Publications by authors named "Marcin Kortylewski"

73 Publications

Large, Anionic Liposomes Enable Targeted Intraperitoneal Delivery of a TLR 7/8 Agonist To Repolarize Ovarian Tumors' Microenvironment.

Bioconjug Chem 2021 Jul 21. Epub 2021 Jul 21.

Ovarian cancer is the most lethal gynecological malignancy in the United States. Current standard of treatment includes surgical debulking and chemotherapy, such as cisplatin and paclitaxel. However, the patients' response rate for chemotherapy in ovarian cancer is not optimal, and they often develop chemoresistance and suffer from side effects. Current clinical trials make extensive use of immune checkpoint blockade (ICB) as a novel cancer immunotherapeutic strategy against ovarian tumors. However, the response rates for ICB antibodies remain limited to 10-20% of treated ovarian cancer patients despite the success of this approach in melanoma, renal, head and neck, and nonsmall cell lung cancers. This lack of efficacy is often attributed to the "cold" immune status of ovarian tumors, as these tumors often have a low number of tumor-infiltrating lymphocytes (TILs) but a high number of suppressive immune cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), or regulatory T cells (Tregs). Repolarizing TAMs could be a promising strategy to reshape the tumor immune microenvironment and promote antitumor activity when combined with ICBs. Toll-like receptor (TLR) 7 and 8 agonists, such as imiquimod and resiquimod, are potent immunostimulatory molecules with potential to repolarize macrophages. However, these small molecules have poor pharmacokinetic profiles and can induce severe side effects when administered systemically. Previously, our group demonstrated that various large, anionic nanomaterials (silica, PLGA, and polystyrene) specifically target TAMs when administered intraperitoneally (IP) to ovarian tumor-bearing mice. In the present study, we demonstrate that large, anionic liposomes administered IP also efficiently localize to TAMs and can be used to target the delivery of resiquimod. Resiquimod delivered in this targeted fashion promoted activation of M1 macrophages and T cell infiltration, while reducing the percentage of Tregs in the tumor microenvironment. Finally, liposome-formulated resiquimod significantly enhanced the efficacy of PD1 blockade against syngeneic ovarian tumors. We anticipate that further optimization of our liposomal delivery strategy can generate a clinically relevant strategy for more effective and safer immunotherapy for ovarian cancer patients.
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http://dx.doi.org/10.1021/acs.bioconjchem.1c00139DOI Listing
July 2021

First Multimodal, Three-Dimensional, Image-Guided Total Marrow Irradiation Model for Preclinical Bone Marrow Transplantation Studies.

Int J Radiat Oncol Biol Phys 2021 Jun 11. Epub 2021 Jun 11.

Department of Radiation Oncology, City of Hope Medical Center, Duarte, California; Beckman Research Institute of City of Hope, Duarte, California; Department of Radiation Oncology, University of Minnesota, Minneapolis, Minnesota. Electronic address:

Purpose: Total marrow irradiation (TMI) has significantly advanced radiation conditioning for hematopoietic cell transplantation in hematologic malignancies by reducing conditioning-induced toxicities and improving survival outcomes in relapsed/refractory patients. However, the relapse rate remains high, and the lack of a preclinical TMI model has hindered scientific advancements. To accelerate TMI translation to the clinic, we developed a TMI delivery system in preclinical models.

Methods And Materials: A Precision X-RAD SmART irradiator was used for TMI model development. Images acquired with whole-body contrast-enhanced computed tomography (CT) were used to reconstruct and delineate targets and vital organs for each mouse. Multiple beam and CT-guided Monte Carlo-based plans were performed to optimize doses to the targets and to vary doses to the vital organs. Long-term engraftment and reconstitution potential were evaluated by a congenic bone marrow transplantation (BMT) model and serial secondary BMT, respectively. Donor cell engraftment was measured using noninvasive bioluminescence imaging and flow cytometry.

Results: Multimodal imaging enabled identification of targets (skeleton and spleen) and vital organs (eg, lungs, gut, liver). In contrast to total body irradiation (TBI), TMI treatment allowed variation of radiation dose exposure to organs relative to the target dose. Dose reduction mirrored that in clinical TMI studies. Similar to TBI, mice treated with different TMI regimens showed full long-term donor engraftment in primary BMT and second serial BMT. The TBI-treated mice showed acute gut damage, which was minimized in mice treated with TMI.

Conclusions: A novel multimodal image guided preclinical TMI model is reported here. TMI conditioning maintained long-term engraftment with reconstitution potential and reduced organ damage. Therefore, this TMI model provides a unique opportunity to study the therapeutic benefit of reduced organ damage and BM dose escalation to optimize treatment regimens in BMT and hematologic malignancies.
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http://dx.doi.org/10.1016/j.ijrobp.2021.06.001DOI Listing
June 2021

Revisiting TLR9 as a target for CLL therapy.

Blood 2021 Jun;137(22):3006-3008

City of Hope Comprehensive Cancer Center.

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http://dx.doi.org/10.1182/blood.2020010618DOI Listing
June 2021

Novel Target Opportunities in Non-Metastatic Castrate Resistant Prostate Cancer.

Cancers (Basel) 2021 May 17;13(10). Epub 2021 May 17.

Department of Urology, SUNY Upstate Medical University, 750 East Adams Str., Syracuse, NY 13010, USA.

Nearly one third of men will incur biochemical recurrence after treatment for localized prostate cancer. Androgen deprivation therapy (ADT) is the therapeutic mainstay; however, some patients will transition to a castrate resistant state (castrate resistant prostate cancer, CRPC). Subjects with CRPC may develop symptomatic metastatic disease (mCRPC) and incur mortality several years later. Prior to metastatic disease, however, men acquire non-metastatic CRPC (nmCRPC) which lends the unique opportunity for intervention to delay disease progression and symptoms. This review addresses current therapies for nmCRPC, as well as novel therapeutics and pathway strategies targeting men with nmCRPC.
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http://dx.doi.org/10.3390/cancers13102426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157020PMC
May 2021

Circulating cytokines associated with clinical response to systemic therapy in metastatic renal cell carcinoma.

J Immunother Cancer 2021 Mar;9(3)

Department of Medical Oncology and Therapeutics Research, City of Hope Comprehensive Cancer Center, Duarte, California, USA

Background: Circulating cytokines and angiogenic factors have been associated with clinical outcomes in patients with metastatic renal cell carcinoma (RCC) receiving systemic therapy. However, none have yet examined cytokine concentrations in parallel cohorts receiving either immunotherapy or targeted therapy.

Methods: In this prospective correlative study, we enrolled 56 patients who were planned for treatment with either a vascular endothelial growth factor-tyrosine kinase inhibitor (VEGF-TKI) or immune checkpoint inhibitor (ICI). Eligibility requirements permitted any RCC histologic subtype, International Metastatic Renal Cell Carcinoma risk classification, and line of therapy. Immunologic profile was assessed at baseline and after 1 month on treatment using a Human Cytokine 30-plex protein assay (Invitrogen). Clinical benefit was defined as complete response, partial response, or stable disease ≥6 months per RECIST (Response Evaluation Criteria in Solid Tumors) V.1.1 criteria.

Results: Clinical benefit was similar between VEGF-TKI and ICI arms (65% vs 54%). Patients with clinical benefit from VEGF-TKIs had lower pretreatment levels of interleukin-6 (IL-6) (p=0.02), IL-1RA (p=0.03), and granulocyte colony-stimulating factor (CSF) (p=0.02). At 1 month, patients with clinical benefit from ICIs had higher levels of interferon-γ (IFN-γ) (p=0.04) and IL-12 (p=0.03). Among patients on VEGF-TKIs, those with clinical benefit had lower 1 month IL-13 (p=0.02) and granulocyte macrophage CSF (p=0.01) as well as higher 1 month VEGF (p=0.04) compared with patients with no clinical benefit.

Conclusion: For patients receiving VEGF-TKI or ICI therapy, distinct plasma cytokines were associated with clinical benefit. Our findings support additional investigation into plasma cytokines as biomarkers in metastatic RCC.
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http://dx.doi.org/10.1136/jitc-2020-002009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944971PMC
March 2021

Cytoplasmic DROSHA and non-canonical mechanisms of MiR-155 biogenesis in FLT3-ITD acute myeloid leukemia.

Leukemia 2021 Aug 15;35(8):2285-2298. Epub 2021 Feb 15.

Gehr Family Center for Leukemia Research, Hematology Malignancies and Stem Cell Transplantation Institute, City of Hope Medical Center, Duarte, CA, USA.

We report here on a novel pro-leukemogenic role of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) that interferes with microRNAs (miRNAs) biogenesis in acute myeloid leukemia (AML) blasts. We showed that FLT3-ITD interferes with the canonical biogenesis of intron-hosted miRNAs such as miR-126, by phosphorylating SPRED1 protein and inhibiting the "gatekeeper" Exportin 5 (XPO5)/RAN-GTP complex that regulates the nucleus-to-cytoplasm transport of pre-miRNAs for completion of maturation into mature miRNAs. Of note, despite the blockage of "canonical" miRNA biogenesis, miR-155 remains upregulated in FLT3-ITD+ AML blasts, suggesting activation of alternative mechanisms of miRNA biogenesis that circumvent the XPO5/RAN-GTP blockage. MiR-155, a BIC-155 long noncoding (lnc) RNA-hosted oncogenic miRNA, has previously been implicated in FLT3-ITD+ AML blast hyperproliferation. We showed that FLT3-ITD upregulates miR-155 by inhibiting DDX3X, a protein implicated in the splicing of lncRNAs, via p-AKT. Inhibition of DDX3X increases unspliced BIC-155 that is then shuttled by NXF1 from the nucleus to the cytoplasm, where it is processed into mature miR-155 by cytoplasmic DROSHA, thereby bypassing the XPO5/RAN-GTP blockage via "non-canonical" mechanisms of miRNA biogenesis.
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http://dx.doi.org/10.1038/s41375-021-01166-9DOI Listing
August 2021

Targeted In Vivo Delivery of NF-κB Decoy Inhibitor Augments Sensitivity of B Cell Lymphoma to Therapy.

Mol Ther 2021 03 26;29(3):1214-1225. Epub 2020 Nov 26.

Department of Immuno-Oncology, Beckman Research Institute, City of Hope, Duarte, CA, USA. Electronic address:

Despite recent advances, non-Hodgkin's B cell lymphoma patients often relapse or remain refractory to therapy. Therapeutic resistance is often associated with survival signaling via nuclear factor κB (NF-κB) transcription factor, an attractive but undruggable molecular target. In this study, we describe a bipartite inhibitor comprising a NF-κB-specific decoy DNA tethered to a CpG oligodeoxynucleotide (ODN) targeting Toll-like receptor-9-expressing B cell lymphoma cells. The Bc-NFκBdODN showed efficient uptake by human diffuse large B cell (U2932, OCI-Ly3), Burkitt (RaJi), and mantle cell (Jeko1) lymphomas, respectively. We confirmed that Bc-NFκBdODN inhibited NF-κB nuclear translocation and DNA binding, resulting in CCND2 and MYC downregulation. Bc-NFκBdODN enhanced radiosensitivity of lymphoma cells in vitro. In xenotransplanted human lymphoma, local injections of Bc-NFκBdODN reduced NF-κB activity in whole tumors. When combined with a local 3-Gy dose of radiation, Bc-NFκBdODN effectively arrested OCI-Ly3 lymphoma progression. In immunocompetent mice, intratumoral injections of Bc-NFκBdODN suppressed growth of directly treated and distant A20 lymphomas, as a result of systemic CD8 T cell-dependent immune responses. Finally, systemic administration of Bc-NFκBdODN to mice bearing disseminated A20 lymphoma induced complete regression and extended survival of most of the treated mice. Our results underscore clinical relevance of this strategy as monotherapy and in support of radiation therapy to benefit patients with resistant or relapsed B cell lymphoma.
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http://dx.doi.org/10.1016/j.ymthe.2020.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934632PMC
March 2021

Myeloid cell-targeted STAT3 inhibition sensitizes head and neck cancers to radiotherapy and T cell-mediated immunity.

J Clin Invest 2021 Jan;131(2)

Department of Immuno-Oncology, Beckman Research Institute at City of Hope, Duarte, California, USA.

The tumor microenvironment affects the outcome of radiotherapy against head and neck squamous cell carcinoma (HNSCC). We recently found that tolerogenic myeloid cells accumulate in the circulation of HNSCC patients undergoing radiotherapy. Here, we analyzed tumor-containing lymph node biopsies collected from these patients. After 2 weeks of radiotherapy, we found an increase in tumor-associated macrophages (TAMs) with activated STAT3, while CD8+ T cells were reduced as detected using multiplex IHC. Gene expression profiling indicated upregulation of M2 macrophage-related genes (CD163, CD206), immunosuppressive mediators (ARG1, LIF, TGFB1), and Th2 cytokines (IL4, IL5) in irradiated tumors. We next validated STAT3 as a potential target in human HNSCC-associated TAMs, using UM-SCC1 xenotransplants in humanized mice. Local injections of myeloid cell-targeted STAT3 antisense oligonucleotide (CpG-STAT3ASO) activated human DCs/macrophages and promoted CD8+ T cell recruitment, thereby arresting UM-SCC1 tumor growth. Furthermore, CpG-STAT3ASO synergized with tumor irradiation against syngeneic HPV+ mEERL and HPV- MOC2 HNSCC tumors in mice, triggering tumor regression and/or extending animal survival. The antitumor immune responses were CD8+ and CD4+ T cell dependent and associated with the activation of antigen-presenting cells (DCs/M1 macrophages) and increased CD8+ to regulatory T cell ratio. Our observations suggest that targeted inhibition of STAT3 in tumor-associated myeloid cells augments the efficacy of radiotherapy against HNSCC.
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http://dx.doi.org/10.1172/JCI137001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810478PMC
January 2021

Biophysical Characterization of the Leukemic Bone Marrow Vasculature Reveals Benefits of Neoadjuvant Low-Dose Radiation Therapy.

Int J Radiat Oncol Biol Phys 2021 01 22;109(1):60-72. Epub 2020 Aug 22.

Department of Radiation Oncology, City of Hope, Duarte, California; Beckman Research Institute of City of Hope, Duarte, California. Electronic address:

Purpose: Although vascular alterations in solid tumor malignancies are known to decrease therapeutic delivery, the effects of leukemia-induced bone marrow vasculature (BMV) alterations on therapeutic delivery are not well known. Additionally, functional quantitative measurements of the leukemic BMV during chemotherapy and radiation therapy are limited, largely due to a lack of high-resolution imaging techniques available preclinically. This study develops a murine model using compartmental modeling for quantitative multiphoton microscopy (QMPM) to characterize the malignant BMV before and during treatment.

Methods And Materials: Using QMPM, live time-lapsed images of dextran leakage from the local BMV to the surrounding bone marrow of mice bearing acute lymphoblastic leukemia (ALL) were taken and fit to a 2-compartment model to measure the transfer rate (K), fractional extracellular extravascular space (ν), and vascular permeability parameters, as well as functional single-vessel characteristics. In response to leukemia-induced BMV alterations, the effects of 2 to 4 Gy low-dose radiation therapy (LDRT) on the BMV, drug delivery, and mouse survival were assessed post-treatment to determine whether neoadjuvant LDRT before chemotherapy improves treatment outcome.

Results: Mice bearing ALL had significantly altered K, increased ν, and increased permeability compared with healthy mice. Angiogenesis, decreased single-vessel perfusion, and decreased vessel diameter were observed. BMV alterations resulted in disease-dependent reductions in cellular uptake of Hoechst dye. LDRT to mice bearing ALL dilated BMV, increased single-vessel perfusion, and increased daunorubicin uptake by ALL cells. Consequently, LDRT administered to mice before receiving nilotinib significantly increased survival compared with mice receiving LDRT after nilotinib, demonstrating the importance of LDRT conditioning before therapeutic administration.

Conclusion: The developed QMPM enables single-platform assessments of the pharmacokinetics of fluorescent agents and characterization of the BMV. Initial results suggest BMV alterations after neoadjuvant LDRT may contribute to enhanced drug delivery and increased treatment efficacy for ALL. The developed QMPM enables observations of the BMV for use in ALL treatment optimization.
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http://dx.doi.org/10.1016/j.ijrobp.2020.08.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736317PMC
January 2021

Automated in vivo Assessment of Vascular Response to Radiation using a Hybrid Theranostic X-ray Irradiator/Fluorescence Molecular Imaging System.

IEEE Access 2020 15;8:93663-93670. Epub 2020 May 15.

Department of Radiation Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010 USA.

Hypofractionated stereotactic body radiotherapy treatments (SBRT) have demonstrated impressive results for the treatment of a variety of solid tumors. The role of tumor supporting vasculature damage in treatment outcome for SBRT has been intensely debated and studied. Fast, non-invasive, longitudinal assessments of tumor vasculature would allow for thorough investigations of vascular changes correlated with SBRT treatment response. In this paper, we present a novel theranostic system which incorporates a fluorescence molecular imager into a commercial, preclinical, microCT-guided, irradiator and was designed to quantify tumor vascular response (TVR) to targeted radiotherapy. This system overcomes the limitations of single-timepoint imaging modalities by longitudinally assessing spatiotemporal differences in intravenously-injected ICG kinetics in tumors before and after high-dose radiation. Changes in ICG kinetics were rapidly quantified by principle component (PC) analysis before and two days after 10 Gy targeted tumor irradiation. A classifier algorithm based on PC data clustering identified pixels with TVR. Results show that two days after treatment, a significant delay in ICG clearance as measured by exponential decay (40.5±16.1% P=0.0405 Paired t-test n=4) was observed. Changes in the mean normalized first and second PC feature pixel values (PC1 & PC2) were found (P=0.0559, 0.0432 paired t-test), suggesting PC based analysis accurately detects changes in ICG kinetics. The PC based classification algorithm yielded spatially-resolved TVR maps. Our first-of-its-kind theranostic system, allowing automated assessment of TVR to SBRT, will be used to better understand the role of tumor perfusion in metastasis and local control.
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http://dx.doi.org/10.1109/access.2020.2994943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295127PMC
May 2020

Evaluating Changes in Immune Function and Bone Microenvironment During Radium-223 Treatment of Patients with Castration-Resistant Prostate Cancer.

Cancer Biother Radiopharm 2020 Sep 5;35(7):485-489. Epub 2020 May 5.

Department of Medicine, USC Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California, USA.

The effects of radium-223 on the immune system and the bone tumor microenvironment are incompletely understood. The authors describe mechanisms by which radium-223 may interact with the immune system, specifically through STAT-3 and impact on tumor and circulating lymphocyte populations. They review mechanisms through which effects of radium-223 and androgen-targeted therapy on bone microenvironment could be better elucidated. These knowledge gaps currently limit development of optimal combination therapy approaches for radium-223. Tissue based studies are currently underway in a prospective clinical trial to enhance therapeutic perspective on radium-223 treatment in the prostate cancer landscape.
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http://dx.doi.org/10.1089/cbr.2019.3397DOI Listing
September 2020

Vaccination against Nonmutated Neoantigens Induced in Recurrent and Future Tumors.

Cancer Immunol Res 2020 07 15;8(7):856-868. Epub 2020 Apr 15.

Department of Microbiology and Immunology, University of Miami, Miller School of Medicine, Miami, Florida.

Vaccination of patients against neoantigens expressed in concurrent tumors, recurrent tumors, or tumors developing in individuals at risk of cancer is posing major challenges in terms of which antigens to target and is limited to patients expressing neoantigens in their tumors. Here, we describe a vaccination strategy against antigens that were induced in tumor cells by downregulation of the peptide transporter associated with antigen processing (TAP). Vaccination against TAP downregulation-induced antigens was more effective than vaccination against mutation-derived neoantigens, was devoid of measurable toxicity, and inhibited the growth of concurrent and future tumors in models of recurrence and premalignant disease. Human CD8 T cells stimulated with TAP dendritic cells elicited a polyclonal T-cell response that recognized tumor cells with experimentally reduced TAP expression. Vaccination against TAP downregulation-induced antigens overcomes the main limitations of vaccinating against mostly unique tumor-resident neoantigens and could represent a simpler vaccination strategy that will be applicable to most patients with cancer.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339786PMC
July 2020

Myeloid cell-targeted miR-146a mimic inhibits NF-κB-driven inflammation and leukemia progression in vivo.

Blood 2020 01;135(3):167-180

Department of Immuno-Oncology, Beckman Research Institute at City of Hope Comprehensive Cancer Center, Duarte, CA.

NF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB-related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.
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http://dx.doi.org/10.1182/blood.2019002045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966933PMC
January 2020

Beclin-1 as a neutrophil-specific immune checkpoint.

J Clin Invest 2019 12;129(12):5079-5081

Department of Immuno-Oncology and.

Neutrophils are early wound healing and inflammation regulators that, due to functional plasticity, can adopt either pro- or antitumor functions. Until recently, beclin-1 was a protein known mainly for its role as a critical regulator of autophagy. In this issue of the JCI, Tan et al. describe the effects of the beclin-1 conditional myeloid cell-specific deletion in mice, in which immunostimulation resulted in hypersensitive neutrophils. The chronic proinflammatory effect of these neutrophils triggered spontaneous B cell malignancies to develop. Such tumorigenic effects were mediated primarily by IL-21 and CD40 signaling, leading to the upregulation of tolerogenic molecules, such as IL-10 and PD-L1. The authors went on to examine samples derived from patient lymphoid malignancies and showed that beclin-1 expression in neutrophils positively correlated with pre-B cell leukemia/lymphoma. Overall, the study provides an elegant model for neutrophil-driven carcinogenesis and identifies potential targets for immunotherapy of B cell malignancies.
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http://dx.doi.org/10.1172/JCI132534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877294PMC
December 2019

Targeted Delivery of miRNA Antagonists to Myeloid Cells In Vitro and In Vivo.

Methods Mol Biol 2019 ;1974:141-150

Department of Immuno-Oncology, Beckman Research Institute at City of Hope, Duarte, CA, USA.

Elevated levels of microRNAs in cancer cells are often associated with oncogenic effects and thus provide potential therapeutic targets. However, the lack of efficient delivery methods for synthetic miRNA inhibitors, antagomiR, or anti-miR oligonucleotides hindered clinical translation of such strategies. We recently developed an approach for targeted delivery of synthetic, 2'-O-methyl-modified antagomiR molecules to normal and malignant myeloid cells and B cells by tethering to the single-stranded, phosphorothioate oligodeoxynucleotides (PSO). The PSO-antagomiR are rapidly internalized through scavenger receptor-mediated endocytosis by human monocytes, dendritic cells, B cells, as well as myeloid leukemia and B-cell lymphoma cells, but not by T cells. Following internalization, the unformulated PSO-antagomiR potently reduces levels of target miRNA and modulates expression of downstream protein targets, both in vitro and in vivo. The simple design of PSO-antagomiR conjugates enable adaptation of this strategy for targeting oncogenic miRNAs in nonmalignant and malignant myeloid cells and B cells.
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http://dx.doi.org/10.1007/978-1-4939-9220-1_10DOI Listing
November 2019

Reduced T-cell Numbers and Elevated Levels of Immunomodulatory Cytokines in Metastatic Prostate Cancer Patients De Novo Resistant to Abiraterone and/or Enzalutamide Therapy.

Int J Mol Sci 2019 Apr 13;20(8). Epub 2019 Apr 13.

Department of Immuno-Oncology, Beckman Research Institute at City of Hope Comprehensive Cancer Center, Duarte, CA 91010, USA.

Currently, there are two Food and Drug Administration (FDA)-approved drugs for androgen deprivation therapy (ADT) of metastatic castration-resistant prostate cancer (mCRPC) patients: abiraterone and enzalutamide. However, our understanding of the effect of these therapies on the immune system in mCRPC patients remains limited. Here, we examined how abiraterone and enzalutamide treatment affects levels of soluble immune mediators in plasma and in circulating immune cells of 44 mCRPC patients. We found that the baseline levels of cytokines fibroblast growth factor (FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10), and IL-6 were significantly lower in ADT-sensitive compared to de novo resistant patients. In addition, resistant patients showed significantly lower T cell frequencies. When comparing the levels of cytokines over the course of treatment, we observed that the levels of proinflammatory mediators, such as interferon-γ (IFN-γ), IL-5, macrophage inflammatory protein 1 alpha (MIP-1α), and tumor necrosis factor alpha (TNFα), were significantly increased in the ADT-sensitive patients. At the same time, the abiraterone/enzalutamide therapy did not reduce the percentage of tolerogenic myeloid cell populations, such as polymorphonuclear myeloid-derived suppressor cells, which retained unaltered expression of programmed death-ligand 1 (PD-L1) and B7-H3. Overall, our results suggest that certain immune markers, such as IL-6 and the frequency of effector T cells, could be predictive of therapeutic response to ADT therapies in mCRPC patients.
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http://dx.doi.org/10.3390/ijms20081831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515443PMC
April 2019

STAT3 Inhibition Combined with CpG Immunostimulation Activates Antitumor Immunity to Eradicate Genetically Distinct Castration-Resistant Prostate Cancers.

Clin Cancer Res 2018 12 18;24(23):5948-5962. Epub 2018 Oct 18.

Department of Immuno-Oncology, Beckman Research Institute at City of Hope Comprehensive Cancer Center, Duarte, California.

Purpose: Prostate cancers show remarkable resistance to emerging immunotherapies, partly due to tolerogenic STAT3 signaling in tumor-associated myeloid cells. Here, we describe a novel strategy combining STAT3 inhibition with Toll-like Receptor 9 (TLR9) stimulation to unleash immune response against prostate cancers regardless of the genetic background.

Experimental Design: We developed and validated a conjugate of the STAT3 antisense oligonucleotide (ASO) tethered to immunostimulatory TLR9 agonist (CpG oligonucleotide) to improve targeting of human and mouse prostate cancer and myeloid immune cells, such as myeloid-derived suppressor cells (MDSC).

Results: CpG-STAT3ASO conjugates showed improved biodistribution and potency of STAT3 knockdown in target cells and . Systemic administration of CpG-STAT3ASO (5 mg/kg) eradicated bone-localized, -driven, and prostate tumors in the majority of treated mice. These antitumor effects were primarily immune-mediated and correlated with an increased ratio of CD8 to regulatory T cells and reduced pSTAT3/PD-L1 MDSCs. Both innate and adaptive immunity contributed to systemic antitumor responses as verified by the depletion of Gr1 myeloid cells and CD8 and CD4 T cells, respectively. Importantly, only the bifunctional CpG-STAT3ASO, but not control CpG oligonucleotides, STAT3ASO alone, or the coinjection of both oligonucleotides, succeeded in recruiting neutrophils and CD8 T cells into tumors. Thus, the concurrence of TLR9 activation with STAT3 inhibition in the same cellular compartment is indispensable for overcoming tumor immune tolerance and effective antitumor immunity against prostate cancer.

Conclusions: The bifunctional, immunostimulatory, and tolerance-breaking design of CpG-STAT3ASO offers a blueprint for the development of effective and safer oligonucleotide strategies for treatment of immunologically "cold" human cancers.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279477PMC
December 2018

SIRT1 Activation Disrupts Maintenance of Myelodysplastic Syndrome Stem and Progenitor Cells by Restoring TET2 Function.

Cell Stem Cell 2018 09 23;23(3):355-369.e9. Epub 2018 Aug 23.

Department of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, Hematologic Malignancies and Stem Cell Transplantation Institute, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010, USA.

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is derived from aberrant clonal hematopoietic stem/progenitor cells (HSPCs) that persist after conventional therapies. Defining the mechanisms underlying MDS HSPC maintenance is critical for developing MDS therapy. The deacetylase SIRT1 regulates stem cell proliferation, survival, and self-renewal by deacetylating downstream proteins. Here we show that SIRT1 protein levels were downregulated in MDS HSPCs. Genetic or pharmacological activation of SIRT1 inhibited MDS HSPC functions, whereas SIRT1 deficiency enhanced MDS HSPC self-renewal. Mechanistically, the inhibitory effects of SIRT1 were dependent on TET2, a safeguard against HSPC transformation. SIRT1 deacetylated TET2 at conserved lysine residues in its catalytic domain, enhancing TET2 activity. Our genome-wide analysis identified cancer-related genes regulated by the SIRT1/TET2 axis. SIRT1 activation also inhibited functions of MDS HSPCs from patients with TET2 heterozygous mutations. Altogether, our results indicate that restoring TET2 function through SIRT1 activation represents a promising means to target MDS HSPCs.
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http://dx.doi.org/10.1016/j.stem.2018.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143172PMC
September 2018

STAT3 in Tumor-Associated Myeloid Cells: Multitasking to Disrupt Immunity.

Int J Mol Sci 2018 Jun 19;19(6). Epub 2018 Jun 19.

Department of Immuno-Oncology, Beckman Research Institute at City of Hope Comprehensive Cancer Center, Duarte, 91010 CA, USA.

Myeloid immune cells, such as dendritic cells, monocytes, and macrophages, play a central role in the generation of immune responses and thus are often either disabled or even hijacked by tumors. These new tolerogenic activities of tumor-associated myeloid cells are controlled by an oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3). STAT3 multitasks to ensure tumors escape immune detection by impairing antigen presentation and reducing production of immunostimulatory molecules while augmenting the release of tolerogenic mediators, thereby reducing innate and adaptive antitumor immunity. Tumor-associated myeloid cells and STAT3 signaling in this compartment are now commonly recognized as an attractive cellular target for improving efficacy of standard therapies and immunotherapies. Hereby, we review the importance and functional complexity of STAT3 signaling in this immune cell compartment as well as potential strategies for cancer therapy.
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http://dx.doi.org/10.3390/ijms19061803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032252PMC
June 2018

SNAIL is a key regulator of alveolar rhabdomyosarcoma tumor growth and differentiation through repression of MYF5 and MYOD function.

Cell Death Dis 2018 05 29;9(6):643. Epub 2018 May 29.

Department of Transplantation, Institute of Pediatrics, Jagiellonian University Medical College, Wielicka 265, Krakow, 30-663, Poland.

Rhabdomyosarcoma (RMS) is a mesenchymal tumor of soft tissue in children that originates from a myogenic differentiation defect. Expression of SNAIL transcription factor is elevated in the alveolar subtype of RMS (ARMS), characterized by a low myogenic differentiation status and high aggressiveness. In RMS patients SNAIL level increases with higher stage. Moreover, SNAIL level negatively correlates with MYF5 expression. The differentiation of human ARMS cells diminishes SNAIL level. SNAIL silencing in ARMS cells inhibits proliferation and induces differentiation in vitro, and thereby completely abolishes the growth of human ARMS xenotransplants in vivo. SNAIL silencing induces myogenic differentiation by upregulation of myogenic factors and muscle-specific microRNAs, such as miR-206. SNAIL binds to the MYF5 promoter suppressing its expression. SNAIL displaces MYOD from E-box sequences (CANNTG) that are associated with genes expressed during differentiation and G/C rich in their central dinucleotides. SNAIL silencing allows the re-expression of MYF5 and canonical MYOD binding, promoting ARMS cell myogenic differentiation. In differentiating ARMS cells SNAIL forms repressive complex with histone deacetylates 1 and 2 (HDAC1/2) and regulates their expression. Accordingly, in human myoblasts SNAIL silencing induces differentiation by upregulation of myogenic factors. Our data clearly point to SNAIL as a key regulator of myogenic differentiation and a new promising target for future ARMS therapies.
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http://dx.doi.org/10.1038/s41419-018-0693-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974324PMC
May 2018

The revival of CpG oligonucleotide-based cancer immunotherapies.

Contemp Oncol (Pozn) 2018 Mar 5;22(1A):56-60. Epub 2018 Mar 5.

Department of Immuno-Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, USA.

The promising results of clinical trials using immune checkpoint inhibitors revived interests in cancer immunotherapy. However, it also became apparent that efficacy of immune checkpoint blockade can benefit from combining it with immunostimulatory strategies. Here, we review prior and re-emerging approaches using Toll-like Receptor 9 (TLR9) agonists, CpG oligodeoxynucleotides (ODNs), focused on the generation of antitumor immune responses in cancer patients. While numerous early clinical trials using TLR9 ligands in monotherapies provided evidence of CpG ODNs tolerability and safety, they failed to demonstrate sufficient antitumor efficacy. Recent studies unraveled multiple levels of negative regulation of immunostimulatory TLR9 signaling in immune cells by the tumor microenvironment that can stifle immune activity in cancer patients. Therefore, CpG ODNs-based strategies can greatly benefit from combination with strategies targeting immune checkpoint regulation. The most recent clinical trials of CpG ODNs together with immune checkpoint inhibitors have a chance to generate novel, more effective and safer cancer immunotherapies.
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http://dx.doi.org/10.5114/wo.2018.73887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885070PMC
March 2018

Combined modality radiation therapy promotes tolerogenic myeloid cell populations and STAT3-related gene expression in head and neck cancer patients.

Oncotarget 2018 Feb 2;9(13):11279-11290. Epub 2018 Feb 2.

Immuno-Oncology Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.

Immunomodulation contributes to the antitumor efficacy of the fractionated radiation therapy (RT). Here, we describe immune effects of RT with concurrent systemic cisplatin or cetuximab treatment of patients with stage III-IV head and neck squamous cell carcinoma (HNSCC). Using longitudinally collected blood samples, we identified significant changes in cytokines/chemokines and immune cell populations compared to immune-related gene expression profiles in peripheral blood mononuclear cells (PBMCs). The 7-week combinatorial RT resulted in gradual elevation of proinflammatory mediators (IFNγ, IL-6, TNFɑ, CCL2), while levels of IL-12, cytokine essential for antitumor immune responses, were decreased. These effects correlated with progressive accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) with detectable activity of STAT3 and PD-L1 expression, underscoring tolerogenic effects of MDSCs. Correspondingly, gene expression analysis of PBMCs harvested after two weeks of combinatorial RT, found upregulation of several immunosuppressive mediators. These included , and , which could represent IL-6/STAT3-driven tolerogenic signaling, which inhibits T cell and NK activity. Overall, our results suggest that potential immunostimulatory effects of combinatorial RT in HNSCC patients are likely limited by tolerogenic STAT3 signaling and PD-L1 upregulation in myeloid immune cells. Further studies will clarify whether STAT3 targeting could augment RT efficacy and durability of antitumor responses.
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http://dx.doi.org/10.18632/oncotarget.24397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834279PMC
February 2018

Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia.

Nat Med 2018 05 5;24(4):450-462. Epub 2018 Mar 5.

Department of Immuno-oncology, City of Hope Medical Center, Duarte, California, USA.

Leukemia stem cells (LSCs) in individuals with chronic myelogenous leukemia (CML) (hereafter referred to as CML LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here we show that although the microRNA (miRNA) miR-126 supported the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels were lower in CML LSCs than in long-term hematopoietic stem cells (LT-HSCs) from healthy individuals. Downregulation of miR-126 levels in CML LSCs was due to phosphorylation of Sprouty-related EVH1-domain-containing 1 (SPRED1) by BCR-ABL, which led to inhibition of the RAN-exportin-5-RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using mouse models of CML in which Mir126a (encoding miR-126) was conditionally knocked out in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment caused an undesired increase in endogenous miR-126 levels, which enhanced LSC quiescence and persistence. Mir126a knockout in LSCs and/or ECs, or treatment with a miR-126 inhibitor that targets miR-126 expression in both LSCs and ECs, enhanced the in vivo anti-leukemic effects of TKI treatment and strongly diminished LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in individuals with CML.
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http://dx.doi.org/10.1038/nm.4499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965294PMC
May 2018

B Cell Lymphoma Immunotherapy Using TLR9-Targeted Oligonucleotide STAT3 Inhibitors.

Mol Ther 2018 03 17;26(3):695-707. Epub 2018 Jan 17.

Department of Immuno-Oncology, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA. Electronic address:

Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expression of downstream target genes, such as MYC and BCL2L1, in human and mouse lymphoma cells. We further demonstrated that injections (i.v.) of CpG-STAT3dODN inhibited growth of human OCI-Ly3 lymphoma in immunodeficient mice. Moreover, systemic CpG-STAT3dODN administration induced complete regression of the syngeneic A20 lymphoma, resulting in long-term survival of immunocompetent mice. Both TLR9 stimulation and concurrent STAT3 inhibition were critical for immune-mediated therapeutic effects, since neither CpG7909 alone nor CpG7909 co-injected with unconjugated STAT3dODN extended mouse survival. The CpG-STAT3dODN induced expression of genes critical to antigen-processing/presentation and Th1 cell activation while suppressing survival signaling. These effects resulted in the generation of lymphoma cell-specific CD8/CD4-dependent T cell immunity protecting mice from tumor rechallenge. Our results suggest that CpG-STAT3dODN as a systemic/local monotherapy or in combination with PD1 blockade can provide an opportunity for treating patients with B cell NHL.
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http://dx.doi.org/10.1016/j.ymthe.2018.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910676PMC
March 2018

Synaptophysin expression on circulating tumor cells in patients with castration resistant prostate cancer undergoing treatment with abiraterone acetate or enzalutamide.

Urol Oncol 2018 04 27;36(4):162.e1-162.e6. Epub 2017 Dec 27.

Departement of Medical Oncology, City of Hope Comprehensive Cancer Center, Duarte, CA. Electronic address:

Background: With the advent of secondary androgen receptor (AR)-targeted therapies in metastatic castration resistant prostate cancer (PC), nonadenocarcinoma PCs are becoming more prevalent. Many of these cancers express neuroendocrine markers, which may provide biomarkers for emergence of this disease state. We aimed to quantify the expression of synaptophysin (Syp) on circulating tumor cells (CTCs) from serial samples of patients being treated with abiraterone acetate or enzalutamide.

Methods: CTCs were isolated from 44 patients with castration resistant PC before starting abiraterone or enzalutamide, at 4, 8, and 12 weeks on therapy, and at progression. Patients were stratified into 3 groups: de novo resistance, short response, and long response. CTCs were enumerated on the CellSearch platform and Syp expression was quantified using the open fluorescent channel on the platform. Correlative analyses were performed.

Results: A baseline CTC count of 5 or greater was associated with a more rapid time to progression and increasing CTC counts correlated with emergence of drug resistance. Syp was readily detectable on the surface of CTCs, and baseline percentage CTC Syp expression was significantly associated with time to progression. Furthermore, in evaluable patients, percent CTC Syp expression increased with the emergence of drug resistance. We also found that prior exposure to AR-targeted therapies was inversely associated with progression free survival.

Conclusions: We have demonstrated that Syp can be quantified on CTCs and that Syp expression correlates with resistance to abiraterone and enzalutamide. Larger studies testing Syp as a biomarker of emergence of nonadenocarcinoma disease and as a marker of response to AR-targeted therapies are warranted.
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http://dx.doi.org/10.1016/j.urolonc.2017.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899661PMC
April 2018

Identification of mechanisms of resistance to treatment with abiraterone acetate or enzalutamide in patients with castration-resistant prostate cancer (CRPC).

Cancer 2018 03 19;124(6):1216-1224. Epub 2017 Dec 19.

Department of Medical Oncology and Therapeutics Research, City of Hope Comprehensive Cancer Center, Duarte, California.

Background: Two androgen receptor (AR)-targeted therapies, enzalutamide and abiraterone acetate plus prednisone (abiraterone), have been approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). Many patients respond to these agents, but both de novo and acquired resistance are common. The authors characterized resistant phenotypes that emerge after treatment with abiraterone or enzalutamide.

Methods: Patients who received abiraterone or enzalutamide in the course of routine clinical care were consented for serial blood collection. A proprietary system (CellSearch) was used to enumerate and enrich circulating tumor cells (CTCs). RNA-sequencing (RNA-seq) was performed on pools of up to 10 epithelial cell adhesion molecule (EpCAM)-positive/CD45-negative CTCs. The impact of gene expression changes observed in CTCs between patients who responded or were resistant to abiraterone/enzalutamide therapies was further explored in a model cell line system.

Results: RNA-seq data from CTCs identified mutations commonly associated with CRPC as well as novel mutations, including several in the ligand-binding domain of AR that could facilitate escape from AR-targeted agents. Ingenuity pathway analysis of differentially regulated genes identified the transforming growth factor β (TGFβ) and cyclin D1 (CCND1) signaling pathways as significantly upregulated in drug-resistant CTCs. Transfection experiments using enzalutamide-sensitive and enzalutamide-resistant LNCaP cells confirmed the involvement of SMAD family member 3, a key mediator of the TGFβ pathway, and of CCND1 in resistance to enzalutamide treatment.

Conclusions: The current results indicate that RNA-seq of CTCs representing abiraterone and enzalutamide sensitive and resistant states can identify potential mechanisms of resistance. Therapies targeting the downstream signaling mediated by SMAD family member 3 (SMAD3) and CCND1, such as cyclin-dependent kinase 4/cyclin-dependent kinase 6 inhibitors, could provide new therapeutic options for the treatment of antiandrogen-resistant disease. Cancer 2018;124:1216-24. © 2017 American Cancer Society.
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http://dx.doi.org/10.1002/cncr.31161DOI Listing
March 2018

Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

Acta Biomater 2017 09 8;59:221-233. Epub 2017 Jul 8.

Chair of Medical Biotechnology, Poznan University of Medical Sciences, 61-701 Poznan, Poland; Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, 61-866 Poznan, Poland. Electronic address:

Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo.

Statement Of Significance: We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics. Although, the siRNA constructs have already given very promising results in the cancer therapy, the in vivo application of RNA-based oligonucleotide therapeutics still is limited due to their sensitivity to serum nucleases and some toxicity. We propose a carrier for RNA-based therapeutics that is made of bioengineered spider silk. We showed that functionalized bioengineered spider silk spheres not only protected RNA-based therapeutics from degradation by serum nucleases, but what is more important the embedding of siRNA into silk spheres delayed and extended target gene silencing compared with naked oligonucleotides. Moreover, we showed that plain silk spheres did not have unspecific effect on target gene levels proving not only to be non-cytotoxic but also very neutral vehicles in terms of TLR9/STAT3 activation in macrophages. We demonstrated advantages of novel delivery technology in safety and efficacy comparing with delivery of naked CpG-STAT3siRNA therapeutics.
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http://dx.doi.org/10.1016/j.actbio.2017.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942204PMC
September 2017

TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs.

J Leukoc Biol 2017 08 22;102(2):423-436. Epub 2017 May 22.

Department of Immuno-Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA;

Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor-propagating potential. We investigated whether cancer cell-intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc-driven (Myc-CaP) prostate cancer cells expressing the tetracycline-inducible gene ( ) or the control ( ). When engrafted into mice and treated with tetracycline, , but not , tumors showed accelerated growth kinetics compared with tumors in PBS-treated mice. upregulation in cancer cells triggered the selective accumulation of CD11bLy6GLy6C myeloid cells, phenotypically similar to PMN-MDSCs. The PMN-MDSCs from tetracycline-treated RM9- tumors increased the immunosuppressive activity of the STAT3 transcription factor, thereby more potently inhibiting T cell proliferation. We identified LIF, an IL-6-type cytokine and STAT3 activator, as a potential mediator of crosstalk between TLR9-expressing prostate cancer cells and PMN-MDSCs. Antibody-mediated LIF neutralization reduced the percentage of tumor-infiltrating PMN-MDSCs and inhibited tumor growth in mice. The clinical relevance of LIF is confirmed by the correlation between and expression in prostate cancer specimens. Furthermore, blood samples from patients with prostate cancer showed elevated levels of LIF and high LIFR expression on circulating PMN-MDSCs. Our results suggest that TLR9 prostate cancers promote immune evasion via LIF-mediated expansion and activation of PMN-MDSCs. Finally, targeting TLR9/LIF/STAT3 signaling using oligonucleotide-based inhibitors, such as CpG-STAT3dODN, can offer new opportunities for prostate cancer immunotherapy.
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http://dx.doi.org/10.1189/jlb.3MA1016-451RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505743PMC
August 2017

Myeloid cells as a target for oligonucleotide therapeutics: turning obstacles into opportunities.

Cancer Immunol Immunother 2017 Aug 18;66(8):979-988. Epub 2017 Feb 18.

Department of Immuno-Oncology at Beckman Research Institute, City of Hope National Medical Center, 1500 East Duarte Rd., Duarte, CA, 91010, USA.

Immunotherapies emerged as an alternative for cancer treatment, yet their clinical efficacies are still limited, especially in case of solid tumors. Myeloid immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), are often hijacked by tumors and become pivotal inhibitors of antitumor immunity. Immunosuppressive functions of tumor-associated myeloid cells result from the activity of Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor with well-defined tumorigenic and tolerogenic roles in human cancers. To overcome challenges in the development of pharmacological STAT3 inhibitors, we recently developed oligonucleotide-based strategies for cell-selective, in vivo STAT3 targeting. Conjugation of a STAT3siRNA or decoy STAT3 inhibitors to synthetic Toll-like Receptor 9 (TLR9) agonists, CpG oligonucleotides, allowed for selective delivery into TLR9-positive cells. Cellular target for CpG-STAT3 inhibitors include non-malignant, tumor-associated myeloid cells, such as polymorphonuclear MDSCs, as well as cancer cells in acute myeloid leukemia, B cell lymphoma and in certain solid tumors. The chemically modified CpG-STAT3 inhibitors resist serum nucleases and thus can be administered intravenously. Their potency relies on the intracellular gain-of-function effect: release of the central immune checkpoint regulator (STAT3) to unleash proinflammatory signaling (CpG/TLR9) in the same antigen-presenting cell. At the cellular level, CpG-STAT3 inhibitors exert two-pronged effect by rescuing T cells from the immune checkpoint control while decreasing survival of cancer cells. In this article, we review the preclinical data on CpG-STAT3 inhibitors and discuss perspectives of using TLR9-targeted delivery of oligonucleotide therapeutics for the generation of novel, more effective and safer cancer immunotherapies.
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http://dx.doi.org/10.1007/s00262-017-1966-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522630PMC
August 2017

STING Pathway Activation Stimulates Potent Immunity against Acute Myeloid Leukemia.

Cell Rep 2016 06 2;15(11):2357-66. Epub 2016 Jun 2.

Department of Medicine, University of Chicago, Chicago, IL 60637, USA; Committee on Immunology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

Type I interferon (IFN), essential for spontaneous T cell priming against solid tumors, is generated through recognition of tumor DNA by STING. Interestingly, we observe that type I IFN is not elicited in animals with disseminated acute myeloid leukemia (AML). Further, survival of leukemia-bearing animals is not diminished in the absence of type I IFN signaling, suggesting that STING may not be triggered by AML. However, the STING agonist, DMXAA, induces expression of IFN-β and other inflammatory cytokines, promotes dendritic cell (DC) maturation, and results in the striking expansion of leukemia-specific T cells. Systemic DMXAA administration significantly extends survival in two AML models. The therapeutic effect of DMXAA is only partially dependent on host type I IFN signaling, suggesting that other cytokines are important. A synthetic cyclic dinucleotide that also activates human STING provided a similar anti-leukemic effect. These data demonstrate that STING is a promising immunotherapeutic target in AML.
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http://dx.doi.org/10.1016/j.celrep.2016.05.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116809PMC
June 2016
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