Targeting the IL1ß Pathway for Cancer Immunotherapy Remodels the Tumor Microenvironment and Enhances Antitumor Immune Responses.

High levels of IL1ß can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1ß could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1ß blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti-programmed cell death protein 1 (anti-PD-1), anti-VEGFα, and anti-TGFß treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1ß blockade enhanced the effectiveness of docetaxel and anti-PD-1. Accompanying these effects, blockade of IL1ß alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1ß inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1ß blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1ß inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.

BRAF-Mutant Transcriptional Subtypes Predict Outcome of Combined BRAF, MEK, and EGFR Blockade with Dabrafenib, Trametinib, and Panitumumab in Patients with Colorectal Cancer.

PURPOSE: The influence of the transcriptional and immunologic context of mutations on therapeutic outcomes with targeted therapy in cancer has not been well defined. BRAF V600E-mutant (BM) colorectal cancer comprises two main transcriptional subtypes, BM1 and BM2. We sought to determine the impact of BM subtype, as well as distinct biological features of those subtypes, on response to BRAF/MEK/EGFR inhibition in patients with colorectal cancer. PATIENTS AND METHODS: Paired fresh tumor biopsies were acquired at baseline and on day 15 of treatment from all consenting patients with BM colorectal cancer enrolled in a phase II clinical trial of dabrafenib, trametinib, and panitumumab. For each sample, BM subtype, cell cycle, and immune gene signature expression were determined using RNA-sequencing (RNA-seq), and a Cox proportional hazards model was applied to determine association with progression-free survival (PFS). RESULTS: Confirmed response rates, median PFS, and median overall survival (OS) were higher in BM1 subtype patients compared with BM2 subtype patients. Evaluation of immune contexture identified greater immune reactivity in BM1, whereas cell-cycle signatures were more highly expressed in BM2. A multivariate model of PFS incorporating BM subtype plus immune and cell-cycle signatures revealed that BM subtype encompasses the majority of the effect. CONCLUSIONS: BM subtype is significantly associated with the outcome of combination dabrafenib, trametinib, and panitumumab therapy and may serve as a standalone predictive biomarker beyond mutational status. Our findings support a more nuanced approach to targeted therapeutic decisions that incorporates assessment of transcriptional context.

A noninvasive multi-analyte diagnostic assay: combining protein and DNA markers to stratify bladder cancer patients.

PURPOSE: The authors recently reported the development of a noninvasive diagnostic assay using urinary matrix metalloproteinases (MMPs) as monitors of disease-free status and bladder cancer in high-risk populations. Using an approach called clinical intervention determining diagnostic (CIDD), they identified with high confidence those patients who could be excluded from additional intervention. To maximize performance, MMPs were combined with DNA-based markers and CIDD was applied to a population of patients undergoing monitoring for recurrence. PATIENTS AND METHODS: Urine samples were obtained from 323 patients, 48 of whom had a recurrence and 275 of whom did not have cancer upon cytoscopic evaluation. Twist1 and Nid2 methylation status was determined using methylation-specific polymerase chain reaction, FGFR3 mutational status by quantitative PCR, and MMP levels by enzyme-linked immunosorbent assay. RESULTS: Using a combination of these DNA and protein markers, the authors identified with high confidence (97% negative predicted value) those patients who do not have cancer. Cutoffs were adjusted such that at 92% sensitivity, 51% of disease-free patients might be triaged from receiving further tests. CONCLUSION: The multi-analyte diagnostic readout assay described here is the first to combine protein and DNA biomarkers into one assay for optimal clinical performance. Using this approach, the detection of FGFR3 mutations and Twist1 and Nid2 methylation in the urine of patients undergoing bladder cancer recurrence screening increase the sensitivity and negative predictive value at an established MMP protein cutoff. This noninvasive urinary diagnostic assay could lead to the more efficient triage of patients undergoing recurrence monitoring.

Detection of low frequency FGFR3 mutations in the urine of bladder cancer patients using next-generation deep sequencing.

Biological fluid-based noninvasive biomarker assays for monitoring and diagnosing disease are clinically powerful. A major technical hurdle for developing these assays is the requirement of high analytical sensitivity so that biomarkers present at very low levels can be consistently detected. In the case of biological fluid-based cancer diagnostic assays, sensitivities similar to those of tissue-based assays are difficult to achieve with DNA markers due to the high abundance of normal DNA background present in the sample. Here we describe a new urine-based assay that uses ultradeep sequencing technology to detect single mutant molecules of fibroblast growth factor receptor 3 (FGFR3) DNA that are indicative of bladder cancer. Detection of FGFR3 mutations in urine would provide clinicians with a noninvasive means of diagnosing early-stage bladder cancer. The single-molecule assay detects FGFR3 mutant DNA when present at as low as 0.02% of total urine DNA and results in 91% concordance with the frequency that FGFR3 mutations are detected in bladder cancer tumors, significantly improving diagnostic performance. To our knowledge, this is the first practical application of next-generation sequencing technology for noninvasive cancer diagnostics.

c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma.

Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1-c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells.

Nuclear ferritin in corneal epithelial cells: tissue-specific nuclear transport and protection from UV-damage.

We have identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form that is indistinguishable from the cytoplasmic form of ferritin found in other cell types. Thus it most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and other tissues were UV irradiated, and damage to DNA was detected by an in situ 3'-end labeling assay. Consistent with the hypothesis, corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than the other cells types examined. However, when the expression of nuclear ferritin was inhibited the cells now became much more susceptible to UV-induced DNA damage. Since ferritin is normally cytoplasmic, corneal epithelial cells must have a mechanism that effects its nuclear localization. We have determined that this involves a nuclear transport molecule which binds to ferritin and carries it into the nucleus. This transporter, which we have termed ferritoid for its similarity to ferritin, has at least two domains. One domain is ferritin-like and is responsible for binding the ferritin; the other domain contains a nuclear localization signal that is responsible for effecting the nuclear transport. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage. In addition, since ferritoid is structurally similar to ferritin, it may represent an example of a nuclear transporter that evolved from the molecule it transports (i.e., ferritin).

Ferritoid, a tissue-specific nuclear transport protein for ferritin in corneal epithelial cells.

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of approximately 50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).