Time-course RNAseq data of murine AB1 mesothelioma and Renca renal cancer following immune checkpoint therapy.

Time-critical transcriptional events in the immune microenvironment are important for response to immune checkpoint blockade (ICB), yet these events are difficult to characterise and remain incompletely understood. Here, we present whole tumor RNA sequencing data in the context of treatment with ICB in murine models of AB1 mesothelioma and Renca renal cell cancer. We sequenced 144 bulk RNAseq samples from these two cancer types across 4 time points prior and after treatment with ICB. We also performed single-cell sequencing on 12 samples of AB1 and Renca tumors an hour before ICB administration. Our samples were equally distributed between responders and non-responders to treatment. Additionally, we sequenced AB1-HA mesothelioma tumors treated with two sample dissociation protocols to assess the impact of these protocols on the quality transcriptional information in our samples. These datasets provide time-course information to transcriptionally characterize the ICB response and provide detailed information at the single-cell level of the early tumor microenvironment prior to ICB therapy.

CD4+ T cells drive an inflammatory, TNF-α/IFN-rich tumor microenvironment responsive to chemotherapy.

While chemotherapy remains the first-line treatment for many cancers, it is still unclear what distinguishes responders from non-responders. Here, we characterize the chemotherapy-responsive tumor microenvironment in mice, using RNA sequencing on tumors before and after cyclophosphamide, and compare the gene expression profiles of responders with progressors. Responsive tumors have an inflammatory and highly immune infiltrated pre-treatment tumor microenvironment characterized by the enrichment of pathways associated with CD4+ T cells, interferons (IFNs), and tumor necrosis factor alpha (TNF-α). The same gene expression profile is associated with response to cyclophosphamide-based chemotherapy in patients with breast cancer. Finally, we demonstrate that tumors can be sensitized to cyclophosphamide and 5-FU chemotherapy by pre-treatment with recombinant TNF-α, IFNγ, and poly(I:C). Thus, a CD4+ T cell-inflamed pre-treatment tumor microenvironment is necessary for response to chemotherapy, and this state can be therapeutically attained by targeted immunotherapy.

Temporally restricted activation of IFNß signaling underlies response to immune checkpoint therapy in mice.

The biological determinants of the response to immune checkpoint blockade (ICB) in cancer remain incompletely understood. Little is known about dynamic biological events that underpin therapeutic efficacy due to the inability to frequently sample tumours in patients. Here, we map the transcriptional profiles of 144 responding and non-responding tumours within two mouse models at four time points during ICB. We find that responding tumours display on/fast-off kinetics of type-I-interferon (IFN) signaling. Phenocopying of this kinetics using time-dependent sequential dosing of recombinant IFNs and neutralizing antibodies markedly improves ICB efficacy, but only when IFNß is targeted, not IFNα. We identify Ly6C+/CD11b+ inflammatory monocytes as the primary source of IFNß and find that active type-I-IFN signaling in tumour-infiltrating inflammatory monocytes is associated with T cell expansion in patients treated with ICB. Together, our results suggest that on/fast-off modulation of IFNß signaling is critical to the therapeutic response to ICB, which can be exploited to drive clinical outcomes towards response.

Comprehensive Testing of Chemotherapy and Immune Checkpoint Blockade in Preclinical Cancer Models Identifies Additive Combinations.

Antibodies that target immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and the programmed cell death protein 1/ligand 1 (PD-1/PD-L1) are now a treatment option for multiple cancer types. However, as a monotherapy, objective responses only occur in a minority of patients. Chemotherapy is widely used in combination with immune checkpoint blockade (ICB). Although a variety of isolated immunostimulatory effects have been reported for several classes of chemotherapeutics, it is unclear which chemotherapeutics provide the most benefit when combined with ICB. We investigated 10 chemotherapies from the main canonical classes dosed at the clinically relevant maximum tolerated dose in combination with anti-CTLA-4/anti-PD-L1 ICB. We screened these chemo-immunotherapy combinations in two murine mesothelioma models from two different genetic backgrounds, and identified chemotherapies that produced additive, neutral or antagonistic effects when combined with ICB. Using flow cytometry and bulk RNAseq, we characterized the tumor immune milieu in additive chemo-immunotherapy combinations. 5-fluorouracil (5-FU) or cisplatin were additive when combined with ICB while vinorelbine and etoposide provided no additional benefit when combined with ICB. The combination of 5-FU with ICB augmented an inflammatory tumor microenvironment with markedly increased CD8+ T cell activation and upregulation of IFNγ, TNFα and IL-1ß signaling. The effective anti-tumor immune response of 5-FU chemo-immunotherapy was dependent on CD8+ T cells but was unaffected when TNFα or IL-1ß cytokine signaling pathways were blocked. Our study identified additive and non-additive chemotherapy/ICB combinations and suggests a possible role for increased inflammation in the tumor microenvironment as a basis for effective combination therapy.

Immune checkpoint inhibitor therapy for malignant pleural mesothelioma.

Mesothelioma is a rare and universally fatal cancer linked to exposure to asbestos. Until recently, standard of care treatment was chemotherapy; a treatment resulting in a minimal survival extension, and not improved upon for almost twenty years. However, the advent of cancer immunotherapy - and in particular the immune checkpoint inhibitor class of drugs - has resulted in recently approved new treatment options, with more currently under investigation. Here, we review clinical trials of both single agent and combination checkpoint inhibitors in mesothelioma, plus studies investigating their combination with chemotherapy. We also describe current advances in biomarker identification regarding prediction of patient response to checkpoint inhibitors. Finally, we assess the probable future direction of the field; including where current and developing technologies are likely to lead - in terms of both biomarker discovery and treatment options.

Sensitizing the Tumor Microenvironment to Immune Checkpoint Therapy.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, providing remarkable clinical responses in some patients. However, the majority of patients do not respond. It is therefore crucial both to identify predictive biomarkers of response and to increase the response rates to immune checkpoint therapy. In this review we explore the current literature about the predictive characteristics of the tumor microenvironment and discuss therapeutic approaches that aim to change this toward a milieu that is conducive to response. We propose a personalized biomarker-based adaptive approach to immunotherapy, whereby a sensitizing therapy is tailored to the patient's specific tumor microenvironment, followed by on-treatment verification of a change in the targeted biomarker, followed by immune checkpoint therapy. By incorporating detailed knowledge of the immunological tumor microenvironment, we may be able to sensitize currently non-responsive tumors to respond to immune checkpoint therapy.

A phase II trial of single oral FGF inhibitor, AZD4547, as second or third line therapy in malignant pleural mesothelioma.

OBJECTIVES: Currently, there is no optimal salvage therapy for patients with malignant pleural mesothelioma (MPM) who relapse after treatment with first-line chemotherapy. In line with the strong preclinical rationale for targeting fibroblast growth factor receptor (FGFR) signalling in malignant mesothelioma, we conducted a phase II study assessing the efficacy of AZD4547, an oral tyrosine multi-kinase FGFR 1-3 inhibitor, as a second or third-line treatment. MATERIALS AND METHODS: We conducted a single-center, open-label, single-arm phase II study of AZD4547 in eligible patients with confirmed, measurable MPM and radiological progression after first or second-line systemic chemotherapy. Patients received continuous, twice-daily oral AZD4547 on a 3-weekly cycle. The primary end point was 6-month progression free survival (PFS6). Response was assessed with CT scan every 6 weeks according to the modified RECIST criteria for mesothelioma (mRECIST) and toxicities were also assessed. The study used a Simon's two-stage design: 26 patients would be recruited to the first stage and more than 7 (27 %) of 26 patients were required to achieve PFS6 to continue to stage two, for a potential total cohort of 55 patients. RESULTS: 3 of 24 patients (12 %) were progression-free at 6 months. Hence, the study fulfilled stopping criteria regardless of further recruitment and warranted discontinuation. The most common toxicities (across all grades) were hyperphosphatemia, xerostomia, mucositis, retinopathy, dysgeusia, and fatigue. Maximum toxicities were grade 2 or below for all patients across all cycles. There was no association between tumour BAP1 protein loss and clinical outcomes. CONCLUSIONS: The FGFR 1-3 inhibitor AZD4547 did not demonstrate efficacy in patients with MPM who had progressed after first line treatment with platinum-based chemotherapy.

Sensitization to immune checkpoint blockade through activation of a STAT1/NK axis in the tumor microenvironment.

Cancer immunotherapy using antibodies that target immune checkpoints has delivered outstanding results. However, responses only occur in a subset of patients, and it is not fully understood what biological processes determine an effective outcome. This lack of understanding hinders the development of rational combination treatments. We set out to define the pretreatment microenvironment associated with an effective outcome by using the fact that inbred mouse strains bearing monoclonal cancer cell line-derived tumors respond in a dichotomous manner to immune checkpoint blockade (ICB). We compared the cellular composition and gene expression profiles of responsive and nonresponsive tumors from mice before ICB and validated the findings in cohorts of patients with cancer treated with ICB antibodies. We found that responsive tumors were characterized by an inflammatory gene expression signature consistent with up-regulation of signal transducer and activator of transcription 1 (STAT1) and Toll-like receptor 3 (TLR3) signaling and down-regulation of interleukin-10 (IL-10) signaling. In addition, responsive tumors had more infiltrating-activated natural killer (NK) cells, which were necessary for response. Pretreatment of mice with large established tumors using the STAT1-activating cytokine interferon-γ (IFNγ), the TLR3 ligand poly(I:C), and an anti-IL-10 antibody sensitized tumors to ICB by attracting IFNγ-producing NK cells into the tumor, resulting in increased cure rates. Our results identify a pretreatment tumor microenvironment that predicts response to ICB, which can be therapeutically attained. These data suggest a biomarker-driven approach to patient management to establish whether a patient would benefit from treatment with sensitizing therapeutics before ICB.

Characterising the binding specificities of the subunits associated with the KMT2/Set1 histone lysine methyltransferase.

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5'-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.