a potential mechanism involved in the immune escape process. Finally, we report the strong rationale for planning strategies of combination therapy with immune checkpoints blockade and adenosine signaling inhibition to overcome immune escape and immunotherapy resistance in EGFR-mutated NSCLC.

Immune Checkpoint Inhibitors: A Promising Choice for Endometrial Cancer Patients?

Although around 80% of endometrial cancers are diagnosed at early stages and present with a 5-year survival rate exceeding 95%, patients with advanced and recurrent disease show a poor prognosis and low response rates to standard chemotherapy. In the era of targeted therapy, the great advances in the understanding of programmed death-ligand 1 (PD-L1) upregulation in cancer cells, which is responsible for tumor immune escape, have contributed to the increasing interest in immune checkpoint inhibitors as a promising strategy for the treatment of several refractory solid malignancies, including endometrial cancer. Several clinical trials have investigated the efficacy and safety of immune checkpoint inhibitors in endometrial cancer, which already led to the approval of the anti-programmed cell death protein 1 (anti-PD-1) antibody pembrolizumab as a satisfactory alternative for selected patients with unresectable or metastatic disease. As the future of cancer treatment will probably rely on combination therapy strategies, currently, innovative ongoing trials are exploring the potential role of immune checkpoint inhibitors associated with chemotherapy, radiotherapy, and other targeted therapies. Moreover, further research is warranted to discover new specific biomarkers that can accurately predict the response to immunotherapy.

THU0163 USE OF BIOLOGICAL AGENT IN MONOTHERAPY IN RHEUMATOID ARTHRITIS IN COMPARISON TO THE ASSOCIATIONS WITH D(ISEASE) M(ODIFIING) A(NTI) R(HEUMATIC) D(RUGS): REVIEW OF LITERATURE AND META-ANALYSIS OF RANDOMIZED TRIALS.

Background:Biologic disease-modifying antirheumatic drugs (bDMARDs) extend the treatment choices for rheumatoid arthritis (RA) patients with suboptimal response or intolerance to conventional synthetic DMARDs (CsDMARDs). Currently, 9 biologic agents are approved in the RA treatment: and among them, three anti TNF agents are also approved in monotherapy (adalimumab, certolizumab and etanercept), but also abatacept, anakinra and tocilizumab. Registries of routine clinical practice treatment indicate that approximately one third of RA patients are being treated with a bDMARD in monotherapy and analyses from health care claims suggest that when methotrexate (MTX) is prescribed in combination with a bDMARD, more than half of the patients do not collect the MTX prescription and overall patients seem to taper MTX intake over time. So it is important to evaluate the benefit and harm associated with use of biological agents as monotherapy, and not only the traditional combination therapy strategies.Objectives:To compare the efficacy and safety of the individual biological agents used in monotherapy in patients with RA than the combination therapy strategy with CsDMARD + bDMARD.Methods:We used The Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, and MEDLINE in order to carry out our research, for published reports from inception of each database through December 2019. Search results were limited to randomised controlled trials (RCTs), with our two arms: biological agent in monotherapy and combination strategie (with any CsDMARDs). Major outcome was the ACR 20 reponse criteria at 24 week. The secondary outcomes were: the ACR 20 at 52 week, ACR 50, 70, 90 reponse criteria, the DAS 28 remission (with CRP and/or ESR), the score sharps modified non progressor, the proportion of patients who withdrawals the study due to adverse events, the proportion of patients who withdrawals the study due to lack of efficacy, the HAQ improvement > 0,22, CDAI and SDAI remission at week 24 and 52 if the data were available. The study of tolerance was also made. To estimate the relative efficacy of treatments whilst preserving the randomized comparisons within each trial, a Bayesian network meta-analysis was conducted in R (version 3.6.1) using fixed and random-effects.Results:The systematic review identified 2566 citations. The analysis comprises 22 trials (6358 patients), including six biological agents approved for RA (abatacept, adalimumab, etanercept, golimumab, rituximab and tocilizumab) as well as two other molecules: Clazakizumab, a humanized monoclonal antibody that binds to the interleukin-6 (IL-6) cytokine and Anbainuo, recombinant human TNFRII:Fc fusion protein. No study satisfyies our search criteria for anakinra, certolizumab and infliximab. Compared to combination therapy with CsDMARD+bDMARD, bDMARD monotherapy has less probability to give a ACR20 response at 24 weeks (RR: 0,92 [0,89 – 0,96]) in fixed or random effect model and this result is similar at 52 weeks (RR: 0,94 [0,89 – 0,99]). For all other outcome mesures, we can see an increased of ACR50–70 and 90 responses, an improve of the DAS 28 remission score, an increase of the proportion of sharp’s score non progressors (<0,5) as well as a decrease of withdrawals for inefficacy without increase of withdrawals for toxicity.Conclusion:Evidence from this meta-analysis suggests that combinaison strategy with bDMARD+CsDMARD remains the most efficacious option, being more effective than the use of biologics in monotherapy. The interest from this point of view is to sensitize prescribers to the use of other CsDMARDs when there is a contraindication or intolerance to MTX, but also to make patients aware of the superiority of the association of biological agents with CsDMARDs.figureDisclosure of Interests:Célia DELPECH: None declared, François-Xavier LABORNE: None declared, Pascal Hilliquin Consultant of: BMS, MSD, Novartis, Roche-Shugai.

Loss of microRNA-21 leads to profound stromal remodeling and short survival in K-Ras-driven mouse models of pancreatic cancer.

The microenvironment of pancreatic cancer adenocarcinoma (PDAC) is highly desmoplastic with distinct tumor-restraining and tumor-promoting fibroblast subpopulations. Re-education rather than indiscriminate elimination of these fibroblasts has emerged as a new strategy for combination therapy. Here, we studied the effects of global loss of profibrotic noncoding regulatory microRNA-21 (miR-21) in K-Ras-driven p53-deleted genetically engineered mouse models of PDAC. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. The absence of tumor-restraining myofibroblasts and a massive infiltrate of immune cells were salient phenotypic features of global miR-21 loss. Stromal miR-21 activity was required for induction of tumor-restraining myofibroblasts in in vivo isograft transplantation experiments. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in The Cancer Genome Atlas PDAC data set (n = 156) mirroring findings in the mouse models. Our results exposed an overall tumor-suppressive function of miR-21 in in vivo PDAC models. These results have important clinical implications for anti-miR-21-based inhibitory therapeutic approaches under consideration for PDAC and other cancer types. Mechanistic dissection of the cell-intrinsic role of miR-21 in cancer-associated fibroblasts and other cell types will be needed to inform best strategies for pharmacological modulation of miR-21 activity to remodel the tumor microenvironment and enhance treatment response in PDAC.

Biodegradable Nanocomposite with Dual Cell‐Tissue Penetration for Deep Tumor Chemo‐Phototherapy

Chemo-phototherapy, as a promising cancer combination therapy strategy, has attracted widespread attention. However, the complex tumor microenvironment restricts the penetration depth of chemo-phototherapy agents in the tumor region. Here, biodegradable amphiphilic gelatin (AG) wrapped nanocomposite (PRDCuS@AG) composed of doxorubicin and copper sulfide (CuS)-loaded dendrimer is designed for deep tumor chemo-phototherapy. PR in PRDCuS@AG represents arginine-conjugated polyamidoamine dendrimer. PRDCuS@AG can rapidly biodegrade into PRDCuS by matrix metalloproteinases under near-infrared light irradiation. The resulted PRDCuS harbors dual cell-tissue penetration ability, which can effectively penetrate deep into the tumor tissue. In particular, PRDCuS@AG achieves photoacoustic imaging-guided synergistic chemo-phototherapy with 97% of tumor inhibition rate. Moreover, PRDCuS@AG can further degrade into 3 nm ultrasmall CuS, which can be eliminated from the body after treatment to avoid side effects. This strategy provides an insight that the development of chemo-phototherapy agents with high penetration ability to overcome the limitation of current deep tumor therapy.

In Vivo Suppression of Autophagy via Lentiviral shRNA Targeting Atg5 Improves Lupus-Like Syndrome.

In both mouse models and clinical patients with lupus, autophagy levels were significantly elevated and correlated with disease activity. Furthermore, autophagy can promote the survival of B and T cells, plasma cell differentiation, and antibody production. These results suggest that autophagy may promote the progression of lupus by regulating the survival of autoreactive immune cells. Therefore, we aimed at studying whether suppressing autophagy can modulate lupus progression in vivo. First, we found that the autophagy levels in splenocytes and lymphocytes of peripheral blood (PB) were elevated and positively correlated with disease severity in lupus-prone mice. The shAtg5-lentivirus, which effectively inhibits autophagy in vitro, was then injected into the lupus-prone mice. Autophagy levels in lymph node cells and PB lymphocytes were reduced following Atg5 suppression. We also found that lymphadenopathy and the numbers of plasma cells, CD4−CD8−, and CD4+ T cells decreased in mice treated with the shAtg5-lentivirus. The mice treated with shAtg5-lentivirus exhibited lower levels of proteinuria, serum anti-dsDNA antibody, B-cell activating factor (BAFF), and glomerular immune complex deposition. Therefore, targeting autophagy to moderate overactivated autophagy in immune cells seems to be a novel strategy for combination therapy of lupus.

Abstract IA30: 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-occurrent 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. Citation Format: Gerard Manning, Nick Lounsbury, Ryan Hartmaier, Sally Trabucco, Ethan Sokol. 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 AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA30.

A review of glioblastoma immunotherapy.

Glioblastoma is a very aggressive cancer with dismal prognosis despite standard of care including surgical resection, radiation therapy, and chemotherapy. There is interest in applying immunotherapy to glioblastoma as this modality has demonstrated remarkable improvements in the management of several solid tumors including melanoma, renal cell carcinoma, and non-small cell lung cancer. This review aims to provide an overview of the current state of glioblastoma immunotherapy. Literature search was performed on PubMed between 1961 and 2020. Initial clinical trials of checkpoint inhibitors and vaccine therapy for glioblastoma have largely been disappointing for both primary and recurrent glioblastoma. This failure has been attributed to glioblastoma's highly immunosuppressive environment and multiple mechanisms of therapy resistance including high tumor heterogeneity, low mutational burden, systemic immunosuppression, and local immune dysfunction. Current clinical trials are exploring combination therapy and novel treatment strategies beyond immune checkpoint therapies and vaccine therapy such as CAR T cells. There is also an effort to establish synergy between immunotherapy and current standard of care. Furthermore, recent advances in personalized neoantigen vaccines suggest a shift towards personalized, patient-specific GBM treatment.

Abstract A43: Identifying a role for Ntrk1 in regulating immune functionality and checkpoint blockade resistance in Kras/p53 mutant lung cancer

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States, with the 5-year survival rate of all lung cancer patients combined at only 18%. The implementation of cancer immunotherapeutics for solid tumors such as lung cancers has shown great promise for improving clinical outcome. Immune checkpoint blocking antibodies targeting the PD-1/PD-L1 axis have been FDA approved for the treatment of late-stage metastatic disease, and clinical trial results indicate durable response in a small percentage of patients (~15-20%) compared to standard-of-care chemotherapy. However, the majority of patients show little to no response, or they acquire resistance during treatment with checkpoint inhibitors delivered as a monotherapy. Therefore, identifying resistance mechanisms and potential combination therapy strategies is imperative to improve response rates to immune checkpoint inhibitors and patient prognosis. To address this, we developed a clinically relevant in vivo shRNA screen that focuses on genes encoding for FDA-approved drug targets (FDAome). We implanted Kras/p53 (KP) mutant murine lung cancer cells expressing the FDAome shRNA library into syngeneic mice treated with isotype control or anti-PD-1 antibody. Sequencing for the unique barcoded shRNAs revealed that neurotrophic receptor tyrosine kinase 1 (Ntrk1) was significantly depleted from tumors challenged with PD-1 blockade, suggesting that it provides a survival advantage to tumor cells when under immune system pressure. To confirm Ntrk1 as a hit from the screen, we analyzed tumors treated with anti-PD-1 antibody and found that transcript levels of Ntrk1 are significantly upregulated with treatment, and cell lines derived from tumors with acquired resistance to anti-PD-1 treatment also have higher levels of Ntrk1 expression. Additionally, in vitro co-culture assays revealed that Ntrk1 overexpressing cells upregulate PD-L1 and other immunosuppressive markers when cultured with splenocytes, and this upregulation was inhibited through pharmacologic targeting of either Trk or Jak signaling. Stable knockdown of Ntrk1 in murine KP mutant lung cancer cells reduced Jak signaling and downregulation of PD-L1. In vivo, Ntrk1 knockdown tumors demonstrated significantly reduced tumor growth, and analysis of tumor-infiltrating T cell populations via flow cytometry revealed significantly fewer CD25+/FoxP3+ T regulatory cells in Ntrk1 knockdown tumors, as well as reduced exhaustion of CD8+ cytotoxic T cells. In contrast, Ntrk1 overexpressing tumors had a significant increase in tumor growth, as well as a hyperexhaustive phenotype in the intratumoral CD8+ T cells. Together, these data suggest that Ntrk1 activity can modulate Jak/Stat signaling to regulate PD-L1 expression as well as the expression of other immunosuppressive molecules, thereby promoting an exhausted and suppressed tumor microenvironment. Citation Format: Jessica Konen, Bertha L. Rodriguez, Laura Gibson, Rosalba Minelli, Jeffrey Kovacs, Alessandro Carugo, Christopher Bristow, Timothy Heffernan, Don L. Gibbons. Identifying a role for Ntrk1 in regulating immune functionality and checkpoint blockade resistance in Kras/p53 mutant lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A43.