Current Treatment Options for Renal Cell Carcinoma: Focus on Cell-Based Immunotherapy.

Renal cell carcinoma (RCC) affects over 400,000 patients globally each year, and 30% of patients present with metastatic disease. Current standard of care therapy for metastatic RCC involve TKIs and ICIs, including combinatorial strategies, but this offers only modest clinical benefit. Novel treatment approaches are warranted, and cell-based immunotherapies for RCC hold significant promise. These are currently being tested in the pre-clinical setting and in early phase clinical trials. Here, we review the landscape of cellular immunotherapy for RCC in the context of currently available therapies, with a particular focus on defining the current best antigenic targets, the range of cell therapy products being explored in RCC, and how advanced engineering solutions may further enhance these therapies in the RCC space.

The Emerging Role of Induced Pluripotent Stem Cells as Adoptive Cellular Immunotherapeutics.

Adoptive cell therapy (ACT) has transformed the treatment landscape for cancer and infectious disease through the investigational use of chimeric antigen receptor T-cells (CAR-Ts), tumour-infiltrating lymphocytes (TILs) and viral-specific T-cells (VSTs). Whilst these represent breakthrough treatments, there are subsets of patients who fail to respond to autologous ACT products. This is frequently due to impaired patient T-cell function or "fitness" as a consequence of prior treatments and age, and can be exacerbated by complex manufacturing protocols. Further, the manufacture of autologous, patient-specific products is time-consuming, expensive and non-standardised. Induced pluripotent stem cells (iPSCs) as an allogeneic alternative to patient-specific products can potentially overcome the issues outlined above. iPSC technology provides an unlimited source of rejuvenated iPSC-derived T-cells (T-iPSCs) or natural killer (NK) cells (NK-iPSCs), and in the context of the growing field of allogeneic ACT, iPSCs have enormous potential as a platform for generating off-the-shelf, standardised, "fit" therapeutics for patients. In this review, we evaluate current and future applications of iPSC technology in the CAR-T/NK, TIL and VST space. We discuss current and next-generation iPSC manufacturing protocols, and report on current iPSC-based adoptive therapy clinical trials to elucidate the potential of this technology as the future of ACT.

Regulome analysis in B-acute lymphoblastic leukemia exposes Core Binding Factor addiction as a therapeutic vulnerability.

The ETV6-RUNX1 onco-fusion arises in utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFß results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFß-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.

In vitro differentiation of human pluripotent stem cells into the B lineage using OP9-MS5 co-culture.

In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting. For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018).

Generation of a cancer testis antigen mCherry reporter HCT116 colorectal carcinoma cell line.

In the context of cancer immunotherapy, agents that target the immune system to cancer cells need to fulfil two criteria: 1) that they are only expressed on the desired target cell and 2) that they can elicit a potent immunological response. Cancer Testis Antigens are a large disparate family of factors ordinarily expressed in the germ-line but aberrantly expressed across multiple types of cancer. The ability to enforce their expression on tumour cells is an attractive strategy that could render such cells potent targets of the immune system, but very little is known about their regulation. We describe the generation of an mCherry reporter cell line using HCT116 colorectal carcinoma cells that we anticipate will be useful for screen-based approaches to identify novel regulators of CTA expression. Discoveries arising from their use could in future be exploited to enhance tumour cell immunogenicity and improve cancer immuno-therapy.

A novel requirement for DROSHA in maintenance of mammalian CG methylation.

In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation.

ATG9A loss confers resistance to trastuzumab via c-Cbl mediated Her2 degradation.

Acquired or de novo resistance to trastuzumab remains a barrier to patient survival and mechanisms underlying this still remain unclear. Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare proteome profiles between trastuzumab sensitive/resistant cells, we identified autophagy related protein 9A (ATG9A) as a down-regulated protein in trastuzumab resistant cells (BT474-TR). Interestingly, ATG9A ectopic expression markedly decreased the proliferative ability of BT474-TR cells but not that of the parental line (BT474). This was accompanied by a reduction of Her2 protein levels and AKT phosphorylation (S473), as well as a decrease in Her2 stability, which was also observed in JIMT1 and MDA-453, naturally trastuzumab-resistant cells. In addition, ATG9A indirectly promoted c-Cbl recruitment to Her2 on T1112, a known c-Cbl docking site, leading to increased K63 Her2 polyubiquitination. Whereas silencing c-Cbl abrogated ATG9A repressive effects on Her2 and downstream PI3K/AKT signaling, its depletion restored BT474-TR proliferative rate. Taken together, our findings show for this first time that ATG9A loss in trastuzumab resistant cells allowed Her2 to escape from lysosomal targeted degradation through K63 poly-ubiquitination via c-Cbl. This study identifies ATG9A as a potentially druggable target to overcome resistance to anti-Her2 blockade.

Dickkopf-3 regulates prostate epithelial cell acinar morphogenesis and prostate cancer cell invasion by limiting TGF-ß-dependent activation of matrix metalloproteases.

Dickkopf-3 (Dkk-3) is a secreted protein whose expression is downregulated in many types of cancer. Endogenous Dkk-3 is required for formation of acini in 3D cultures of prostate epithelial cells, where it inhibits transforming growth factor (TGF)-ß/Smad signaling. Here, we examined the effects of Dkk-3 on the expression and activity of matrix metalloproteases (MMPs), which mediate the effects of TGF-ß on extracellular matrix disassembly during tissue morphogenesis and promote invasion of tumor cells. Silencing of Dkk-3 in prostate epithelial cells resulted in increased expression and enzyme activity of MMP-2 and MMP-9. Inhibition of MMP-9 partially restored normal acinar morphogenesis in Dkk-3-silenced RWPE-1 prostate epithelial cells. In PC3 prostate cancer cells, Dkk-3 inhibited TGF-ß-dependent migration and invasion. Inhibition was mediated by the Dkk-3 C-terminal cysteine-rich domain (Cys2), which also inhibited TGF-ß-induced expression of MMP9 and MMP13. In contrast, Dkk-3, but not Cys2, increased formation of normal acini in Dkk-3-silenced prostate epithelial cells. These observations highlight a role for Dkk-3 in modulating TGF-ß/MMP signals in the prostate, and suggest that the Dkk-3 Cys2 domain can be used as a basis for therapies that target the tumor promoting effects of TGF-ß signaling in advanced prostate cancer.