New Class Of Cancer Therapeutics Research Articles

Structure-Based Discovery of Selective Histone Deacetylase 8 Degraders with Potent Anticancer Activity.

Inducing protein degradation by proteolysis targeting chimeras has gained tremendous momentum as a promising novel therapeutic strategy. Here, we report the design, synthesis, and biological characterization of highly potent proteolysis targeting chimeric small molecules targeting the epigenetic regulator histone deacetylase 8 (HDAC8). We developed potent and effective HDAC8 degraders, as exemplified by SZUH280 (16e), which effectively induced HDAC8 protein degradation and inhibited cancer cell growth even at low micromolar concentrations. Our preliminary mechanistic studies revealed that SZUH280 hampers DNA damage repair in cancer cells, promoting cellular radiosensitization. In mice, a single SZUH280 dose induced rapid and prolonged HDAC8 protein degradation in xenograft tumor tissues. Moreover, SZUH280 alone or in combination with irradiation resulted in long-lasting tumor regression in an A549 tumor mouse model. Our findings qualify a new chemical tool for HDAC8 knockdown and may lead to the development of a new class of cancer therapeutics.

Abstract 2644: Therapeutic inhibition of RNA polymerase I reveals vulnerability of mismatch repair defective cancers

Abstract RNA polymerase I inhibitors represent a new class of cancer therapeutics with potential application in many cancer types. By curbing the transcription of ribosomal RNAs they block the rate-limiting step in ribosome biogenesis and protein synthesis. We have recently identified specific and selective first-in-class inhibitors of Pol I. This class of Pol I inhibitors, represented by small-molecule BMH-21, blocks transcription initiation and elongation and causes the destruction of the enzyme catalytic subunit. BMH-21 activity is genetically dependent on Pol I in cancer cells and in yeast confirming the targeting specificity. Our earlier mechanistic and efficacy studies with BMH-21 have demonstrated the utility of the approach in targeting colon and prostate cancers and melanoma. The goal of this study was to pinpoint cancer types with specific sensitivity to these inhibitors and to identify markers of this sensitivity. To do so, we undertook unbiased large-scale cancer cell line screens with our Pol I inhibitors and conducted correlative analyses using open source DepMap and Achilles databases to identify cancer types and features that sensitize cancer cells to these inhibitors. The most sensitive lineages were colorectal, endometrial, esophageal, leukemia, myeloma, and ovarian. Comparison of the cancer cell line responses to the Achilles gene essentiality database showed high correlation to genetic knock-out of three Pol I subunits. This unbiased finding strongly supports identification of Pol I as the target. Further, we identified two ribosomal proteins, RPL22 and RPL22L1 as the top genetic and expression markers denoting sensitivity, and MDM4, a p53 transcriptional repressor, as the top protein marker. Together, these three markers conform to a new p53 regulatory pathway. These findings were validated using genetic and mechanistic analyses. We show that Pol I inhibition alters the MDM4 splicing and decreases the expression of MDM4 isoform that represses p53, and that this activity is dependent on the RPL22/RPL22L1 proteins. Notably, perturbation of the biomarkers is strongly associated with mismatch repair defective (MMRd) cancers. We show the efficacy of the Pol I inhibitors in xenograft and patient-derived MMRd models. Our data suggest that RPL22mutation could serve as tumor-agnostic biomarker for targeting cancers with the Pol I inhibitors. Furthermore, this work identifies a new Pol I transcription-dependent ribosomal protein-governed pathway that converges on the regulation of p53. Citation Format: Wenjun Fan, Hester Liu, Asma Begum, Stephanie Pitts, Brittany Ford, Tony Dorado, Pablo de Leon, Rajeshkumar NV, James C. Barrow, Marikki Laiho. Therapeutic inhibition of RNA polymerase I reveals vulnerability of mismatch repair defective cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2644.

Abstract 1754: Amatoxin-based antibody-drug conjugates induce immunogenic cell death and improve the anti-tumor efficacy of immune checkpoint inhibitors in humanized mouse models

Abstract Background: Amatoxin-based antibody-drug conjugates (ATACs) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as a toxic payload. Amanitin binds to the RNA polymerase II and thereby efficiently inhibits the cellular transcription process. In the present study, we show that ATACs belong to the class of immune activating drugs which exhibit synergistic anti-tumor efficacy with immune checkpoint inhibitors (ICIs) in vivo by induction of immunogenic cell death (ICD). ICIs are a new class of cancer therapeutics utilizing patients` immune system to kill cancer cells. ICIs rely on the activity of the immune system to develop their full potential. Therefore, drugs that are heating up cold tumors and make them visible to the patients` immune system and by that enhancing the anti-tumor efficacy of ICIs, e.g., ATACs, are on high demand for the treatment of tumor patients. Material and methods: Cell line: Her2+ cell lines: NCI-N87; CD19+ cell line: Raji Antibody: anti-Her2 and anti-CD19 engineered monoclonal antibody produced at Heidelberg Pharma Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibody. Animal models: Raji cells with human peripheral blood mononuclear cells (PBMCs), NCI-N87 cells or HCBx-10/HCBx-11 PDX (Xentech, France) tumor pieces were implanted subcutaneously in mice. Treatment: ATAC: single dose i.v.; immune checkpoint inhibitors: Q3Dx6 or Q3Dx5. Results: In vivo treatment of heterogenous Her2low PDX tumors with an anti-Her2-ATAC led to a significant tumor growth delay and complete tumor remission. Furthermore, increased expression of HMGB1 and surface exposure of calreticulin (CRT), two hallmarks of ICD, was observed in the same PDX tumors as well as in a Her2+ CDX (NCI-N87) tumor after the treatment with an anti-HER2 ATAC. In addition, the combined treatment of ATACs and different ICIs targeting CTLA-4 (Ipilimumab), PD-L1 (Pembrolizumab) or PD-1 (Avelumab) had a synergistic effect in a humanized lymphoma CDX model in the presence of human PBMCs. Combination treatment led to enhanced tumor growth inhibition and more tumor free animals as compared to single treatments with ATAC or ICI. Conclusions: The strong anti-tumor efficacy of ATACs even in heterogenous patient-derived xenograft models in combination with the induction of ICD in vitro and in vivo suggest that the anti-tumor effect of ATACs is accompanied by the activation of the immune system. This hypothesis is strengthened by the finding that ATACs and ICIs show a synergistic effect in vivo. The presented data highlights the general concept of the synergistic effect of ATACs and ICIs which applies to several types of ICIs thus strengthening the scientific rationale for combination treatments in clinical trials. Citation Format: Christian Orlik, Franziska Ebeling, Kristin Decker, Irina Dranova, Anikó Pálfi, Christoph Mueller, Torsten Hechler, Andreas Pahl, Michael Kulke. Amatoxin-based antibody-drug conjugates induce immunogenic cell death and improve the anti-tumor efficacy of immune checkpoint inhibitors in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1754.

SLC46A3 is a lysosomal proton-coupled steroid conjugate and bile acid transporter involved in transport of active catabolites of T-DM1.

Antibody-drug conjugates (ADCs) represent a new class of cancer therapeutics that enable targeted delivery of cytotoxic drugs to cancer cells. Although clinical efficacy has been demonstrated for ADC therapies, resistance to these conjugates may occur. Recently, SLC46A3, a lysosomal membrane protein, was revealed to regulate the efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC that has been widely used for treating breast cancer. However, the role of SLC46A3 in mediating T-DM1 cytotoxicity remains unclear. In this study, we discovered the function of SLC46A3 as a novel proton-coupled steroid conjugate and bile acid transporter. SLC46A3 preferentially recognized lipophilic steroid conjugates and bile acids as endogenous substrates. In addition, we found that SLC46A3 directly transports Lys-SMCC-DM1, a major catabolite of T-DM1, and potent SLC46A3 inhibitors attenuate the cytotoxic effects of T-DM1, suggesting a role in the escape of Lys-SMCC-DM1 from the lysosome into the cytoplasm. Our findings reveal the molecular mechanism by which T-DM1 kills cancer cells and may contribute to the rational development of ADCs that target SLC46A3.

Cell-Free RNA as a Novel Biomarker for Response to Therapy in Head & Neck Cancer.

Liquid biopsies are gaining more traction as non-invasive tools for the diagnosis and monitoring of cancer. In a new paradigm of cancer treatment, a synergistic botanical drug combination (APG-157) consisting of multiple molecules, is emerging as a new class of cancer therapeutics, targeting multiple pathways and providing a durable clinical response, wide therapeutic window and high level of safety. Monitoring the efficacy of such drugs involves assessing multiple molecules and cellular events simultaneously. We report, for the first time, a methodology that uses circulating plasma cell-free RNA (cfRNA) as a sensitive indicator of patient response upon drug treatment. Plasma was collected from six patients with head and neck cancer (HNC) and four healthy controls receiving three doses of 100 or 200 mg APG-157 or placebo through an oral mucosal route, before treatment and on multiple points post-dosing. Circulating cfRNA was extracted from plasma at 0-, 3- and 24-hours post-treatment, followed by RNA sequencing. We performed comparative analyses of the circulating transcriptome and were able to detect significant perturbation following APG-157 treatment. Transcripts associated with inflammatory response, leukocyte activation and cytokine were upregulated upon treatment with APG-157 in cancer patients, but not in healthy or placebo-treated patients. A platelet-related transcriptional signature could be detected in cancer patients but not in healthy individuals, indicating a platelet-centric pathway involved in the development of HNC. These results from a Phase 1 study are a proof of principle of the utility of cfRNAs as non-invasive circulating biomarkers for monitoring the efficacy of APG-157 in HNC.

A Combined Ultrafiltration/Diafiltration Process for the Purification of Oncolytic Measles Virus.

Measles virus (MV) is an important representative of a new class of cancer therapeutics known as oncolytic viruses. However, process intensification for the downstream purification of this fragile product is challenging. We previously found that a mid-range molecular weight cut-off (300 kDa) is optimal for the concentration of MV. Here, we tested continuous and discontinuous diafiltration for the purification of MV prepared in two different media to determine the influence of high and low protein loads. We found that a concentration step before diafiltration improved process economy and MV yield when using either serum-containing or serum-free medium. We also found that discontinuous diafiltration conferred a slight benefit in terms of the permeate flow, reflecting the repetitive dilution steps and the ability to break down parts of the fouling layer on the membrane. In summary, the combined ultrafiltration/diafiltration process is suitable for the purification of MV, resulting in the recovery of ~50% infectious virus particles with a total concentration factor of 8 when using 5 diavolumes of buffer.

Characterization of Cutaneous Adverse Events Associated With PI3K Inhibitors in 11 Patients

Phosphoinositide 3-kinase (PI3K) inhibitors are a new class of cancer therapeutics that inhibits one or more enzymes in the PI3K/AKT/mTOR tumor growth pathway. As compared to other tyrosine kinase inhibitors, there is evidence that PI3K inhibitors have a higher incidence of severe cutaneous adverse events (CAEs) ranging from 2-21%. There is a lack of further characterization of clinical trials and management options for these CAEs. A retrospective chart review of our institution's records between January 2015 and May 2019 was conducted; electronic medical records were queried by using a pharmacy database and ICD-10 codes for patients receiving PI3K inhibitor who experienced CAEs during therapy. These CAEs were characterized by two board-certified dermatologists at a major cancer center. Eleven patients were identified as having 12 cumulative CAEs. Average time to rash onset was 4 weeks, and the most common identified rashes were eczematous (25%) and morbilliform (17%). Four patients experienced a dose delay, and one patient immediately discontinued their PI3K inhibitor. Although most CAEs caused by PI3K inhibitors in this study were limited to grade 1-2 and were controlled with topical corticosteroids and oral antihistamines, a number of patients experienced dose impact. This highlights the dermatologist's role in managing and minimizing interruption of therapy while maintaining quality of life.

Abstract 4067: Inhibition of XBP1 expression in murine macrophages by miR-214 delivered by extracellular vesicles

Abstract Recent advances in cancer therapy have focused on dysregulated local immunity, with paradigm-shifting discoveries like immune checkpoint inhibitors fundamentally altering the way we think about treating cancer. It is now well appreciated that local immune dysregulation is a viable target for cancer therapy, offering many advantages over traditional approaches. One mechanism at the origin of immune dysregulation is a sustained activation of the unfolded protein response (UPR) in the endoplasmic reticulum. The UPR is activated under conditions of stress, including hypoxia, DNA damage, and aneuploidy- stressors present within the tumor microenvironment. Unsurprisingly, the UPR is commonly dysregulated in cancer, acting to promote tumor growth. Multiple lines of evidence support this: TCGA data shows that UPR genes are upregulated in human cancer, and animal models show that tumor formation and growth rates are lower in mice lacking key UPR genes such as IRE1α and XBP1. The UPR promotes tumor growth not only cell-intrinsically, via cancer cell cytoprotection, but also cell-extrinsically, by polarizing tumor-infiltrating macrophages to a pro-tumor inhibitory/ immune stimulatory (IIS) phenotype. We recently found that the IRE1α-XBP1 branch of the UPR is responsible for the IIS signature that occurs in ER stressed macrophages. Here, we sought to develop a novel strategy to target the UPR in macrophages for the treatment of cancer. We focused on microRNAs (miRNA), since no approved small molecule inhibitors of the UPR exist at present for use in cancer patients due to toxicity. We used a proprietary technology developed by our lab that allows for the generation of “induced” extracellular vesicles (iEVs) from B cells that contain a microRNA of choice as cargo. Previously, we reported that transfection of B cells with plasmid DNA encoding for a pre-miRNA loop results in the secretion of large numbers of iEVs that are enriched in the corresponding mature miRNA. Using this technology, we engineered a plasmid DNA to code for miR-214, a miRNA known to target the 3'-UTR of XBP1 mRNA, and used this plasmid to transfected B cells, yielding iEV-214. iEV-214 were highly enriched in miR-214 relative to control iEVs, which include EVs from B cells that were not transfected (iEV-empty), transfected with a scrambled miRNA (iEV-scr), or transfected with an un-related miRNA (iEV-1291). Bone marrow-derived macrophages (BMDM) treated with iEV-214 were enriched in miR-214 relative to all of the controls, demonstrating that iEVs can transfer their miRNA payload to BMDM. When we induced ER stress in BMDM treated with iEV-214, XBP1 transcription was reduced over 50% compared to iEV-scr. Together, these data show that iEVs can transfer miR-214 into macrophages and reduce expression of XBP1 mRNA. We propose iEV-214 as a prototype of a new class of cancer therapeutics to correct UPR-mediated macrophage dysregulation. Citation Format: Stephen Searles, Gonzalo Alamanza, Maurizio Zanetti. Inhibition of XBP1 expression in murine macrophages by miR-214 delivered by extracellular vesicles [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 4067.

Immune checkpoint inhibitors in women with gynecologic cancers: Practical considerations

Immune checkpoint inhibitors are an exciting new class of cancer therapeutics. Recently, a PD-1 inhibitor has been approved by the Food and Drug Administration for several indications that are relevant to patients with gynecologic malignancies. In this review, we explore the clinical considerations for the use of checkpoint inhibitor therapy in this population. Specifically, we will discuss the approved indications, recommended dosing, clinical monitoring while on treatment, common adverse events, and treatment of adverse events should they arise. Additionally, we will review mechanisms of resistance and other challenges associated with the use of checkpoint inhibitors. We will conclude with a discussion of possible future directions for immunotherapy in women with gynecologic cancers.

Targeted Metabolic Reprogramming to Improve the Efficacy of Oncolytic Virus Therapy.

Oncolytic viruses (OVs) represent a promising new class of cancer therapeutics and cause antitumor effects by two major mechanisms: (1) directly killing cancer cells in a process known as oncolysis, or (2) initiating a powerful antitumor immune response. Interestingly, energy metabolism, within either cancer cells or immune cells, plays a pivotal role in defining the outcome of OV-mediated antitumor effects. Following therapeutic administration, OVs must hijack host cell metabolic pathways to acquire building blocks such as nucleotides, lipids, and amino acids for the process of replication that is necessary for oncolysis. Additionally, OV-stimulated antitumor immune responses are highly dependent on the metabolic state withinthe tumor microenvironment. Thus, metabolic reprogramming strategies bear the potential to enhance the efficacyofboth OV-mediated oncolysis and antitumor immune responses.

Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics.

MAIT Cells Promote Tumor Initiation, Growth, and Metastases via Tumor MR1

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that require MHC class I-related protein 1 (MR1) for their development. The role of MAIT cells in cancer is unclear, and to date no study has evaluated these cells in vivo in this context. Here, we demonstrated that tumor initiation, growth, and experimental lung metastasis were significantly reduced in Mr1 -/- mice, compared with wild-type mice. The antitumor activity observed in Mr1 -/- mice required natural killer (NK) and/or CD8+ T cells and IFNγ. Adoptive transfer of MAIT cells into Mr1 -/- mice reversed metastasis reduction. Similarly, MR1-blocking antibodies decreased lung metastases and suppressed tumor growth. Following MR1 ligand exposure, some, but not all, mouse and human tumor cell lines upregulated MR1. Pretreatment of tumor cells with the stimulatory ligand 5-OP-RU or inhibitory ligand Ac-6-FP increased or decreased lung metastases, respectively. MR1-deleted tumors resulted in fewer metastases compared with parental tumor cells. MAIT cell suppression of NK-cell effector function was tumor-MR1-dependent and partially required IL17A. Our studies indicate that MAIT cells display tumor-promoting function by suppressing T and/or NK cells and that blocking MR1 may represent a new therapeutic strategy for cancer immunotherapy. SIGNIFICANCE: Contradicting the perception that MAIT cells kill tumor cells, here MAIT cells promoted tumor initiation, growth, and metastasis. MR1-expressing tumor cells activated MAIT cells to reduce NK-cell effector function, partly in a host IL17A-dependent manner. MR1-blocking antibodies reduced tumor metastases and growth, and may represent a new class of cancer therapeutics.This article is highlighted in the In This Issue feature, p. 1.

A-to-I-edited miRNA-379-5p inhibits cancer cell proliferation through CD97-induced apoptosis.

Both miRNAs and A-to-I RNA editing, a widespread nucleotide modification mechanism, have recently emerged as key players in cancer pathophysiology. However, the functional impact of RNA editing of miRNAs in cancer remains largely unexplored. Here, we focused on an ADAR2-catalyzed RNA editing site within the miR-379-5p seed region. This site was under-edited in tumors relative to normal tissues, with a high editing level being correlated with better patient survival times across cancer types. We demonstrated that in contrast to wild-type miRNA, edited miR-379-5p inhibited cell proliferation and promoted apoptosis in diverse tumor contexts in vitro, which was due to the ability of edited but not wild-type miR-379-5p to target CD97. Importantly, through nanoliposomal delivery, edited miR-379-5p mimics significantly inhibited tumor growth and extended survival of mice. Our study indicates a role of RNA editing in diversifying miRNA function during cancer progression and highlights the translational potential of edited miRNAs as a new class of cancer therapeutics.

Abstract 3077: Preclinical studies of SR-8314, a highly selective ENPP1 inhibitor and an activator of STING pathway

Abstract Purpose: STING (stimulator of interferon genes) plays an important role in innate immunity by activating type I interferons in response to cytosolic nucleic acid ligands such as cyclic dinucleotides (CDNs). In recent years, STING has become an attractive therapeutic target for cancer immune therapy and hydrolysis resistant CDNs have been developed as a new class of cancer therapeutics. These CDNs have been shown to possess potent preclinical efficacy but early results from phase I trials have been disappointing. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1 or NPP1) is natural antagonist of STING pathway. ENPP1 constitutively hydrolyzes 2′3′-cGAMP, a CDN that is the natural ligand for STING. Previously, we reported that SR-8291, a highly selective ENPP1 inhibitor, induces STING activity and demonstrates anti-tumor activity in B16F10 melanoma and CT26 colorectal models. In this study, we extend these findings and report the discovery of SR-8314, an analog of SR-8291 that shows improved physiochemical and developability properties. Methods: With the application of computational techniques (ICM, Maestro), human NPP1 homology model was built by utilizing the crystal structure of mouse NPP1 (PDB: 4GTW). Series of docking simulations on built in ligands were performed within the substrate binding pocket and that led to the identification of lead NPP1 inhibitor SR-8314. Binding of SR-8314 to ENPP1 was evaluated using thermal shift assay. Activity of recombinant human ENPP1 using ATP as a substrate was measured using Cell Titer Glo (Promega) reagent. IRF-Lucia reporter activity, RT-PCR and Western Blotting were performed to evaluate the effect of SR-8314 in 2′3′-cGAMP primed THP1 dual reporter cells (Invivogen). In vivo efficacy studies of intraperitoneally-dosed SR-8314 and SR-8291 were performed in a syngeneic murine tumor model. Tumor T cell infiltration, tumor and plasma pharmacokinetics were assessed by flow cytometry and mass-spectrometry, respectively. Results: SR-8314 has higher binding affinity towards ENPP1 and it potently inhibits ENPP1 activity with a Ki value of 0.079µM. A significant increase in gene expression of IFNβ, ISG15 and CXCL10 along with an increase in the secretion of IFNβ was observed in SR-8314 treated THP1 cells. We show anti-tumor activity as well as an increase in CD3+, CD4+ and CD8+ T cells in both SR-8314 and SR-8291 treated tumors. In addition, there was a decrease in tumor associated macrophages in SR-8314 treated tumors. Conclusions: In summary, we show SR-8314 as a potent inhibitor of ENPP1 that promotes STING activity in vitro. SR-8314 displays promising anti-tumor activity and has ideal candidate properties that warrant further evaluation. Citation Format: Alexis Weston, Trason Thode, Ruben Munoz, Sherin Daniel, Raffaella Soldi, Mohan Kaadige, Haiyong Han, Hariprasad Vankayalapti, Sunil Sharma. Preclinical studies of SR-8314, a highly selective ENPP1 inhibitor and an activator of STING pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3077.

Emerging Cellular Therapies for Cancer.

Genetically engineered T cells are powerful new medicines, offering hope for curative responses in patients with cancer. Chimeric antigen receptor (CAR) T cells were recently approved by the US Food and Drug Administration and are poised to enter the practice of medicine for leukemia and lymphoma, demonstrating that engineered immune cells can serve as a powerful new class of cancer therapeutics. The emergence of synthetic biology approaches for cellular engineering provides a broadly expanded set of tools for programming immune cells for enhanced function. Advances in T cell engineering, genetic editing, the selection of optimal lymphocytes, and cell manufacturing have the potential to broaden T cell-based therapies and foster new applications beyond oncology, in infectious diseases, organ transplantation, and autoimmunity.

Abstract IA20: Updates on CAR T Cells

Abstract The emergence of immune-oncology as the first broadly successful strategy for metastatic cancer will require clinicians to integrate this new pillar of medicine with the pillars of chemotherapy, radiation, and targeted small-molecule compounds. Chimeric antigen receptor (CAR) T cells have proven that engineered immune cells can serve as a powerful new class of cancer therapeutics. Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. Clinical experience has helped to define the major challenges that must be met to make engineered T cells a reliable, safe, and effective platform that can be deployed against a broad range of tumors. The emergence of synthetic biology approaches for cellular engineering provides the field with a broadly expanded set of tools for programming immune cells. In this presentation, I will discuss how these tools could be used to design the next generation of smart T-cell precision therapeutics. We have been exploring in preclinical models and clinical trials methods to synthetically enhance T-cell antitumor efficacy by transfer of genetically engineered T cells. Infusions of chimeric antigen receptor (CAR) T cells can result in remissions in patients with hematologic malignancies, but efficacy is often limited by the extent of expansion and persistence of engineered lymphocytes. In a patient with a delayed clinical response, we show that a complete and durable response to CAR T cell therapy resulted from a clonal expansion of a single CAR T cell. At the peak of the antitumor response, 94 percent of CD8+ CTL019 cells originated from a single clone in which proviral insertion disrupted the gene encoding the methylcytosine dioxygenase TET2. We conclude that loss of function of TET2 secondary to insertional mutagenesis promoted T-cell proliferation, and that the progeny of a single CAR T cell induced durable remission in refractory leukemia. In solid tumors, we have observed antitumor activity in patients with ovarian cancer, pancreatic ductal adenocarcinoma, pleural mesothelioma, and glioblastoma following infusion of CAR T cells expressing scFv specific for mesothelin or EGFRvIII. However, this approach has not yet resulted in complete tumor eradication. Using genome-edited T cells, it may be possible to enhance and prolong the activity of T cells that have disrupted immune and metabolic checkpoints. In preclinical studies, we show that TCR-specific T cells have enhanced antitumor activity following disruption of TCR alpha and beta genes and the PD1 gene using CRISPR/Cas9. This approach is just entering a clinical trial. These findings provide insights into the immunobiology of effector T cells and demonstrate the potential of multiplexed CRISPR/Cas9 genome editing to synthetically enhance the efficacy of immunotherapy.

Abstract 3106: Generation and characterization of IDH1R132H and IDH2R140Q in vitro models for drug discovery and development

Abstract Isocitrate dehydrogenase (IDH) is a metabolic enzyme that converts isocitrate to α-ketoglutarate. This process leads to NADPH production, and is involved in the control of oxidative cellular damage. Mutations in this enzyme have been linked to human cancers such as glioma and acute myeloid leukemia (AML). While there are three isoforms of IDH, mutations that lead to cancer have only been identified in IDH1 and IDH2, which result in simultaneous loss of normal catalytic activity, the production of α-ketoglutarate (α-KG), and gain of a new function, the production of 2-hydroxyglutarate (2-HG). 2-HG is structurally similar to α-KG, and acts as an α-KG antagonist to competitively inhibit multiple α-KG-dependent dioxygenases, including lysine histone demethylases (KDM) and DNA hydroxylases (TET), causing widespread changes in histone and DNA methylation and potentially promoting tumorigenesis. A number of pre-clinical and clinical studies from the mutant IDH inhibitors indicate that IDH is a valid target for a new class of cancer therapeutics. However, there is a lack of well-established and characterized in vitro models containing IDH mutants. The most prominent IDH1 mutation takes place at residue R132H and plays a role in the development of gliomas, while the majority of IDH2 mutations take place at residue R140Q, linked to AML. Given the prevalence of these mutations, we sought to use CRISPR/Cas9 gene editing technology to create two in vitro disease models harboring either the IDH1 or IDH2 mutations. To develop the most clinically relevant models we selected highly appropriate parental cell lines from our extensive portfolio. An IDH1R132H mutation was introduced in the malignant glioblastoma U-87 MG cell line, and an IDH2R140Q mutation was introduced in the TF-1 erythroblast cell line derived from an AML patient. The introduction of the IDH mutations in the respective cell lines was then confirmed via sequencing at the genomic and transcriptional levels. Furthermore, to validate the isogenic IDH mutations confer gain-of-function in vitro, we tested the intracellular and extracellular levels of 2-hydroxyglutarate (2-HG). Bio-functional evaluation data indicated that IDH1R132H U-87 MG cells showed an increase in cellular 2-HG and elevated level of histone methylation. In IDH2R140Q TF-1 cells, an increase in cellular 2-HG was also observed. In response to IDH2 specific inhibitors, AG-221 and AGI-6780, we demonstrated that IDH2R140Q TF-1 cells exhibited decreases in both cellular 2-HG and histone methylation levels. Taken together, these isogenic in vitro models are valuable tools for elucidating mechanisms involved in cancer-associated tumorigenesis and use in screening anti-cancer compounds for drug discovery and development. Citation Format: Diana Douglas, Lysa-Anne Volpe, Luping Chen, Metewo S. Enuameh, Fang Tian, Weiguo Shu. Generation and characterization of IDH1R132H and IDH2R140Q in vitro models for drug discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3106.

PO-354 Star-shaped gene silencing nanoparticles– an advanced approach to treating medulloblastoma

IntroductionMedulloblastoma is the most common malignant brain tumour in children. Chemoresistance and treatment toxicity are major problems for this disease. New effective and less toxic therapies are needed.Short-Interfering-RNA’s (siRNA) are gene silencing molecules which have potential as a new class of cancer therapeutics. siRNA can be designed to inhibit any gene of choice including those which promote tumour growth and chemoresistance and are difficult to target using chemical agents. However, siRNA requires a delivery vehicle to enable it to enter cells and be released into the cytoplasm. The aim of this study was to design and generate nanoparticles which can deliver and release therapeutic siRNA into medulloblastoma cells and silence a gene (polo-like kinase-1, PLK1) which plays a major role in regulating their growth and chemosensitivity.Material and methodsStar-shaped nanoparticles (Star) were synthesised using RAFT polymerisation chemistry. Star size, surface charge and ability to package siRNA were assessed using dynamic light scattering, transmission electron microscopy and gel electrophoresis. Star-siRNA cellular internalisation and gene silencing activity was examined using correlative light electron microscopy, confocal microscopy, qPCR, western blotting and colony formation assay.Results and discussionsStar nanoparticles packaged siRNA with very high efficiency (>90% encapsulation efficiency, n=3) to form uniform nano-complexes (19.1 nm ±0.7) with a near neutral charge (5.8 mV ±0.2). Star-siRNA complexes were stable in human and mouse serum. Star-siRNA was internalised into medulloblastoma cells via endocytosis and siRNA was trafficked to the cytoplasm. Star-siRNA inhibited PLK1 protein expression by 70% (p<0.001, n=3) when compared to control cells (star-non-functional siRNA). Knockdown of PLK1 resulted in G2 cell cycle arrest (p<0.001) and DNA damage. This led to the induction of apoptosis and cell death (p<0.001). Star-PLK1 siRNA also sensitised cells to cisplatin.ConclusionThis is the first study to demonstrate that star nanoparticles can deliver siRNA to medulloblastoma cells. This nanomedicine may be a new therapeutic tool which has potential to revolutionise medulloblastoma treatment and could enhance precision-medicine where it can be used to inhibit the expression of any tumour-promoting gene or cocktail of genes personalised to the genetic profile of a patient’s tumour.

Treatment of Urothelial Cancer in Elderly Patients: Focus on Immune Checkpoint Inhibitors.

Urothelial carcinoma, or bladder cancer, is a malignancy that most commonly affects older patients. The median age at diagnosis is 73years, and care of these patients requires consideration not just of the disease-related factors such as stage and histology, but also of patient-related factors. Many of these patients have concurrent medical morbidities and additional changes related to the aging process. Older patients with cancer are a unique population requiring additional considerations and assessment in treatment decision-making. It is important to look beyond chronologic age. The traditional treatment for advanced disease has relied on platinum-based chemotherapy. These multi-agent regimens require consideration of baseline organ function as well as competing conditions that may heighten toxicity. The advent of a new class of cancer therapeutics, the immune checkpoint inhibitors, has changed the care of patients with advanced disease considerably. These immunotherapeutics have been approved for treating patients with disease progression on chemotherapy, or those who are ineligible (or unfit) to receive cisplatin-based therapy. This expansion of the population of patients eligible for treatment has great applicability to the unique considerations in an older patient population. In general, these new immunotherapies are well tolerated and effective in this group of patients.

The target invites a foe: antibody-drug conjugates in gynecologic oncology.

Antibody-drug conjugates (ADCs) represent a promising new class of cancer therapeutics. Currently more than 60 ADCs are in clinical development, however, only very few trials focus on gynecologic malignancies. In this review, we summarize the most recent advances in ADC drug development with an emphasis on how this progress relates to patients diagnosed with gynecologic malignancies and breast cancer. The cytotoxic payloads of the majority of the ADCs that are currently in clinical trials for gynecologic malignancies or breast cancer are auristatins (MMAE, MMAF), maytansinoids (DM1, DM4), calicheamicin, pyrrolobenzodiazepines and SN-38. Both cleavable and noncleavable linkers are currently being investigated in clinical trials. A number of novel target antigens are currently being validated in ongoing clinical trials including folate receptor alpha, mesothelin, CA-125, NaPi2b, NOTCH3, protein tyrosine kinase-like 7, ephrin-A4, TROP2, CEACAM5, and LAMP1. For most ADCs currently in clinical development, dose-limiting toxicities appear to be unrelated to the targeted antigen but more tightly associated with the payload. Rational drug design involving optimization of the antibody, the linker and the conjugation chemistry is aimed at improving the therapeutic index of new ADCs. Antibody-drug conjugates can increase the efficacy and decrease the toxicity of their payloads in comparison with traditional cyctotoxic agents. A better and quicker translation of recent scientific advances in the field of ADCs into rational clinical trials for patients diagnosed with ovarian, endometrial or cervical cancer could create real improvements in tumor response, survival and quality of life for our patients.