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Investigations into the function of nonpromoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated nonpromoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 260 genes exhibiting a correlation of > 0.5 between gene body methylation and gene expression in at least one tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in kidney renal cell carcinoma (KIRC) and lung adenocarcinoma (LUAD) tumor tissues in comparison with respective control samples; and is significantly associated with lower overall survival in KIRC. Contrary to its canonical function in lymphocyte NFκB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy. Furthermore, demethylation of a CpG island within the gene body of CARD11 decreases gene expression. Collectively, our study highlights how DNA methylation outside the promoter region can impact tumor progression.Implications:Our study describes a novel regulatory role of gene body DNA methylation–dependent CARD11 expression on mTOR signaling and its impact on tumor progression.

S1: A) Agarose gels showing PCR amplification of a region within HIF-2α exon 2 from genomic DNA from 786-0 HIF-2α deletion mutants, EPAS1-/- #4 and EPAS1-/-#11 compared to 786-0 cells transfected with Nickase control (NicCon). Clone #4 and #11 were generated using separate pairs of guide strands targeting different regions within exon 2. B) PCR primers used for amplification. C) Western blot showing HIF-2α protein levels in 786-0 Crispr clones compared to Nickase Control. D) Luciferase assay showing luciferase activity of a transiently transfected hypoxia-responsive element (HRE) - luciferase reporter normalized to a co-transfected beta-actin Renilla luciferase reporter in 786-0 Crispr clones compared to parental 786-0 cells. E) Genetic characterization of EPAS1-/-#4 aligned to EPAS1 NCBI RefSeq NG_016000.1. Clone #4 is a clonal population of cells with a 70bp deletion within one EPAS1 (HIF-2α) allele, and multiple indels within the corresponding region of the second allele: 5 insertions (underlined), 8 mutations (caps). F) Genetic characterization of EPAS-/- #11 aligned to EPAS1 NCBI RefSeq NG_016000.1. Clone #11 is a polyclonal cell population containing at least three variants of EPAS1 (HIF-2α), each with multiple mutations/indels within the targeted region. No wild-type allele was detected. S2: A) Growth curves of 786-0 Vec and HAF overexpressing CCRCC cells in normoxia. B) Normalized densitometry values of Western blots shown in Fig. 5A S3: Western blot showing expression levels of total PDGFR in ACHN, 786-0 and Caki-1 cells. S4: Western blot showing dose-dependent effect of SCH772984 on levels of phosphorylated and total p42/p44 ERK, and their downstream targets, the p90 ribosomal S6 kinases (RSK1/2/3) in 786-0 cells in normoxia. S5: Quantitation of HAF nuclear positivity in CCRCC tumor microarray comprising tumor cores of patients treated with sorafenib. Representative tumor cores image with low and high HAF levels with Aperio quantitation of HAF staining (given by % positivity) are shown. S6: HAF (SART1) expression data from TCGA-KIRC with associated survival data and HAF expression level group stratification. To show survival in months (in Figure 7), survival in days was divided by an average of 30 days per month. S7: A) Western blot showing levels of phosphorylated and total p70 ribosomal S6 protein (S6 ribosomal protein) in 786-0 cells stably expressing HAF or HAF DM after 24 hours' exposure to normoxia or hypoxia. B) Impact of 24 hours' rapamycin treatment on levels of phospho- and total mTOR, S6RP and ERK in 786-0 cells stably expressing empty vector or HAF.

Supplementary Table from Gene Body Methylation of the Lymphocyte-Specific Gene CARD11 Results in Its Overexpression and Regulates Cancer mTOR Signaling

Low oxygen or hypoxia is a feature of all solid tumors and has been associated with aggressive disease. Here, we describe a novel mechanism for the hypoxia-dependent degradation of the Ras-GTPase–activating protein neurofibromin, by hypoxia-associated factor (HAF). We have previously characterized HAF as an oxygen-independent ubiquitin ligase for HIF-1α. Here, we show that HAF promotes neurofibromin ubiquitination and degradation independently of oxygen and pVHL, resulting in Ras–ERK pathway activation. Hypoxia enhanced HAF:neurofibromin binding independently of HAF-SUMOylation, whereas HAF knockdown increased neurofibromin levels primarily in hypoxia, supporting the role of HAF as a hypoxia-specific neurofibromin regulator. HAF overexpression increased p-ERK levels and promoted resistance of clear cell kidney cancer (ccRCC) cells to sorafenib and sunitinib in both normoxia and hypoxia. However, a greater-fold increase in sorafenib/sunitinib resistance was observed during hypoxia, particularly in pVHL-deficient cells. Intriguingly, HAF-mediated resistance was HIF-2α–dependent in normoxia, but HIF-2α–independent in hypoxia indicating two potential mechanisms of HAF-mediated resistance: a HIF-2α–dependent pathway dominant in normoxia, and the direct activation of the Ras–ERK pathway through neurofibromin degradation dominant in hypoxia. Patients with ccRCC with high HAF transcript or protein levels showed significantly decreased overall survival compared with those with low HAF. Thus, we establish a novel, nonmutational pathway of neurofibromin inactivation through hypoxia-induced HAF-mediated degradation, leading to Ras–ERK activation and poor prognosis in ccRCC.Implications:We describe a novel mechanism of neurofibromin degradation induced by hypoxia that leads to activation of the prooncogenic Ras–ERK pathway and resistance to therapy.

Supplementary Figures 1-7

CCR Translation for This Article from Rapid Angiogenesis Onset after Discontinuation of Sunitinib Treatment of Renal Cell Carcinoma Patients

Figure legends for Figures S1-S10

Chromophobe renal cell carcinoma (ChRCC) is characterized by major changes in chromosomal copy number (CN). No model is available to precisely elucidate the molecular drivers of this tumor type. HNF1B is a master regulator of gene expression. Here, we report that the transcription factor HNF1B is downregulated in the majority of ChRCC and that the magnitude of HNF1B loss is unique to ChRCC. We also observed a strong correlation between reduced HNF1B expression and aneuploidy in ChRCC patients. In murine embryonic fibroblasts or ACHN cells, HNF1B deficiency reduced expression of the spindle checkpoint proteins MAD2L1 and BUB1B, and the cell-cycle checkpoint proteins RB1 and p27. Furthermore, it altered the chromatin accessibility of Mad2l1, Bub1b, and Rb1 genes and triggered aneuploidy development. Analysis of The Cancer Genome Atlas database revealed TP53 mutations in 33% of ChRCC where HNF1B expression was repressed. In clinical specimens, combining HNF1B loss with TP53 mutation produced an association with poor patient prognosis. In cells, combining HNF1B loss and TP53 mutation increased cell proliferation and aneuploidy. Our results show how HNF1B loss leads to abnormal mitotic protein regulation and induction of aneuploidy. We propose that coordinate loss of HNF1B and TP53 may enhance cellular survival and confer an aggressive phenotype in ChRCC. Cancer Res; 77(19); 5313–26. ©2017 AACR.

XLSX file - 12K, Predicted percentages of samples having FGFR2 IIIc as the major isoform in multiple cancers

XLSX file - 41K, Top candidate exons differentially expressed between normal kidney and ccRCCs

PDF file - 318K, Validation of FGFR2-IIIc isoform switch in ccRCC tumor samples and cell lines by PCR sequencing

PDF file - 497K, Gene copy number profiles of individual FGFR2-IIIb-1/2 RCCs and FGFR2-IIIc ccRCCs

PDF file - 44K

Supplemental Methods 1. Exome sequencing pipeline Supplemental Methods 2. RNA seq pipeline Supplemental Methods 3. DNA methylation profiling pipeline Supplemental Methods 4. Fluorescence in situ hybridization Supplemental Methods 5. TCGA samples with possible copy neutral loss of heterozygosity in 3p21 and 3p25 regions

PDF file - 431K, FGFR2 promoter DNA methylation near exon 1 correlated with reduced FGFR2 expression

Supplemental Figure Legends

Working model.

Knockdown of Hnf1b led to polyploidy, CIN and checkpoint protein expression reduction in immortalized MEFs.

PDF file - 420K, Correlation of ESRP1/GRHL2 gene expression level to FGFR2 gene expression level in normal kidney and ccRCC

PDF file - 604K, Alternative splicing of the FGFR2 mRNA in normal kidney and clear cell renal cell carcinomas (ccRCC).

Purpose: Sarcomatoid renal cell carcinoma (SRCC) ranks among the most aggressive clinicopathologic phenotypes of RCC. However, the paucity of high-quality, genome-wide molecular examinations of SRCC has hindered our understanding of this entity.Experimental Design: We interrogated the mutational, copy number, and transcriptional characteristics of SRCC and compared these data with those of nonsarcomatoid RCC (RCC). We evaluated whole-exome sequencing, single-nucleotide polymorphism, and RNA sequencing data from patients with SRCC (n = 65) and RCC (n = 598) across different parent RCC subtypes, including clear-cell RCC, papillary RCC, and chromophobe RCC subtypes.Results: SRCC was molecularly discrete from RCC and clustered according to its parent RCC subtype, though with upregulation of TGFβ signaling across all subtypes. The epithelioid (E-) and spindled (S-) histologic components of SRCC did not show differences in mutational load among cancer-related genes despite a higher mutational burden in S-. Notably, sarcomatoid clear-cell RCC (SccRCC) showed significantly fewer deletions at 3p21-25, a lower rate of two-hit loss for VHL and PBRM1, and more mutations in PTEN, TP53, and RELN compared with ccRCC. A two-hit loss involving VHL predicted for ccRCC and a better prognosis, whereas mutations in PTEN, TP53, or RELN predicted for SccRCC and worse prognosis.Conclusions: SRCC segregates by parent subtype, and SccRCC has a fundamentally different early molecular pathogenesis, usually lacking the classic 3p21-25 deletion and showing distinctive mutational and transcriptional profiles. These features prompt a more precise molecular classification of RCC, with diagnostic, prognostic, and therapeutic implications. Clin Cancer Res; 23(21); 6686–96. ©2017 AACR.See related commentary by Bergerot et al., p. 6381

FLCN mRNA level was not decreased in sporadic ChRCC.

Purpose: We aim to identify tumor-specific alternative splicing events having potential applications in the early detection, diagnosis, prognosis, and therapy for cancers.Experimental Design: We analyzed RNA-seq data on 470 clear cell renal cell carcinomas (ccRCC) and 68 kidney tissues to identify tumor-specific alternative splicing events. We further focused on the fibroblast growth factor receptor 2 (FGFR2) isoform switch and characterized ccRCCs expressing different FGFR2 isoforms by integrated analyses using genomic data from multiple platforms and tumor types.Results: We identified 113 top candidate alternatively spliced genes in ccRCC. Prominently, the FGFR2 gene transcript switched from the normal IIIb isoform (“epithelial”) to IIIc isoform (“mesenchymal”) in nearly 90% of ccRCCs. This switch is kidney specific as it was rarely observed in other cancers. The FGFR2-IIIb ccRCCs show a transcriptome and methylome resembling those from normal kidney, whereas FGFR2-IIIc ccRCCs possess elevated hypoxic and mesenchymal expression signatures. Clinically, FGFR2-IIIb ccRCCs are smaller in size, of lower tumor grade, and associated with longer patient survival. Gene set enrichment and DNA copy number analyses indicated that FGFR2-IIIb ccRCCs are closely associated with renal oncocytomas and chromophobe RCCs (chRCC). A reexamination of tumor histology by pathologists identified FGFR2-IIIb tumors as chRCCs and clear cell papillary RCCs (ccpRCC).Conclusions: FGFR2 IIIb RCCs represent misdiagnosed ccRCC cases, suggesting FGFR2 isoform testing can be used in the diagnosis of RCC subtypes. The finding of a prevalent isoform switch of FGFR2 in a tissue-specific manner holds promise for the future development of FGFR2-IIIc as a distinct early detection biomarker and therapeutic target for ccRCC. Clin Cancer Res; 19(9); 2460–72. ©2013 AACR.

PDF file - 461K, Isoform switch in FGFR1, 3 is not detected in ccRCC, and FGFR2 switch is not found in breast cancer

Additional methods

Figure S1. Biphasic histologic components of sarcomatoid renal cell carcinoma. The macrodissected paired epithelioid or carcinomatous (E) and spindled or sarcomatoid (S) components of clear cell RCC (upper panel), Papillary RCC (middle panel), and chromophobe RCC (lower panel). H&E stain, scale bar 200 �m. Figure S2. Sarcomatoid ccRCC shows fewer VHL deletions. (A) Clear cell RCC (H&E stain, scale bar 100 µm) with (B) Fluorescence in situ hybridization (FISH) image showing paired CEN3q signals (green) and a single VHL signal (red). (C) Sarcomatoid ccRCC (H&E stain, scale bar 100 µm) with (D) FISH image showing balanced CEN3q (green) and VHL (red) signals. (E) Box plot showing significantly higher VHL/3q ratios associated with sarcomatoid histology, P

XLSX file - 75K, Computational prediction of FGFR2 IIIb/c isoforms in normal kidney and ccRCC

Supplementary Figure S1 from Cytoplasmic Sequestration of p27 via AKT Phosphorylation in Renal Cell Carcinoma

Depletion of Hnf1b did not affect time from NEB to anaphase.

Loss of both Bub1b and Rb1 produced a polynuclear phenotype and larger cell size.

Purpose: To investigate the angiogenic changes in primary tumor tissue of renal cell carcinoma (RCC) patients treated with VEGF-targeted therapy.Experimental Design: Phase II trials of VEGF pathway–targeted therapy given before cytoreductive surgery were carried out with metastatic RCC patients with the primary tumor in situ to investigate the necessity of nephrectomy. Primary tumor tissues were obtained and assessed for angiogenesis parameters. Results were compared with similar analyses on untreated tumors.Results: Sunitinib or bevacizumab pretreatment resulted in a significant reduction of microvessel density in the primary tumor. Also, an increase in vascular pericyte coverage was found in sunitinib-pretreated tumors, consistent with efficient angiogenesis inhibition. Expression of several key regulators of angiogenesis was found to be suppressed in pretreated tissues, among which VEGFR-1 and VEGFR-2, angiopoietin-1 and angiopoietin-2 and platelet-derived growth factor-B. In addition, apoptosis in tumor and endothelial cells was induced. Interestingly, in sunitinib-pretreated tissues a dramatic increase of the number of proliferating endothelial cells was observed, which was not the case in bevacizumab-pretreated tumors. A positive correlation with the interval between halting the therapy and surgery was found, suggesting a compensatory angiogenic response caused by the discontinuation of sunitinib treatment.Conclusion: This study describes, for the first time, the angiostatic response in human primary renal cancers at the tissue level upon treatment with VEGF-targeted therapy. Discontinuation of treatment with tyrosine kinase inhibitors leads to accelerated endothelial cell proliferation. The results of this study contribute important data to the ongoing discussion on the discontinuation of treatment with kinase inhibitors. Clin Cancer Res; 18(14); 3961–71. ©2012 AACR.

Table S7a. Ingenuity Pathway Analysis of SRCC vs RCC Table S7b. Ingenuity Pathway Analysis of SccRCC vs advanced stage ccRCC Table S7c. Ingenuity Pathway Analysis of E vs S components of ccRCC

Postulated HNF1B consensus-binding sequences in Mad1l1, Mad2l1, Bub1b, Rb1 and Cdkn1b genes and affected postulated substrates' chromatin access after depletion of Hnf1b.

Purpose:Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel–Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC.Experimental Design:To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC.Results:HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs.Conclusions:These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

Purpose: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy.Experimental Design: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated.Results: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not.Conclusions: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.

Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, driving constitutive activation of hypoxia-inducible factor-1 (HIF1) and HIF2. However, whereas HIF1 has a tumor-suppressor role, HIF2 plays a distinct role in driving CRCC. In this study, we show that the HIF1α E3 ligase hypoxia-associated factor (HAF) complexes with HIF2α at DNA to promote HIF2-dependent transcription through a mechanism relying upon HAF SUMOylation. HAF SUMOylation was induced by hypoxia, whereas HAF-mediated HIF1α degradation was SUMOylation independent. HAF overexpression in mice increased CRCC growth and metastasis. Clinically, HAF overexpression was associated with poor prognosis. Taken together, our results show that HAF is a specific mediator of HIF2 activation that is critical for CRCC development and morbidity. Cancer Res; 75(2); 316–29. ©2014 AACR.

XLSX file - 8KB, Missense Mutations of VHL in Renal Cell Carcinoma in the TCGA dataset.

XLSX file - 13KB, Folding Free Energy (deltadeltaG) of VHL missense mutations computed using the PoPMuSiC program.

Supplemental methods. A, B) Sequences for the A) P117 Luc, and B) P117 HRE reporters containing the HAF binding site, the hypoxia responsive element (HRE) and the HIF ancillary sequence (HAS), with the restriction enzyme sites used for ligation into the pGL4.17 construct. C) PCR primers used in ChIP assays. Supplementary data S1. A) Quantitative TaqMan RT-PCR showing the effect of transient HAF overexpression on the transcription of HIF-2 target genes in pVHL-deficient A498 CRCC cells B) Quantitative TaqMan RT-PCR showing the effect of transient HAF overexpression on the transcription of OCT-3/4. SOX2 and NANOG in normoxia and 24hours' hypoxia in A498 CRCC cells. Supplementary data S2. A) Effects of HAF overexpression on luciferase activity of co-transfected P117 Luc or P117 containing a deletion of the HAF site (P117D), in PANC1 cells in normoxia. B) Western blot showing knockdown specificity of siRNAs targeting HIF-2α (siH2_1, siH2_2) or HAF (siHAF_1, siHAF_2) when transfected with the P117 reporter in 786-0 cells. Supplementary data S3. A) Effect of hypoxia on SUMO-2/3 modification of HAF in 786-0 cells. B) Effect of hypoxia on SUMO-1 modification of HAF in PANC-1 cells. C) Western blot showing immunoprecipitation of HIF-2α from lysates of PANC-1 cells. Supplementary data S4. A-B) Immunocytochemistry confirming specificities of A) HAF, and B) HIF-2α antibodies. Supplementary data S5. A) Growth curve of HAF overexpressing 786-0 cells in normoxia. B) Western blot showing GFP expression in suspected lesions observed in the GI tracts of mice bearing renal orthotopic 786-0 HAF or vector expressing CRCC cells. Supplementary data S6. A) Table showing multiple disease parameters of the patients represented on the CRCC TMA. B) Scatterplot showing correlation between nuclear and cytoplasmic HAF localization in CRCC TMA. Supplementary data S7. Kaplan-Meier curves showing relationship between good versus intermediate Motzer criteria in grade III/IV CRCC. Supplementary data S8. A-B) Kaplan-Meier curves showing relationships between levels of A) HIF-2α or B) HIF-1α and progression free survival (PFS) in grade III/IV CRCC.

Aberrant von Hippel Lindau (VHL) protein function is the underlying driver of VHL-related diseases, including both sporadic and inherited clear cell renal cell carcinoma (ccRCC). About one third of VHL mutations are missense point mutations, with R167Q being the most common VHL point mutation in hereditary VHL disease. Although it has been studied extensively, the ability of VHL-R167Q to downregulate hypoxia-inducible factor 2α (HIF2α) is still controversial. In addition, the manner in which the mutation contributes to tumorigenesis is not fully understood. No therapeutic approach is available to target VHL-R167Q and similar missense point mutations. We analyzed VHL-R167Q proteostasis and function at normoxia, at hypoxia with different oxygen pressure, and in a xenograft mouse model. We showed that the protein levels of VHL-R167Q dictate its ability to downregulate HIF2α and suppress tumor growth. Strikingly, the proteasome inhibitors bortezomib and carfilzomib, which are currently in clinical use, stabilize VHL-R167Q and increase its ability to downregulate HIF2α. VHL-R167Q binds elongin C and elongin B with considerably less avidity than wild-type VHL does but retains residual capacity to generate a VHL-elongin C-elongin B complex, downregulate HIF2α, and suppress tumorigenesis, which could be rescued by increase of VHL-R167Q levels. Finally, we used in silico approaches and identified other missense VHL mutants in addition to VHL-R167Q that might be rescued by similar strategies. Thus, our studies revealed detailed information describing how VHL-R167Q contributes to tumorigenesis and identified a potential targeted therapy for ccRCC and other VHL-related disease in patients carrying VHL-R167Q or similar missense mutations. Cancer Res; 74(11); 3127–36. ©2014 AACR.

Investigations into the function of nonpromoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated nonpromoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 260 genes exhibiting a correlation of > 0.5 between gene body methylation and gene expression in at least one tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in kidney renal cell carcinoma (KIRC) and lung adenocarcinoma (LUAD) tumor tissues in comparison with respective control samples; and is significantly associated with lower overall survival in KIRC. Contrary to its canonical function in lymphocyte NFκB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy. Furthermore, demethylation of a CpG island within the gene body of CARD11 decreases gene expression. Collectively, our study highlights how DNA methylation outside the promoter region can impact tumor progression.Implications:Our study describes a novel regulatory role of gene body DNA methylation–dependent CARD11 expression on mTOR signaling and its impact on tumor progression.

Rationale: Immune checkpoint blockade (ICB) demonstrates durable clinical benefit only in a minority of renal cell carcinoma (RCC) patients. Identifying molecular features that determine response and developing approaches to enhance the response remain an urgent clinical need. Targeting S phase DNA damage repair (S-DDR) network has been reported to activate cGAS signaling and convert a non-immunogenic tumor microenvironment to an immunogenic tumor microenvironment, thus improving immunotherapy response in breast cancer and small cell lung cancer. SETD2 has been known to be required for ATM-mediated DNA damage response, and we hypothesize that SETD2 loss confers greater dependence on ATR activity and provides vulnerability to ATR inhibitors, which may further potentiate the response to immunotherapy. Methods: Applying molecular, cellular, and bioinformatic approaches, we comprehensively investigated the effects of SEDT2/Setd2 loss on the cytosolic DNA damage response pathway, the cGAS-mediated cytosolic DNA sensing pathway, the tumor immune microenvironment, and immunotherapy response in RCC cell lines, in the Renca-BALB/c immune competent murine model, and in RCC patients cohorts with or without ICB therapy. Results: We found that, in multiple RCC cell lines, pharmacological inhibition of the ATR-CHK1 axis activated the cGAS-IRF3-dependent cytosolic DNA sensing pathway, resulting in inflammatory cytokine expression. SETD2 mutated RCC cell lines or tumor samples were associated with preferential ATR-CHK1 activation over ATM-CHK2 activation. SETD2 knockdown promoted the cytosolic DNA sensing pathway and conferred greater sensitivity to ATR inhibition. In murine Renca tumors, Setd2 knockdown and the ATR inhibitor VE822 synergistically activated the cytosolic DNA sensing pathway, immune cell infiltration, and immune checkpoint protein expression. Setd2 deficient Renca tumors demonstrated greater vulnerability to ICB monotherapy or in combination with ATRi than Setd2 proficient tumors. SETD2 mutations were associated with a higher response rate and prolonged overall survival in ICB-treated RCC patients, but not in non-ICB-treated RCC patients. Conclusion: This study reveals that SETD2/Setd2 loss and ATR inhibitor synergize to enhance cGAS signaling and RCC tumor immune responsiveness, and provides mechanism-based guidance to develop more personalized combination therapy regimens for RCC patients with SETD2 mutations. Citation Format: Xiande Liu, Yanting Zhang, Xuesong Zhang, Daniel McGrail, Truong Lam, Anh Hoang, Elshad Hasanov, Ganiraju Manyam, Christine B. Peterson, Haifeng Zhu, Shwetha V. Kumar, Rehan Akbani, Nizar M. Tannir, Guang Peng, Eric Jonasch. SETD2 loss and ATR inhibition synergistically promote cGAS signaling and immunotherapy response in renal cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4153.

Purpose: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel–Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. Experimental Design: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. Results: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. Conclusions: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

Low oxygen or hypoxia is a feature of all solid tumors and has been associated with aggressive disease. Here, we describe a novel mechanism for the hypoxia-dependent degradation of the Ras-GTPase–activating protein neurofibromin, by hypoxia-associated factor (HAF). We have previously characterized HAF as an oxygen-independent ubiquitin ligase for HIF-1α. Here, we show that HAF promotes neurofibromin ubiquitination and degradation independently of oxygen and pVHL, resulting in Ras–ERK pathway activation. Hypoxia enhanced HAF:neurofibromin binding independently of HAF-SUMOylation, whereas HAF knockdown increased neurofibromin levels primarily in hypoxia, supporting the role of HAF as a hypoxia-specific neurofibromin regulator. HAF overexpression increased p-ERK levels and promoted resistance of clear cell kidney cancer (ccRCC) cells to sorafenib and sunitinib in both normoxia and hypoxia. However, a greater-fold increase in sorafenib/sunitinib resistance was observed during hypoxia, particularly in pVHL-deficient cells. Intriguingly, HAF-mediated resistance was HIF-2α–dependent in normoxia, but HIF-2α–independent in hypoxia indicating two potential mechanisms of HAF-mediated resistance: a HIF-2α–dependent pathway dominant in normoxia, and the direct activation of the Ras–ERK pathway through neurofibromin degradation dominant in hypoxia. Patients with ccRCC with high HAF transcript or protein levels showed significantly decreased overall survival compared with those with low HAF. Thus, we establish a novel, nonmutational pathway of neurofibromin inactivation through hypoxia-induced HAF-mediated degradation, leading to Ras–ERK activation and poor prognosis in ccRCC. Implications: We describe a novel mechanism of neurofibromin degradation induced by hypoxia that leads to activation of the prooncogenic Ras–ERK pathway and resistance to therapy.

Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability, but lacks mutations in canonical DNA damage response (DDR) genes. Bi-allelic loss of VHL is a near ubiquitous, truncal event in ccRCC development. Herein, we performed preclinical and clinical analysis to understand how VHL loss impacts DDR pathway activity and how specific DDR deficiencies (DDR-D) may be targeted in ccRCC. Methods: We performed DNA whole exome sequencing (WES) and reverse phase protein array (RPPA) early stage ccRCC tumors. All patients were consented under an IRB-approved protocol for the use of tissue. Transcriptomic and protein analyses of select DDR pathways was performed on in silico data (TCGA). DDR functional assays and ATR inhibitor treatment were performed on preclinical cell line models, and in vivo ATR inhibitor treatment in ccRCC xenografts with varying VHL status. Results: Bi-allelic loss of VHL via mutation in one allele and copy number loss of chromosome 3p was observed in 13/13 patient tumors. Mutations in other genes associated with ccRCC and/or canonical DDR genes were not observed. Despite VHL loss being the only driver mutation, these early stage tumors displayed tumor mutational burden (TMB) similar to ccRCC across all tumor stages (1.1 ± 0.5 mut/Mb). Proteomic analysis of patient tumor versus normal tissue (N=12) revealed significantly different (P Conclusions: Here we show in patient tissue and preclinical models that VHL biallelic loss results in genomic instability and functional impairment of ATM kinase activity; furthermore, we provide preclinical evidence for selectively targeting the ATR signaling cascade in patients with VHL-deficient kidney cancer. Clinical trials of ATR inhibitor based therapy in patients with advanced cancer, including ccRCC, are underway (e.g. NCT04266912). Citation Format: Patrick G. Pilie, Lijun Zhou, Yan-Ting Zhang, Christine Peterson, Daniel McGrail, Yang Peng, Guang Peng, Xiande Liu, Xuesong Zhang, Eric Jonasch. VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition [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 LB039.

Chromophobe renal cell carcinoma (ChRCC) is characterized by major changes in chromosomal copy number (CN). No model is available to precisely elucidate the molecular drivers of this tumor type. HNF1B is a master regulator of gene expression. Here, we report that the transcription factor HNF1B is downregulated in the majority of ChRCC and that the magnitude of HNF1B loss is unique to ChRCC. We also observed a strong correlation between reduced HNF1B expression and aneuploidy in ChRCC patients. In murine embryonic fibroblasts or ACHN cells, HNF1B deficiency reduced expression of the spindle checkpoint proteins MAD2L1 and BUB1B, and the cell-cycle checkpoint proteins RB1 and p27. Furthermore, it altered the chromatin accessibility of Mad2l1, Bub1b, and Rb1 genes and triggered aneuploidy development. Analysis of The Cancer Genome Atlas database revealed TP53 mutations in 33% of ChRCC where HNF1B expression was repressed. In clinical specimens, combining HNF1B loss with TP53 mutation produced an association with poor patient prognosis. In cells, combining HNF1B loss and TP53 mutation increased cell proliferation and aneuploidy. Our results show how HNF1B loss leads to abnormal mitotic protein regulation and induction of aneuploidy. We propose that coordinate loss of HNF1B and TP53 may enhance cellular survival and confer an aggressive phenotype in ChRCC. Cancer Res; 77(19); 5313–26. ©2017 AACR.

In the original version of [this article][1] ([1][2]), the wrong files were supplied for Figs. 5B and C. The figures and related article text have been corrected in the latest online HTML and PDF versions of this article. The authors regret these errors. 1. 1.[↵][3]1. Cowman SJ, 2. Fuja

PBRM1 is a subunit of a SWI/SNF chromatin remodeling complex, and more than 30% of clear cell RCCs show PBRM1 mutations. The function of PBRM1 in tumor immune microenvironment, tumor prognosis and response to immune checkpoint therapies still remain unclear. Here we found that Pbrm1 knockout in mouse Renca RCC cells decreased the activity of IFNγ-STAT1 signaling pathway and consequently the expression of chemokines that recruit effector CD8+ T cells. Pbrm1 deficient murine RCC tumors showed reduced infiltration of CD8+ T cells, which was associated with less PD-1 expression. Pbrm1 deficient tumors demonstrated longer latency but they were more resistant to anti-PD1 treatment. This study provided mechanistic insights and therapeutic guideline to manage ccRCC associated with PBRM1 inactivation. Citation Format: Xian-De Liu, Wen Kong, Anh Hoang, Xuesong Zhang, Lijun Zhou, Patrick G. Pilie, Sevinj Isgandrova, Margie M. Moczygemba, Eric Jonasch. PBRM1 loss promotes resistance to immunotherapy in RCC [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B48.

PBRM1 is a subunit of SWI/SNF complex that regulates chromatin accessibility for transcription factors, and more than 30% of clear cell renal cell carcinoma (RCC) contain PBRM1 mutations. Nivolumab (anti-PD-1) showed profound and prolonged response only in a subset of patients with advanced RCC. The impact of PBRM1 loss on response to immunotherapy in RCC patients still remains contradictory. It is important to develop immune competent murine RCC models that can precisely isolate the influence of PBRM1 loss on the tumor microenvironment. IFNγ-related gene expression profiles have been reported to predict response to anti-PD-1 blockade. We analyzed the effect of PBRM1 on the IFNγ STAT1 signaling pathway in isogenic murine RCC Renca cells. We observed that Pbrm1 knockout reduced IFNγ-induced STAT1 phosphorylation, and consequently the expression of downstream genes including Irf1, Cxc9 and Stat1 itself. Considering the critical role of CXCL9 in T cell recruitment, we found that Pbrm1 deficiency reduced accessibility to the Cxcl9 promoter by STAT1 and BRG1, the ATP-dependent enzyme subunit of the SWI/SNF complex. Furthermore, a bioinformatics analysis of the Cancer Genome Atlas of human RCC and our murine RCC RNAseq database showed that PBRM1inactivation was associated with reduced expression of an IFNγ-related mRNA profile, including checkpoint genes CTLA4 and PDCD1. These results revealed that PBRM1 loss reduced IFNγ signaling pathway activation and chromatin accessibility by the transcription factor STAT1. Since Pbrm1 deficiency reduced IFNγ-STAT1 activity and consequently Cxcl9 expression, we then compared the immune cell infiltration in both Pbrm1 proficient and deficient Renca tumors in BalB/C mice. As expected, Pbrm1 deficient tumors harbored fewer infiltrating CD3+ and CD8+ T cells, and expressed less PD-1. It has previously been reported that response to immune checkpoint therapy requires pre-existing T cells, and their intrinsic anti-tumor activity potentiated by blocking immune checkpoints. We found that Pbrm1 deficient tumors demonstrated longer latency but more resistance to immunotherapy with PD-1 blockade. We previously showed that antiangiogenic therapy increased T cell infiltration associated with PD-L1 upregulation, and proposed that antiangiogenic agents could promote the response to checkpoint blockade. Here we confirmed that antiangiogenic agent axitinib reduced Pbrm1 proficient tumor growth, and its combination with anti-PD-1 blockade exhibited synergistic efficacy. In contrast, neither axitinib monotherapy nor combination therapy with PD-L1 antibody exerted obvious control on Pbrm1 deficient tumor, indicating patients with PBRM1 functional mutation were more resistant to immunotherapy and antiangiogenic therapy. This study provides mechanistic insights into therapeutic response of PBRM1 mutated RCC. Citation Format: Xiande Liu, Wen Kong, Christine Peterson, Anh Hoang, Xuesong Zhang, Lijun Zhou, Patrick Pilie, Truong Lam, Haifeng Zhu, Sevinj Isgandrova, Margie Moczygemba, Eric Jonasch. PBRM1 loss reduces IFNγ-STAT1 activity and promotes resistance to immunotherapy and antiangiogenic therapy in renal cell carcinoma [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 945.

Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability across all tumor stages, indicative of increased replicative stress and defects in DNA damage response (DDR) pathways including homologous repair (HR); however ccRCC does not display mutations in canonical DDR genes. We hypothesized that biallelic VHL loss is sufficient to cause HR deficiency (HRD) in ccRCC via direct regulation of DDR pathways. Experimental Procedures: We performed whole-exome (WES) sequencing of 15 small ccRCC tumors. We performed in silico genomic, transcriptomic, and proteomic analysis of tumors in KIRC TCGA to assess for HRD, using published HRD signatures. We assessed HR efficiency as a product of biallelic VHL loss in engineered cell line models. We studied the VHL-dependent DNA damage repair molecular mechanisms with cell line models, by HR and nonhomologous end joining (NHEJ) using reporter gene assays. We then assessed the status of TIP60 activation in Vhl deficient murine embryo fibroblasts, isogenic VHL deficient and proficient cell lines by determining DSB induced tip60 acetylation and foci formation. DSB induced HR and NHEJ activation were analyzed by assessing the induction of marker proteins including γH2AX, RAD51, and 53BP1. To examine VHL-protein interaction, green-fluorescent protein (GFP)-trap with GFP-tagged VHL and co-immunoprecipitation with specific antibodies were used. Results: 15/15 early stage ccRCC had biallelic VHL mutations and approximately 100 additional mutations per tumor, but no driver mutations typically associated with HRD. Nonetheless, in silico analysis showed 67% of KIRC TCGA displayed an HRD gene signature, and this signature was significantly higher in stage I disease (p=2.21e-08). Patients with VHL-mutated tumors were more commonly HRD than HRI (p=0.03), with frameshift/nonsense variants in VHL more likely to result in HRD than missense variants (p=0.02). Multivariate analysis showed HRD predicted for better overall survival compared to an HR intact (HRI) state (p Citation Format: Lijun Zhou, Patrick G. Pilie, Christine B. Peterson, Xian-de Liu, Xuesong Zhang, Eric Jonasch. Tip60 dependent DNA homologous recombination repair is impaired in VHL-deficient clear cell renal cell carcinoma [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 1364.