Tumor-Infiltrating Myeloid Cells Confer De Novo Resistance to PD-L1 Blockade through EMT-Stromal and Tgfß-Dependent Mechanisms.

SIGNIFICANCE: Most patients with bladder cancer do not respond to ICB targeting of the PD-L1 signaling axis. Our modeling applied a de novo resistance signature to show that tumor-infiltrating myeloid cells promote poor treatment response in a TGFß-dependent mechanism.

World Trade Center dust exposure promotes cancer in PTEN-deficient mouse prostates.

During the 9/11 attacks individuals were exposed to World Trade Center (WTC) dust which contained a complex mixture of carcinogens. Epidemiological studies have revealed the increased incidence of prostate and thyroid cancer in WTC survivors and responders. While reports have shown that WTC-dust associates with the increased prevalence of inflammatory related disorders, studies to date have not determined whether this exposure impacts cancer progression. In this study, we have used genetically engineered mouse (GEM) models with prostate specific deletion of the PTEN tumor suppressor to study the impact of WTC-dust exposure on deposition of dust particles, inflammation, and cancer progression. In normal C57/BL6 mice, dust exposure increased cellular expression of inflammatory genes with highest levels in the lung and peripheral blood. In normal and tumor bearing GEM mice, increased immune cell infiltration to the lungs was observed. Pathological evaluation of mice at different time points showed that WTC-dust exposure promoted PI3K-AKT activation, increased epithelial proliferation and acinar invasion in prostates with heterozygous and homozygous Pten loss. Using autochthonous and transplant GEM models of prostate cancer we demonstrated that dust exposure caused reduced survival as compared to control cohorts. Finally, we used imaging mass cytometry (IMC) to detect elevated immune cell infiltration and cellular expression of inflammatory markers in prostate tumors isolated from human WTC survivors. Collectively, our study shows that chronic inflammation, induced by WTC dust exposure, promotes more aggressive cancer in genetically predisposed prostates and potentially in patients.

Dynamic plasticity of prostate cancer intermediate cells during androgen receptor-targeted therapy.

Treatment-emergent small cell neuroendocrine prostate cancer (t-SCNC) is associated with an epithelial lineage switch from an androgen receptor (AR)-positive to neuroendocrine (NE)-marker-positive status. Understanding the potential for reversibility of this aggressive disease state has been hampered by the paucity of models suitable for studying rate-limiting, transitional, or intermediate tumor cell subpopulations. We define a dual reporter model that measures acute transcriptional changes in response to castration or AR targeting agents. We identify steady-state transcriptional heterogeneity in AR and NE biomarkers, including intermediate subpopulations that are coordinately high for prostate-specific antigen (PSA) and neuron-specific enoclase (NSE) promoter activity. In the presence of castration or AR inhibitors, intermediate cells were necessary and sufficient for therapy-induced conversion of human PC cells to an NSE-high transcriptional status. Using hormone add-back studies, treatment-induced PSA-NSE transcriptional plasticity was reversible in PTEN-deficient PC cells but not in the presence of secondary genetic driver genes, including MYCN.

Global DNA methylation of WTC prostate cancer tissues show signature differences compared to non-exposed cases.

There is increased incidence of prostate cancer (PC) among World Trade Center (WTC)-exposed responders and community members, with preliminary evidence suggestive of more aggressive disease. While previous research is supportive of differences in DNA methylation and gene expression as a consequence of WTC exposure, as measured in blood of healthy individuals, the epigenetics of WTC PC tissues has yet to be explored. Patients were recruited from the World Trade Center Health Program. Non-WTC PC samples were frequency matched on age, race/ethnicity and Gleason score. Bisulfite-treated DNA was extracted from tumor tissue blocks and used to assess global DNA methylation with the MethylationEPIC BeadChip. Differential and pathway enrichment analyses were conducted. RNA from the same tumor blocks was used for gene expression analysis to further support DNA methylation findings. Methylation data were generated for 28 samples (13 WTC and 15 non-WTC). Statistically significant differences in methylation were observed for 3,586 genes; on average WTC samples were statistically significantly more hypermethylated (P = 0.04131). Pathway enrichment analysis revealed hypermethylation in epithelial mesenchymal transition (EMT), hypoxia, mitotic spindle, TNFA signaling via NFKB, WNT signaling, and TGF beta signaling pathways in WTC compared to non-WTC samples. The androgen response, G2M and MYC target pathways were hypomethylated. These results correlated well with RNA gene expression. In conclusion, long-term epigenic changes associated with WTC dust exposure were observed in PC tissues. These occurred in genes of critical pathways, likely increasing prostate tumorigenesis potential. This warrants analysis of larger WTC groups and other cancer types.

Tumor Heterogeneity and Consequences for Bladder Cancer Treatment.

Acquired therapeutic resistance remains a major challenge in cancer management and associates with poor oncological outcomes in most solid tumor types. A major contributor is tumor heterogeneity (TH) which can be influenced by the stromal; immune and epithelial tumor compartments. We hypothesize that heterogeneity in tumor epithelial subpopulations-whether de novo or newly acquired-closely regulate the clinical course of bladder cancer. Changes in these subpopulations impact the tumor microenvironment including the extent of immune cell infiltration and response to immunotherapeutics. Mechanisms driving epithelial tumor heterogeneity (EpTH) can be broadly categorized as mutational and non-mutational. Mechanisms regulating lineage plasticity; acquired cellular mutations and changes in lineage-defined subpopulations regulate stress responses to clinical therapies. If tumor heterogeneity is a dynamic process; an increased understanding of how EpTH is regulated is critical in order for clinical therapies to be more sustained and durable. In this review and analysis, we assess the importance and regulatory mechanisms governing EpTH in bladder cancer and the impact on treatment response.

Peroxisomal L-bifunctional protein (EHHADH) deficiency causes male-specific kidney hypertrophy and proximal tubular injury in mice.

BACKGROUND: Proximal tubular (PT) cells are enriched in mitochondria and peroxisomes. Whereas mitochondrial fatty acid oxidation (FAO) plays an important role in kidney function by supporting the high-energy requirements of PT cells, the role of peroxisomal metabolism remains largely unknown. EHHADH, also known as L-bifunctional protein, catalyzes the second and third step of peroxisomal FAO. METHODS: We studied kidneys of WT and Ehhadh KO mice on a C57BL/6N background using histology, immunohistochemistry, immunofluorescence, immunoblot, RNA-sequencing, and metabolomics. To assess the role of androgens in the kidney phenotype of Ehhadh KO mice, mice underwent orchiectomy. RESULTS: We observed male-specific kidney hypertrophy and glomerular filtration rate reduction in adult Ehhadh KO mice. Transcriptome analysis unveiled a gene expression signature similar to PT injury in acute kidney injury mouse models. This was further illustrated by the presence of KIM-1 (kidney injury molecule-1), SOX-9, and Ki67-positive cells in the PT of male Ehhadh KO kidneys. Male Ehhadh KO kidneys had metabolite changes consistent with peroxisomal dysfunction as well as an elevation in glycosphingolipid levels. Orchiectomy of Ehhadh KO mice decreased the number of KIM-1 positive cells to WT levels. We revealed a pronounced sexual dimorphism in the expression of peroxisomal FAO proteins in mouse kidney, underlining a role of androgens in the kidney phenotype of Ehhadh KO mice. CONCLUSIONS: Our data highlight the importance of EHHADH and peroxisomal metabolism in male kidney physiology and reveal peroxisomal FAO as a sexual dimorphic metabolic pathway in mouse kidneys.

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

Leflunomide triggers synthetic lethality in PTEN-deficient prostate cancer.

BACKGROUND: The loss of PTEN function presents in up to 50% of late-stage prostate cancers, and is therefore a potential target for therapeutics. PTEN-deficient cells depend on de novo pyrimidine synthesis, a feature that can present a vulnerability. METHODS: We utilized in vitro growth assays and in vivo xenograft models to test the effect of de novo pyrimidine synthesis inhibition on prostate cell lines. RESULTS: Here, we demonstrate that PTEN-deficient prostate cancer cell lines are susceptible to inhibition of de novo pyrimidine synthesis by leflunomide. Tumor growth inhibition was observed in vitro and in vivo following leflunomide treatment, and is likely due to an overwhelming accumulation of DNA damage. CONCLUSIONS: Our work highlights that synthetic lethality arises upon the combination of PTEN loss and leflunomide treatment in prostate cancer, and may present a therapeutic opportunity for this patient population.

Epithelial plasticity can generate multi-lineage phenotypes in human and murine bladder cancers.

Tumor heterogeneity is common in cancer, however recent studies have applied single gene expression signatures to classify bladder cancers into distinct subtypes. Such stratification assumes that a predominant transcriptomic signature is sufficient to predict progression kinetics, patient survival and treatment response. We hypothesize that such static classification ignores intra-tumoral heterogeneity and the potential for cellular plasticity occurring during disease development. We have conducted single cell transcriptome analyses of mouse and human model systems of bladder cancer and show that tumor cells with multiple lineage subtypes not only cluster closely together at the transcriptional level but can maintain concomitant gene expression of at least one mRNA subtype. Functional studies reveal that tumor initiation and cellular plasticity can initiate from multiple lineage subtypes. Collectively, these data suggest that lineage plasticity may contribute to innate tumor heterogeneity, which in turn carry clinical implications regarding the classification and treatment of bladder cancer.

Complementary biobank of rodent tissue samples to study the effect of World Trade Center exposure on cancer development.

World Trade Center (WTC) responders were exposed to mixture of dust, smoke, chemicals and carcinogens. New York University (NYU) and Mount Sinai have recreated WTC exposure in rodents to observe the resulting systemic and local biological responses. These experiments aid in the interpretation of epidemiological observations and are useful for understanding the carcinogenesis process in the exposed human WTC cohort. Here we describe the implementation of a tissue bank system for the rodents experimentally exposed to WTC dust. NYU samples were experimentally exposed to WTC dust via intratracheal inhalation that mimicked conditions in the immediate aftermath of the disaster. Tissue from Mount Sinai was derived from genetically modified mice exposed to WTC dust via nasal instillation. All processed tissues include annotations of the experimental design, WTC dust concentration/dose, exposure route and duration, genetic background of the rodent, and method of tissue isolation/storage. A biobank of tissue from rodents exposed to WTC dust has been compiled representing an important resource for the scientific community. The biobank remains available as a scientific resource for future research through established mechanisms for samples request and utilization. Studies using the WTC tissue bank would benefit from confirming their findings in corresponding tissues from organs of animals experimentally exposed to WTC dust. Studies on rodent tissues will advance the understanding of the biology of the tumors developed by WTC responders and ultimately impact the modalities of treatment, and the probability of success and survival of WTC cancer patients.

Fibroblast Growth Factor Receptor 3 Alterations and Response to PD-1/PD-L1 Blockade in Patients with Metastatic Urothelial Cancer.

Prior studies have demonstrated that fibroblast receptor 3 (FGFR3)-mutant urothelial cancers (UCs) are associated with decreased T-cell infiltration. As FGFR3 mutations are enriched in luminal-like UC and luminal-like UC has been shown to be relatively less responsive to PD-1/PD-L1 inhibition (checkpoint inhibition [CPI]), these data have led to the speculation that FGFR3 mutations may be causally related to poor T-cell infiltration and that UC patients harboring FGFR3 mutations may be suboptimal candidates for CPI. Using data derived from two clinical trials exploring CPI in metastatic UC, we demonstrate no statistically significant difference in response rates in patients with FGFR3-mutant versus wild-type UC. We present hypothesis-generating data, suggesting that similar response rates may be explained by a "balancing out" of previously identified independent positive and negative predictors of CPI sensitivity; that is, compared with FGFR3 wild-type UC, FGFR3-mutant UC is associated with a similar tumor mutational burden, lower T-cell infiltration, but also lower stromal/transforming growth factor beta (TGF-ß) signals. Based on our findings, FGFR3 mutation status is not a biomarker of resistance to CPI. Indeed, the single-agent activity of both FGFR3 inhibitors and CPI in FGFR3-mutant UC, and potential non-cross resistance provide a strong pragmatic rationale for combination approaches. PATIENT SUMMARY: In this report, we examined the impact of a mutated gene found in a subset of urothelial cancers on response to treatment with immunotherapy. We found that patients with tumors harboring mutations in the gene FGFR3 respond to immunotherapy similarly to patients without such mutations.

EMT- and stroma-related gene expression and resistance to PD-1 blockade in urothelial cancer.

Cancers infiltrated with T-cells are associated with a higher likelihood of response to PD-1/PD-L1 blockade. Counterintuitively, a correlation between epithelial-mesenchymal transition (EMT)-related gene expression and T-cell infiltration has been observed across tumor types. Here we demonstrate, using The Cancer Genome Atlas (TCGA) urothelial cancer dataset, that although a gene expression-based measure of infiltrating T-cell abundance and EMT-related gene expression are positively correlated, these signatures convey disparate prognostic information. We further demonstrate that non-hematopoietic stromal cells are a major source of EMT-related gene expression in bulk urothelial cancer transcriptomes. Finally, using a cohort of patients with metastatic urothelial cancer treated with a PD-1 inhibitor, nivolumab, we demonstrate that in patients with T-cell infiltrated tumors, higher EMT/stroma-related gene expression is associated with lower response rates and shorter progression-free and overall survival. Together, our findings suggest a stroma-mediated source of immune resistance in urothelial cancer and provide rationale for co-targeting PD-1 and stromal elements.

PRL­3 increases the aggressive phenotype of prostate cancer cells in vitro and its expression correlates with high-grade prostate tumors in patients.

The increased expression of phosphatase of regenerating liver-3 (PRL­3) has been shown to be associated with the aggressive and metastatic phenotype of different solid tumors. However, it is not known whether PRL­3 plays a similar role in the progression of prostate cancer (PCa). In this study, immunoblot analysis of androgen receptor (AR)-positive PCa lines (LNCaP and LNCaP­SF) revealed the constitutive cytoplasmic expression of PRL­3, and stimulation with R1881 (AR agonist) rapidly increased the nuclear translocation of PRL­3. The AR-negative cell lines exhibited negligible PRL­3 expression, and the ectopic overexpression of PRL­3 increased both the proliferative and invasive potential of PC3 and DU145 cells. In addition, we measured PRL­3 protein expression in human prostate tumor sections. A high-density prostate tumor microarray (TMA) was immunostained to assess whether PRL­3 expression and its subcellular localization (cytoplasmic and nuclear levels) is associated with the Gleason score (GS), Gleason grade (GG) and tumor stage (T-stage). Digital image analysis (DIA) revealed that PRL­3 expression was significantly higher in the malignant cores, as compared to the non­malignant areas. Increases in both total and nuclear PRL­3 levels were also associated with a higher GS and GG. Metastatic tumors (T4­stage) had lower cytoplasmic, but higher nuclear PRL­3 levels. Furthermore, the nuclear/cytoplasmic ratio for PRL­3 in the tumors graded as GS7 could effectively distinguish between indolent (3+4) and aggressive (4+3) disease. Thus, our experiments using PCa lines suggested that PRL­3 is an AR-regulated gene and its androgen-induced nuclear localization may increase the aggressive behavior of PCa cells. Furthermore, the digital analysis of immunostained tumor sections suggested that PRL­3 may be an effective biomarker of high-grade PCa, and its nuclear/cytoplasmic ratio may be used to distinguish between indolent vs. aggressive tumors.

Overcoming Oncogenic Mediated Tumor Immunity in Prostate Cancer.

Immunotherapy is being tested intensively in clinical trials for prostate cancer; it includes immune checkpoint inhibition, prostate specific antigen (PSA) vaccines and dendritic cell-based strategies. Despite increasing evidence for clinical responses, the consensus of multiple trials is that prostate cancers are poorly responsive to immunotherapy. Prostate cancer has a high degree of pathological and genetic heterogeneity compared to other cancer types, which may account for immunotherapeutic resistance. This hypothesis also implies that select types of prostate tumors may be differentially responsive to immune-based strategies and that the clinical stage, pathological grade and underlying genetic landscape may be important criteria in identifying tumors that respond to immune therapies. One strategy is to target oncogenic driver pathways in combination with immunotherapies with the goal of overcoming tumor immunity and broadening the number of patients achieving a clinical response. In this analysis, we address the hypothesis that driver oncogenic signaling pathways regulate cancer progression, tumor immunity and resistance to current immune therapeutics in prostate cancer. We propose that increased responsiveness may be achieved through the combined use of immunotherapies and inhibitors targeting tumor cell autonomous pathways that contribute towards anti-tumor immunity in patients with prostate cancer.

Strategies to avoid treatment-induced lineage crisis in advanced prostate cancer.

The increasing potency of therapies that target the androgen receptor (AR) signalling axis has correlated with a rise in the proportion of patients with prostate cancer harbouring an adaptive phenotype, termed treatment-induced lineage crisis. This phenotype is characterized by features that include soft-tissue metastasis and/or resistance to standard anticancer therapies. Potent anticancer treatments might force cancer cells to evolve and develop alternative cell lineages that are resistant to primary therapies, a mechanism similar to the generation of multidrug- resistant microorganisms after continued antibiotic use. Herein, we assess the hypothesis that treatment-adapted phenotypes harbour reduced AR expression and/or activity, and acquire compensatory strategies for cell survival. We highlight the striking similarities between castration-resistant prostate cancer and triple-negative breast cancer, another poorly differentiated endocrine malignancy. Alternative treatment paradigms are needed to avoid therapy-induced resistance. Herein, we present a new clinical trial strategy designed to evaluate the potential of rapid drug cycling as an approach to delay the onset of resistance and treatment-induced lineage crisis in patients with metastatic castration-resistant prostate cancer.

A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.

Inhibition of Pten deficient Castration Resistant Prostate Cancer by Targeting of the SET - PP2A Signaling axis.

The PP2A signaling axis regulates multiple oncogenic drivers of castration resistant prostate cancer (CRPC). We show that targeting the endogenous PP2A regulator, SET (I2PP2A), is a viable strategy to inhibit prostate cancers that are resistant to androgen deprivation therapy. Our data is corroborated by analysis of prostate cancer patient cohorts showing significant elevation of SET transcripts. Tissue microarray analysis reveals that elevated SET expression correlates with clinical cancer grading, duration of neoadjuvant hormone therapy (NHT) and time to biochemical recurrence. Using prostate regeneration assays, we show that in vivo SET overexpression is sufficient to induce hyperplasia and prostatic intraepithelial neoplasia. Knockdown of SET induced significant reductions in tumorgenesis both in murine and human xenograft models. To further validate SET as a therapeutic target, we conducted in vitro and in vivo treatments using OP449 - a recently characterized PP2A-activating drug (PAD). OP449 elicits robust anti-cancer effects inhibiting growth in a panel of enzalutamide resistant prostate cancer cell lines. Using the Pten conditional deletion mouse model of prostate cancer, OP449 potently inhibited PI3K-Akt signaling and impeded CRPC progression. Collectively, our data supports a critical role for the SET-PP2A signaling axis in CRPC progression and hormone resistant disease.

Identification of Androgen Receptor Splice Variants in the Pten Deficient Murine Prostate Cancer Model.

Androgen receptor (AR) variants are associated with resistance to anti androgen therapy both in human prostate cancer cell lines and clinical samples. These observations support the hypothesis that AR isoform accumulation is a consequence of selective therapeutic pressure on the full length AR. The Pten deficient prostate cancer model proceeds with well-defined kinetics including progression to castration resistant prostate cancer (CRPC). While surgical castration and enzalutamide treatments yield an initial therapeutic response, Pten-/-epithelia continue to proliferate yielding locally invasive primary tumor pathology. That most epithelium remains AR positive, but ligand independent, suggests the presence of oncogenic AR variants. To address this hypothesis, we have used a panel of recently described Pten-/- tumor cell lines derived from both from hormone intact (E4, E8) and castrated Pten mutants (cE1, cE2) followed by RACE PCR to identify and characterize three novel truncated, amino terminus containing AR variants (mAR-Va, b, c). Variants appear not only conserved throughout progression but are correlated with nearly complete loss of full length AR (AR-FL) at castrate androgen levels. The overexpression of variants leads to enhanced transcriptional activity of AR while knock down studies show reduced transcriptional output. Collectively, the identification of truncated AR variants in the conditional PTEN deletion model supports a role for maintaining the CRPC phenotype and provides further therapeutic applications of this preclinical model.

Idiopathic Intracranial Hypertension in the Northwest of Northern Ireland: Epidemiology and Clinical Management.

BACKGROUND: There is limited literature on the epidemiology of idiopathic intracranial hypertension (IIH). The diagnosis and management of IIH require a multidisciplinary approach. We sought to study the incidence as well as prevalence of IIH and to evaluate the current management of IIH in the northwest of Northern Ireland. METHODS: Medical records of patients diagnosed with IIH between 2007 and 2014 in a general hospital in Northern Ireland were reviewed. Clinical and outcome data were retrieved. RESULTS: There were 45 patients with IIH, 44 women: 1 man. The mean age at presentation was 29.4 (SD 9.8) years and mean body mass index (BMI) 39.8 (SD 9.5) kg/ m(2). All patients had neuroimaging, 44 (98%) had CT/MR venogram and 41 (91%) had visual perimetry. The crude incidence of IIH was 2.36 per 100,000 (95% CI 1.65-3.37). For women, the incidence was 4.65 per 100,000/year (95% CI 3.25-6.66). The prevalence was 14.3 per 100,000 overall (95% CI 9.72-20.9) but 28.1 per 100,000 in women (95% CI 19.2-41.2). Visual field defects were identified in 25 of 41 (61%); 4 patients (9%) required shunting procedures. At follow-up, the mean BMI decreased by 1.6 kg/m(2) (p = 0.024). CONCLUSIONS: The incidence of IIH in the northwest of Northern Ireland is among the highest ever reported and probably reflects the known increase in obesity.