Periodic Reporting for period 1 - VulneraBAP1 (Mechanism and vulnerability of BAP1 loss in tumor metastasis)
Periodo di rendicontazione: 2018-07-01 al 2020-06-30
Kidney cancer is among the ten most prevalent cancers arising in Western countries, with clear-cell renal cell carcinoma (ccRCC) being the most frequent subtype (75%). About 30% of ccRCC patients present with metastatic disease at diagnosis, and another 30% will develop metastases after surgery. When metastatic, ccRCC remains largely incurable.
I discovered that the tumor suppressor BAP1 (BRCA1-associated protein 1) is inactivated in 15% of ccRCCs (Peña-Llopis et al. Nat. Genet. 2012). Notably, I found that mutations in BAP1 are mutually exclusive with mutations of the tumor suppressor gene PBRM1, and loss of BAP1 was associated with higher tumor grade, activation of mTORC1, and poorer overall patient survival, whereas tumors with PBRM1 loss were associated with lower tumor grade and better overall survival. This first molecular genetic classification of ccRCC may have tangible clinical implications, since tumors with BAP1 loss display in general more aggressive pathological features and are more prone to metastasize. However, the molecular mechanism through which BAP1 loss induces metastasis and tumor aggressiveness remains elusive.
In this study, we aimed to investigate the molecular mechanism of repression of a miRNA cluster involved in metastasis by BAP1 and to identify therapeutic opportunities. Specifically, (1) I supervised a PhD student (supported by a grant I was recently been awarded) in the identification and characterization of the regulation by BAP1 of the expression of a miRNA cluster; and (2) we are deciphering the genetic vulnerabilities of BAP1 loss by a synthetic lethality strategy. These studies were fundamental to support my Junior Group Leader position.
I discovered that the tumor suppressor BAP1 (BRCA1-associated protein 1) is inactivated in 15% of ccRCCs (Peña-Llopis et al. Nat. Genet. 2012). Notably, I found that mutations in BAP1 are mutually exclusive with mutations of the tumor suppressor gene PBRM1, and loss of BAP1 was associated with higher tumor grade, activation of mTORC1, and poorer overall patient survival, whereas tumors with PBRM1 loss were associated with lower tumor grade and better overall survival. This first molecular genetic classification of ccRCC may have tangible clinical implications, since tumors with BAP1 loss display in general more aggressive pathological features and are more prone to metastasize. However, the molecular mechanism through which BAP1 loss induces metastasis and tumor aggressiveness remains elusive.
In this study, we aimed to investigate the molecular mechanism of repression of a miRNA cluster involved in metastasis by BAP1 and to identify therapeutic opportunities. Specifically, (1) I supervised a PhD student (supported by a grant I was recently been awarded) in the identification and characterization of the regulation by BAP1 of the expression of a miRNA cluster; and (2) we are deciphering the genetic vulnerabilities of BAP1 loss by a synthetic lethality strategy. These studies were fundamental to support my Junior Group Leader position.
The researcher of this project, Dr. Samuel Peña-Llopis, started a Junior Group Leader position in July 1st, 2018, at the same time as the MSCA-IF started. However, the remodeling of the wet laboratories did not finish until around October/November 2018. A PhD student was recruited with funds from a German Research Foundation (DFG) grant and started working in October 15th, 2018, spearheading Objective 1 (WP1-3). Another PhD student was recruited with funds from the German Cancer Consortium (DKTK) and started spearheading Objective 2 (WP4-6) in November 1st, 2018. Anyway, the authorization for performing experiments in S1 and S2 safety laboratories were approved in November 21st, 2018, delaying almost 5 months the beginning of the experiments for the VulneraBAP1 project. Here are the Work Packages (WP), Major Deliverables and Milestones achieved:
Objective 1
WP1: Serially truncated regions of the miRNA cluster were generated and analyzed by dual-luciferase assay to pinpoint the key region that confers BAP1 repression in cell lines reconstituted with wild-type or mutant BAP1.
Major Deliverable 1: The minimal region of binding of BAP1 to the miRNA cluster promoter was identified.
Major Milestone 1: Minimal promoter DNA was used for DNA pull-down assay (TIGR) by Proteomics group.
WP2: In collaboration with Prof. Jeroen Krijgsveld at DKFZ, we aimed to identifying the proteins that bind to the miRNA cluster using a custom-made DNA pull-down assay. Unfortunately, results suggest that BAP1 is not directly regulating the promoter region of the miRNA cluster and, therefore, we are exploring alternative regulatory mechanisms.
Objective 2
WP4: A third experiment for the synthetic lethality screen has been completed. Data analysis identified several genes involved in DNA repair whose knockdown is potentially synthetic lethal with BAP1 loss.
Major Deliverable 4: The synthetic lethality screen for BAP1 loss reveals candidate hits after proper comparisons.
Major Milestone 2: Decision to start the in vitro validation of the hits.
WP5: In vitro validation of three hits using the renal cell carcinoma BAP1 deficient cell lines UM-RC-6 and the wild-type BAP1 cell lines 786-O, Caki-1, Caki-2 and RCC4 was completed. Five different shRNAs were tested for each of the three hits and the two most efficient shRNAs were selected for the in vitro assays using cell proliferation and colony formation.
Major Deliverable 5: Validation of candidate hits that selectively kill BAP1-mutant but not wild-type human cells.
Major Milestone 4: Decision to validate the successful in vitro candidate hit(s) in mouse models of metastasis.
WP6: Unfortunately, ethics approvals for animal experiments were significantly delayed and by the time everything was in place, the COVID-19 pandemic took hold, preventing the execution of the mouse experiments. Currently, the situation in the lab is much better and we have started the experiments with mouse models of metastasis.
The work carried out can bring important benefits for society by using the loss of BAP1 in immunohistochemistry to identify patients with mutations in this tumor suppressor gene who could benefit from novel treatments that we are developing in the lab.
Objective 1
WP1: Serially truncated regions of the miRNA cluster were generated and analyzed by dual-luciferase assay to pinpoint the key region that confers BAP1 repression in cell lines reconstituted with wild-type or mutant BAP1.
Major Deliverable 1: The minimal region of binding of BAP1 to the miRNA cluster promoter was identified.
Major Milestone 1: Minimal promoter DNA was used for DNA pull-down assay (TIGR) by Proteomics group.
WP2: In collaboration with Prof. Jeroen Krijgsveld at DKFZ, we aimed to identifying the proteins that bind to the miRNA cluster using a custom-made DNA pull-down assay. Unfortunately, results suggest that BAP1 is not directly regulating the promoter region of the miRNA cluster and, therefore, we are exploring alternative regulatory mechanisms.
Objective 2
WP4: A third experiment for the synthetic lethality screen has been completed. Data analysis identified several genes involved in DNA repair whose knockdown is potentially synthetic lethal with BAP1 loss.
Major Deliverable 4: The synthetic lethality screen for BAP1 loss reveals candidate hits after proper comparisons.
Major Milestone 2: Decision to start the in vitro validation of the hits.
WP5: In vitro validation of three hits using the renal cell carcinoma BAP1 deficient cell lines UM-RC-6 and the wild-type BAP1 cell lines 786-O, Caki-1, Caki-2 and RCC4 was completed. Five different shRNAs were tested for each of the three hits and the two most efficient shRNAs were selected for the in vitro assays using cell proliferation and colony formation.
Major Deliverable 5: Validation of candidate hits that selectively kill BAP1-mutant but not wild-type human cells.
Major Milestone 4: Decision to validate the successful in vitro candidate hit(s) in mouse models of metastasis.
WP6: Unfortunately, ethics approvals for animal experiments were significantly delayed and by the time everything was in place, the COVID-19 pandemic took hold, preventing the execution of the mouse experiments. Currently, the situation in the lab is much better and we have started the experiments with mouse models of metastasis.
The work carried out can bring important benefits for society by using the loss of BAP1 in immunohistochemistry to identify patients with mutations in this tumor suppressor gene who could benefit from novel treatments that we are developing in the lab.
Our discovery that mutations in BAP1 and PBRM1 can predict prognosis in renal cell carcinoma patients is currently being used in clinical practice for patient risk stratification. Patients with mutations in PBRM1 correlate with responses to immune checkpoint inhibitors. However, there are no specific treatments for patients with BAP1 mutations. We aim to uncover the mechanism of metastasis caused by BAP1 loss and identify a potential gene that is synthetic lethal with mutations in BAP1. If successful, not only renal cell carcinoma patients, but also uveal melanoma, mesothelioma and cholangiocarcinoma, among other cancer patients, will benefit of novel therapies.