PBRM1 Loss Research Articles

PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

SUMMARYPBRM1, a subunit of the PBAF coactivator complex that transcription factors use to activate target genes, is genetically inactivated in almost all clear cell renal cell cancers (RCCs). Using unbiased proteomic analyses, we find that PAX8, a master transcription factor driver of proximal tubule epithelial fates, recruits PBRM1/PBAF. Reverse analyses of the PAX8 interactome confirm recruitment specifically of PBRM1/PBAF and not functionally similar BAF. More conspicuous in the PAX8 hub in RCC cells, however, are corepressors, which functionally oppose coactivators. Accordingly, key PAX8 target genes are repressed in RCC versus normal kidneys, with the loss of histone lysine-27 acetylation, but intact lysine-4 trimethylation, activation marks. Re-introduction of PBRM1, or depletion of opposing corepressors using siRNA or drugs, redress coregulator imbalance and release RCC cells to terminal epithelial fates. These mechanisms thus explain RCC resemblance to the proximal tubule lineage but with suppression of the late-epithelial program that normally terminates lineage-precursor proliferation.

Undifferentiated large cell/rhabdoid carcinoma presenting in the intestines of patients with concurrent or recent non-small cell lung cancer (NSCLC): clinicopathologic and molecular analysis of 14 cases indicates an unusual pattern of dedifferentiated metastases

Undifferentiated carcinoma metastatic to the bowel is uncommon in surgical pathology practice and might be confused with primary gastrointestinal carcinoma, melanoma, lymphoma, and others. We present 14 cases of uni- (n = 9) or multifocal (n = 5) undifferentiated large cell/rhabdoid carcinoma presenting in the bowel of patients with concurrent (n = 9) or recent (diagnosed 1 to 25 months earlier; median, 4) non-small cell lung cancer (NSCLC). Patients were 6 females and 8 males, aged 52 to 85 years. Primary NSCLC was verified histologically in 10 cases and by imaging in 4. The undifferentiated histology was present in the lung biopsy in 4/10 patients (as sole pattern in 3 and combined with adenocarcinoma in 1) and was limited to the intestinal metastases in the remainder. PDL1 was strongly expressed in 7/9 cases (CPS: 41 to 100). Loss of at least one SWI/SNF subunit was detected in 7/13 cases (54%). SMARCA2 loss (n = 6) was most frequent and was combined with SMARCA4 loss in one case. PBRM1 loss was observed in one tumor. Successful molecular testing of 11 cases revealed BRAF mutations in 4 (3 were non-V600E variants), KRAS mutations in 3, and wildtype in 4. None had EGFR mutations. Analysis of 4 paired samples revealed concordant KRAS (2) and BRAF (1) mutations or wildtype (1). Our study indicates that undifferentiated carcinoma within the intestines of patients with concurrent/recent NSCLC represents dedifferentiated metastasis from the NSCLC. Recognition of this unusual presentation is cardinal to avoid misdiagnosis with inappropriate therapeutic and prognostic implications.

Abstract 1312: Identifying the genomic correlates of clinical benefit (CB) from immunotherapies (IO) and vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF-TKI) in metastatic renal cell carcinoma (mRCC)

Abstract Background: Along with the expanding treatment landscape of mRCC, a substantial need for ways to predict individual patients' (pts) benefit from different therapies has emerged. Recent studies highlight the clinical significance of tumor-specific genomic alterations, revealing a prognostic and/or predictive role of PBRM1 mutations with IO in mRCC (Miao et al Nature 2018). Employing a large institutional database, we aimed to identify genomic correlates of CB from IO and VEGF-TKIs in mRCC. Methods: Consecutive pts who underwent genomic profiling (GP) as part of routine clinical care at the City of Hope Comprehensive Cancer Center were retrospectively identified. GP included the GEM ExTra assay, a clinical tumor-normal whole exome sequencing and tumor whole transcriptome sequencing test performed at Ashion Analytics (Phoenix, AZ), a CAP-accredited, CLIA-certified laboratory. The clinical database included detailed information regarding demographics, treatment, response and survival outcomes. Pts who had complete response (CR), partial response (PR) or stable disease (SD) for >6 months were considered as having CB. Progressive disease (PD) as best response was considered no clinical benefit (NCB). Genomic findings were compared between pts with CB and NCB in the IO and VEGF-TKI cohorts. Results: Among the 58 (45:13 M:F) pts, 17 received sequencing treatment involving both a VEGF-TKI and IO, resulting in 32 pts in the IO cohort and 43 pts in the VEGF-TKI cohort. The most commonly used IO and VEGF-TKIs were nivolumab (59%) and sunitinib (40%). CB rate and median progression free survival were 59.4% (CR: 3.1%, PR: 18.8%, SD: 37.5%) and 15.6 months (95%CI NR-NR) in the IO cohort, and 86% and 14.2 months (95%CI 9.0 - 18.5) in the VEGF-TKI cohort. The most frequently detected alterations in the overall cohort were in VHL (64%), PBRM1 (38%), SETD2 (24%), KDM5C (17%) and TERT (12%). Interestingly, TERT promoter mutations and PBRM1 mutations were found to be mutually exclusive. In both IO and VEGF-TKI cohorts, tumor mutational burden did not differ between CB and NCB pts. No single genes were significantly associated with CB from VEGF-TKIs. While PBRM1 loss of function (LOF) was more common in IO pts with CB, and SETD2, KDM5C, TP53 alteration were more common in IO pts with NCB, there was no statistical significance observed (p values > 0.05). TERT promoter mutations were associated with NCB in the IO cohort (p=0.038). TERT promoter mutant tumors did not have CB with IO. Conclusion: Our analysis found that TERT promoter mutations are enriched in pts with NCB from IO and were mutually exclusive with PBRM1 LOF which had previously been shown to be an indicator of IO sensitivity. These results suggest that TERT promoter mutations may be a negative predictor of IO outcomes, which warrants future investigations with a larger sample size. Citation Format: Nazli Dizman, Sara Byron, Yung Lyou, Paulo Gustavo Bergerot, JoAnn Hsu, Andre-Philippe Sam, Denise Phan Trieu, Jeffrey Trent, Sumanta Kumar Pal. Identifying the genomic correlates of clinical benefit (CB) from immunotherapies (IO) and vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF-TKI) in metastatic renal cell carcinoma (mRCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1312.

Immune Checkpoint Inhibitors in the Treatment of Renal Cancer: Current State and Future Perspective.

Systemic treatment of renal cancer (RCC) has undergone remarkable changes over the past 20 years with the introduction of immunotherapeutic agents targeting programmed cell death (PD-1)/programmed death-ligand 1 (PD-L1) axis, as a single-agent or combined with anti-CTLA-4 monoclonal antibodies (MoAbs) or a multi-target vascular endothelial growth factor-(VEGF) tyrosine kinase inhibitor (TKI). In this paper, we review the main evidence on the use of Immune Checkpoint Inhibitors (ICIs) for RCC treatment from the first demonstration of activity of a nivolumab single agent in a phase I trial to the novel combination strategies (anti-PD-1 plus anti-CTLA4 or anti-PD-1 plus TKI). In addition, we discuss the use of anti-PD-1/PD-L1 agents in patients with non-clear cells and rare histological subtype RCC. Then, we critically examine the current findings in biomarkers that have been proposed to be prognostic or predictive to the response of immunotherapy including immune gene expression signature, B7-H1 expression, PBRM1 loss of function, PD-L1 expression, frame shift indel count, mutations in bromodomain-containing genes in patients with MiT family translocation RCC (tRCC), high expression of the T-effector gene signature, and a high myeloid inflammation gene expression pattern. To date, a single biomarker as a predictor of response has not been established. Since the dynamic behavior of the immune response and the different impact of ICI treatment on patients with specific RCC subtypes, the integration of multiple biomarkers and further validation in clinical trials are needed.

Exploring Biological Predictive Factors of Progression After Surgery in High-Risk Renal Cell Carcinoma: Results From the French Cohort of the Randomized S-TRAC Trial Patients.

Objective: We aimed to explore biological predictive factors of progression after surgery in nonmetastatic renal cell carcinoma (RCC) using the collected tumors in the French cohort of the randomized S-TRAC trial patients.Patients and Methods: We analyzed the tumors of the French cohort of STRAC that included 44 cases of clear cell RCC (ccRCC) that were collected from six centers. The main objective was to explore biological predictive factors of progression (defined as PFS) to sunitinib. Broad-spectrum analysis including immunohistochemistry, fluorescent in situ hybridization (FISH), comparative genomic hybridization (CGH) array, and transcriptomic analyses were performed on the tumors.Results:Analysis of vascular density showed type 1 vascular stroma corresponding to high vascular density was associated with progression (p < 0.034). Loss of poly bromo-1 expression showed a distinct profile: a highly histopathological aggressive tumor with a marked angiogenic profile (vascular endothelial growth factor overexpression and immature vascular stroma type 2), no PD1 or PDL1 expression, and wild-type (WT) status of the VHL gene. There were 27 chromosome regions gained in patients with progression (on chromosomes 7 and 16, and to a lesser extent 8, 12, 17, 17, 19, 20 corresponding to 605 associated genes) and 10 regions lost in these same patients on chromosomes 8 and 9, and to a lesser extent 2 and 21 corresponding to 25 associated genes.Conclusion:We found that an angiogenic phenotype defined by a high vascular density with a vascular type 2 stroma was a predictive factor of sunitinib resistance. Regardless of adjuvant treatment, chromosomal gains and losses and genomic alterations including PBRM1 loss were associated with worse outcomes.Clinical Trial Registration: ClinicalTrials.gov number, NCT00375674.

Genomic and transcriptomic correlates of clinical benefit from immunotherapy and targeted therapy among patients with metastatic renal cell carcinoma (mRCC).

5076 Background: Previous studies have delineated PBRM1 alterations as a prognostic indicator for response to nivolumab in mRCC (Miao et al Science 2018). However, further clinically-relevant biomarkers remain elusive in mRCC. Herein, we utilized genomic profiling to detect correlates of clinical benefit (CB) among patients (pts) with mRCC receiving VEGF-directed targeted therapy, immunotherapy, or both. Methods: Pts with mRCC who underwent clinical tumor-normal whole exome and whole transcriptome RNA sequencing (Ashion Analytics, Phoenix, AZ) were retrospectively identified at a single institution. Pts’ demographics and clinical variables including treatment type, treatment response, and survival outcomes were collected from an institutional mRCC database. Pts who had received immunotherapy or VEGF-directed therapy (or both) were eligible for analysis. CB was defined as pts who experienced a complete response, partial response, or maintained stable disease for at least 6 months on therapy. Two-tailed Fisher’s exact test was used to assess characteristics of genomic alterations and clinical benefit. Results: Out of 155 mRCC pts with genomic profiling, 58 pts with evaluable response and sufficient follow-up information were included in the analysis. The median age of the cohort was 63 years and 74% were male. The majority (81%) had clear cell histology. 43 pts received targeted therapy and 32 received immunotherapy (17 pts received both). No singular gene was associated with clinical benefit in the targeted therapy cohort. PBRM1 loss of function mutations were more frequent in pts on immunotherapy who experienced CB (p &gt; 0.05). TERT promoter mutations were enriched in pts who experienced no CB on immunotherapy (p = 0.04). Differential gene expression analysis of the transcriptomes found 135 genes that were significantly upregulated in TERT-mutated samples, including SST, CYSLTR2, WNK2, and PTGES (adjusted p-value &lt; 0.05). ENRICHR analysis found that MYC and KAT2A were key transcription factor pathways significantly enriched in pts with TERT mutation who experienced no CB from immunotherapy. Conclusions: These data support previous observations regarding the prognostic nature of PBRM1 in mRCC and offer novel findings associating TERT mutations with a lack of CB from immunotherapy. Our transcriptomic analysis implies that MYC-targeting agents and epigenetic modifiers (directed at KAT2A) could overcome immunotherapy resistance.

Pbrm1 Steers Mesenchymal Stromal Cell Osteolineage Differentiation by Integrating PBAF-Dependent Chromatin Remodeling and BMP/TGF-β Signaling

Bone morphogenic protein (BMP)/transforming growth factor β (TGF-β) signaling determines mesenchymal-stromal-cell (MSC) osteolineage commitment and tissue identity. However, molecular integration of developmental signaling with MSC-intrinsic chromatin regulation remains incompletely understood. SWI/SNF-(BAF) is an ATP-dependent chromatin remodeler implicated in multi-cellular development. We show that BMPs and long-term osteogenic signals in MSCs selectively induce expression of polybromo BAF (PBAF) components Pbrm1, Arid2, and Brd7. Loss of Pbrm1/Arid2/Brd7 profoundly impairs osteolineage gene expression and osteogenesis without compromising adipogenesis. Pbrm1 loss attenuates MSC invivo ossification. Mechanistically, Pbrm1/PBAF deficiency impairs Smad1/5/8 activation through locus-specific epi-genomic remodeling, involving Pbrm1 bromodomains, along with transcriptional downregulation of Bmpr/TgfβrII affecting BMP-early-responsive gene expression. Gain of function of BmprIβ, TgfβrII in PBAF-deficient MSCs partly restores Smad1/5/8 activation and osteogenesis. Pbrm1 loss further affects hematopoietic stem and progenitor activity through non-cell-autonomous regulation of microenvironment and niche-factor expression. Together, these findings reveal a link illustrating epi-genomic feedforward control of BMP/TGF-β signaling to transcriptional and cellular plasticity in the mesenchymal microenvironment and account for stromal-SWI/SNF in hematopoiesis.

Abstract B48: PBRM1 loss promotes resistance to immunotherapy in RCC

Abstract 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.

NIVOREN GETUG-AFU 26 translational study: Association of PD-1, AXL, and PBRM-1 with outcomes in patients (pts) with metastatic clear cell renal cell carcinoma (mccRCC) treated with nivolumab (N).

618 Background: The NIVOREN GETUG-AFU 26 study reported safety and efficacy of N in mccRCC pts in a “real world setting”. A translationnal research program was launched to characterize immune cell populations in the tumor by immunohistochemistry (IHC) and correlate them with outcome on N. Methods: All pts treated with N in the GETUG AFU 26 NIVOREN trial who consented for translational program and with available archived paraffin- embedded (FFPE) tumor tissue samples were eligible. Tumor were centrally reviewed. Using IHC we quantified main immune populations (B-cells, CD8 T-cells, macrophages), and immune checkpoints receptors (TIM-3, LAG-3, PD-1) at the invasive margin (IM) and at the core of the tumor (CT). We also identified AXL and PBRM1/BAP1 expression. Results: Overall 324 pts were included. Pts had similar baseline characteristics (IMDC Good, Intermediate, Poor in 18%, 60% and 22%, respectively) and comparable outcomes than overall trial population (PFS/OS = 4.5 / 25.4 months). PD-1 (IM) expression was associated with better PFS whereas AXL expression by tumor cells (TC) was associated with worse PFS (table). LAG-3 expression tend to be associated with worse OS. PBRM-1 loss (15%) was associated with better OS and PFS and with a higher density of CD8 T-cells (p = 0.001) and CD163-macrophages (p = 0.01) (CT) and a higher expression of LAG-3 (CT) (p = 0.01) and PD-1 (CT) (p = 0.02). BAP-1 loss was not associated with PFS (p = 0.6) nor OS (p = 0.9) in this cohort. Conclusions: We report the largest translational analysis supporting that PD-1 and AXL expression are associated with PFS in pts with mccRCC receiving N. We further confirm that PBRM-1 loss is a strong prognostic factor in this setting.[Table: see text]

PBRM1 Loss in Kidney Cancer Unbalances the Kidney Lineage Master Transcription Factor Hub to Repress Terminal Epithelial Fates

PBRM1 is the 2nd-most frequently deleted and mutated gene in clear cell renal cell cancers (RCC). How this loss promotes malignancy is poorly understood. PBRM1 is a subunit of the PBAF coactivator complex that transcription factors recruit to reposition nucleosomes for gene activation. Identifying transcription factors using PBRM1/PBAF might therefore indicate gene expression programs repressed by PBRM1 loss. We therefore used affinity-purification mass spectrometry to identify, in unbiased fashion, transcription factors recruiting PBRM1 in kidney lineage cells. These unbiased proteomic analyses identified PAX8, a master transcription factor essential for kidney epithelial fates, as the most prominent PBRM1 interaction partner. Reverse proteomic analyses of the PAX8 protein hub confirmed recruitment specifically of PBRM1/PBAF, and not the BAF coactivator complex. More conspicuous in the PAX8 hub in RCC cells, however, were several corepressors, e.g. DNMT1, which oppose coactivators to repress instead of activate genes. Accordingly, key PAX8 target genes, e.g., GATA3, LHX1, WT1, and >500 other kidney epithelial genes, were repressed and hyper-methylated in RCC versus normal kidney, with the greatest repression in cases with bi-allelic PBRM1 inactivation. PBRM1 re-introduction into RCC cells, or depletion of the corepressor DNMT1 using a clinical drug decitabine, rebalanced both composition and function of the PAX8 transcription factor hub toward coactivators, activating terminal epithelial fates in vitro and in vivo. Thus, PBRM1 coactivator loss skews composition of the PAX8 kidney lineage master transcription factor hub toward corepressors and repression instead of activation of kidney epithelial-fates. Sensitivity of the transcription factor hub to coactivator/corepressor stoichiometry, however, also enables correction by pharmacologic corepressor inhibition.

Abstract 945: PBRM1 loss reduces IFNγ-STAT1 activity and promotes resistance to immunotherapy and antiangiogenic therapy in renal cell carcinoma

Abstract 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.

The impact of PBRM1 mutations on overall survival in greater than 2,100 patients treated with immune checkpoint blockade (ICB).

666 Background: PBRM1 is the second most commonly mutated gene in clear cell renal cell carcinoma (ccRCC). We have previously shown favorable outcomes in PBRM1-mutated ccRCC tumors treated with vascular endothelial growth factor (VEGF) inhibitors. Recent data suggested PBRM1 mutations may sensitize ccRCC and non RCC malignancies to ICB therapy. We queried the impact of PBRM1 loss on overall survival (OS) across 2,152 patients treated with ICB. Methods: PBRM1 mutations were assessed in metastatic ccRCC patients who received first line (n = 82) or second line (n = 61) ICB or ICB/VEGF combinations. Additionally, 41 cohorts of non-RCC malignancies treated with ICB and combination (n = 2,009) were analyzed. Mutations were assessed by next generation targeted sequencing using archival tissue. Association of mutation status and overall survival (OS) was tested by multivariate Cox regression analysis (MVA) and adjusted for tumor mutation burden (TMB), copy number alterations (CNA), loss of function(LOF) mutations (non RCC cohort) and IMDC risk (for ccRCC patients). Results: PBRM1 mutations were not associated with improved OS in ICB the entire ccRCC cohort (HR 1.37; CI 0.79-2.4; p = 0.265), the first line (p = 0.624) or second line setting (p = 0.39) or as combination with VEGF inhibitors (p = 0.2). Several RCC subgroups were investigated (see Table at bottom). In the non-RCC cohorts (n = 2,009) PBRM1 mutations were not significantly associated with OS on univariate analysis (HR = 0.73, p = 0.22 for LOF and HR = 0.84,p = 0.34 for non LOF), and remained insignificant after adjusting for TMB, total CNA, and drug class (CTLA4, PD-1/PDL-1 and combinations) (HR = 1.07, p = 0.78 for LOF and HR = 1.08,p = 0.67 for non LOF). Conclusions: Neither in ccRCC nor in the pan-cancer cohort did PBRM1 mutations appear to be associated with improved overall survival with ICB therapy.[Table: see text]

Duration of treatment (DOT) with targeted therapies (TT) or immunotherapy (IO) in PBRM1 mutated metastatic renal cell carcinoma (mRCC).

622 Background: Current evidence indicates improved outcome with IO in mRCC patients (pts) with PBRM1 loss of function mutations (Miao et al., Nature, 2018). We seek to demonstrate an association between PBRM1 mutation and treatment duration with IO and TT in a retrospective cohort. Methods: Consecutive patients with mRCC who had genomic profiling in the course of routine clinical care were identified from an institutional database. GP assessments included testing either tissue or blood with 1 of the 3 CLIA certified commercial panels (Foundation Medicine, Cambridge, MA; Ashion Analytics, Phoenix, AZ; Guardant Health, Redwood City, CA). Information regarding systemic treatment was collected. Median DOT with first targeted therapy and first immunotherapy received was calculated for each patient. DOT was compared across treatment groups in PBRM1+ and PBRM1- patients. Only PBRM1 mutations with functional significance documented in COSMIC were considered. Results: Among 104 pts (72:32 M:F) with mRCC, 82 pts received TT, 35 pts received IO, and 45 pts received both. GP was performed in blood and tissue in 84 and 63 pts, respective, and 25 pts (24%) with PBRM1 mutations were identified. Among PBRM1+ pts, median DOT was 8.8 months (95% CI, 7.6-9.6) mos and 2.3 mos (95% CI, 1.7 – 2.8) mos with TT and IO, respectively (p=0.049). Among PBRM1- pts, median DOT was 5.5 mos and 2.8 mos with TT and IO, respectively (p=0.544). There were 11 PBRM1+ pts and 34 PBRM- pts who received both TT and IO. The ratio of DOT on IO to DOT on TT (DOTIO/TT) was higher in PBRM1- pts than PBRM1+ pts (0.76 versus 0.37 respectively, p=0.014). Conclusions: We failed to replicate the results from Miao et al, suggesting clinical benefit with IO in PBRM1 mutated patients. PBRM1 mutation did appear to predict benefit with TT versus IO. Although limited by sample size, the contrasting results of the current study with literature highlight the importance of clinical validation in a large and prospective setting.

Mechanisms of acquired resistance to rapalogs in metastatic renal cell carcinoma.

The mechanistic target of rapamycin (mTOR) is an established therapeutic target in renal cell carcinoma (RCC). Mechanisms of secondary resistance to rapalog therapy in RCC have not been studied previously. We identified six patients with metastatic RCC who initially responded to mTOR inhibitor therapy and then progressed, and had pre-treatment and post-treatment tumor samples available for analysis. We performed deep whole exome sequencing on the paired tumor samples and a blood sample. Sequence data was analyzed using Mutect, CapSeg, Absolute, and Phylogic to identify mutations, copy number changes, and their changes over time. We also performed in vitro functional assays on PBRM1 in RCC cell lines. Five patients had clear cell and one had chromophobe RCC. 434 somatic mutations in 416 genes were identified in the 12 tumor samples. 201 (46%) of mutations were clonal in both samples while 129 (30%) were acquired in the post-treatment samples. Tumor heterogeneity or sampling issues are likely to account for some mutations that were acquired in the post-treatment samples. Three samples had mutations in TSC1; one in PTEN; and none in MTOR. PBRM1 was the only gene in which mutations were acquired in more than one post-treatment sample. We examined the effect of PBRM1 loss in multiple RCC cell lines, and could not identify any effect on rapalog sensitivity in in vitro culture assays. We conclude that mTOR pathway gene mutations did not contribute to rapalog resistance development in these six patients with advanced RCC. Furthermore, mechanisms of resistance to rapalogs in RCC remain unclear and our results suggest that PBRM1 loss may contribute to sensitivity through complex transcriptional effects.

Abstract 405: PBRM1 re-introduction in PBRM1-mutant kidney cancer cell lines drives an Interferon-γ expression signature

Abstract Introduction Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer and is driven by loss of chromosome 3p. The 3p tumor suppressor PBRM1 is mutated in about 1/3 of tumors. VHL loss is well-known to drive the hypoxic response, but the role of PBRM1 loss is less well understood. PBRM1 is part of the SWI-SNF complex, an ancient chromatin modifying machine, so we hypothesized that its loss would alter chromatin profiles and subsequently expression of SWI-SNF target genes, and would result therapeutic susceptibility to histone H3-K27 methyltransferase inhibitors. Therefore we sought to reintroduce PBRM1 back into ccRCC cell lines which carry PBRM1 truncations in conjunction with 3p LOH and to characterize these cells in detail. Methods All cell lines harbored chromosome 3p loss with truncation of PBRM1 in the remaining allele. Tet-inducible PBRM1 or GFP was introduced into RCC-ER (D251fs), RCC-FG2 (Q445*), and RCC-MF (N496fs). Western blot and immunohistochemistry were used to confirm BAF180 (the protein encoded by PBRM1) and p21. CellTiter Glo was used to measure cell viability to drug treatments. RNA seq was used followed by gene-set enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) for gene expression profiling. Results BAF180 and p21 (a BAF180 target gene) were re-expressed in a time- and dose-dependent manner in all 3 cell lines. BAF180 was localized to the nucleus. EZH2 inhibitor UNC1999 completely ablated clonogenic formation in all 3 cell lines but there was not differential sensitivity between PBRM1-induced and uninduced cells. All cell lines were resistant to EPZ11011989, a different EZH2 inhibitor. We also characterized sensitivity to everolimus, an MTOR inhibitor, and sunitinib, an antiangiogenic multi-tyrosine kinase inhibitor. BAF180 expression did not alter sensitivity to these drugs either. Therefore, we performed RNA sequencing to evaluate the numerous signaling or epigenetic pathways which may have been altered in cells re-expressing BAF180. Using IPA, we saw that several immune signaling pathways were increased in cells upon reintroduction of PBRM1 including Interferon-γ signaling (RCC-ER and RCC-FG2), JAK/STAT signaling, and IL6 pathway. GSEA confirmed the finding that Interferon-γ pathway is activated in each cell line after 7 days of BAF180 induction. These findings were confirmed using Western blot for STAT1/2/3 and their phosphoepitopes, JAK1 and phospo-JAK1, IRF1 and IRF9. Conclusions Our hypothesis that PBRM1 loss would lead to a targeted vulnerability with EZH2 inhibitors was not supported. However, our finding that Interferon-γ signaling is increased when PBRM1 is reintroduced is significant. Given the prominence of Interferon signaling in the response to PD1 and PDL1 inhibitors, somatic PBRM1 inactivation in ccRCC may have significant biological and clinical implications for triaging patients into immune checkpoint therapy. Citation Format: Rahmat K. Sikder, Wafik S. El-Deiry, Philip H. Abbosh. PBRM1 re-introduction in PBRM1-mutant kidney cancer cell lines drives an Interferon-γ expression signature [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 405.

SWI/SNF protein expression status in fumarate hydratase–deficient renal cell carcinoma: immunohistochemical analysis of 32 tumors from 28 patients

Fumarate hydratase-deficient renal cell carcinoma (FH-RCC) is a rare, aggressive RCC type, originally described in the setting of hereditary leiomyomatosis and RCC syndrome, which is defined by germline FH gene inactivation. Inactivation of components of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex is involved in renal medullary carcinoma (SMARCB1/INI1 loss), clear cell RCC (PBRM1 loss), and subsets of dedifferentiated RCC of clear cell, chromophobe, and papillary types (loss of different SWI/SNF components). FH-RCC and SWI/SNF-deficient RCC share anaplastic nuclear features and highly aggressive course. We analyzed 32 FH-RCCs from 28 patients using 7 commercially available SWI/SNF antibodies (SMARCB1/INI1, SMARCA2, SMARCA4, SMARCC1, SMARCC2, PBRM1, and ARID1A). Variable loss of SMARCB1, ARID1A, and SMARCC1 was observed in 1 of 31, 2 of 31, and 1 of 29 evaluable cases, respectively; 3 of these 4 SWI/SNF-deficient tumors had confirmed FH mutations. No correlation of SWI/SNF loss with solid or sarcomatoid features was observed. Two tumors with SMARCB1 and ARID1A deficiency had available SWI/SNF molecular data; both lacked SMARCB1 and ARID1A mutations. The remaining 5 SWI/SNF components were intact in all cases. Especially PBRM1 seems not to be involved in the pathogenesis or progression of FH-RCC. Our data showed that a subset of FH-RCC (12%) have a variable loss of SWI/SNF complex subunits, likely as secondary genetic events. This should not be confused with SWI/SNF-deficient RCC of other types. Evaluation of FH and SWI/SNF together with comprehensive molecular genetic profiling is needed to explore possible prognostic implications of FH/SWI-SNF double deficiency and to better understand the somatic mutation landscape in high-grade RCC.

Kidney cancer: PBRM1 loss promotes tumour response to immunotherapy.

Kidney cancer: PBRM1 loss promotes tumour response to immunotherapy.

Kidney cancer: PBRM1 loss promotes tumour response to immunotherapy.

Kidney cancer: PBRM1 loss promotes tumour response to immunotherapy.

Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma.

Immune checkpoint inhibitors targeting the programmed cell death 1 receptor (PD-1) improve survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). To identify genomic alterations in ccRCC that correlate with response to anti-PD-1 monotherapy, we performed whole-exome sequencing of metastatic ccRCC from 35 patients. We found that clinical benefit was associated with loss-of-function mutations in the PBRM1 gene (P = 0.012), which encodes a subunit of the PBAF switch-sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. We confirmed this finding in an independent validation cohort of 63 ccRCC patients treated with PD-1 or PD-L1 (PD-1 ligand) blockade therapy alone or in combination with anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) therapies (P = 0.0071). Gene-expression analysis of PBAF-deficient ccRCC cell lines and PBRM1-deficient tumors revealed altered transcriptional output in JAK-STAT (Janus kinase-signal transducers and activators of transcription), hypoxia, and immune signaling pathways. PBRM1 loss in ccRCC may alter global tumor-cell expression profiles to influence responsiveness to immune checkpoint therapy.

Modeling Renal Cell Carcinoma in Mice: Bap1 and Pbrm1 Inactivation Drive Tumor Grade.

Clear cell renal cell carcinoma (ccRCC) is characterized by BAP1 and PBRM1 mutations, which are associated with tumors of different grade and prognosis. However, whether BAP1 and PBRM1 loss causes ccRCC and determines tumor grade is unclear. We conditionally targeted Bap1 and Pbrm1 (with Vhl) in the mouse using several Cre drivers. Sglt2 and Villin proximal convoluted tubule drivers failed to cause tumorigenesis, challenging the conventional notion of ccRCC origins. In contrast, targeting with PAX8, a transcription factor frequently overexpressed in ccRCC, led to ccRCC of different grades. Bap1-deficient tumors were of high grade and showed greater mTORC1 activation than Pbrm1-deficient tumors, which exhibited longer latency. Disrupting one allele of the mTORC1 negative regulator, Tsc1, in Pbrm1-deficient kidneys triggered higher grade ccRCC. This study establishes Bap1 and Pbrm1 as lineage-specific drivers of ccRCC and histologic grade, implicates mTORC1 as a tumor grade rheostat, and suggests that ccRCCs arise from Bowman capsule cells.Significance: Determinants of tumor grade and aggressiveness across cancer types are poorly understood. Using ccRCC as a model, we show that Bap1 and Pbrm1 loss drives tumor grade. Furthermore, we show that the conversion from low grade to high grade can be promoted by activation of mTORC1. Cancer Discov; 7(8); 900-17. ©2017 AACR.See related commentary by Leung and Kim, p. 802This article is highlighted in the In This Issue feature, p. 783.