Gene Targeting Facility

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

As part of the strategic vision to leverage the wide expertise present in the MCGP to foster closer collaborations between basic and clinical scientists we have initiated a close collaboration with MyPart (My Pediatric and Adult Rare Tumor network; see below details under "Strategic Vision, Goals and Plans") to generate a new mouse model of a rare type of gastrointestinal stromal tumors (GISTs). Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms occurring in the gastrointestinal tract (GI) with approximately 5000 cases per year in the United States. Although GISTs can occur anywhere in the GI tract, they are found most often in the stomach (about 60%) and in the small intestine (about 35%). GISTs are thought to be originating from interstitial cells of Cajal (ICCs), known also as "pacemaker" cells of the gastrointestinal tract, although this has not yet been firmly established . The majority of GISTs (about 85%) are characterized by activating mutations in the KIT or PDGFRA genes, while approximately 15% of GISTs are negative for KIT and PDGFRA mutations and they are denominated as "wild-type" GISTs. Because of the lack of activating mutations in KIT and PDGFRA these rare WT-GISTs are generally not responding to standard therapies with tyrosine-kinase inhibitors which are commonly used for treating non-WT-GISTs. Loss of succinate dehydrogenase (SDH) enzyme activity and/or expression (mitochondria complex II) is among the most common molecular alterations associated with WT-GISTs, especially in pediatric GISTs (SDH-deficient GISTs) (Miettinen and Lasota 2014). Loss of the enzyme activity can be due to mutation and/or epigenetic alteration of SDHA, SDHB, SDHC or SDHD, which are the genes encoding for the units forming the enzyme complex. Other characteristic mutations found in WT-GISTs include BRAF activating mutation (V600E) which is one of the most frequent and occurs in about 10-15% of cases and NF1 mutations. Because of the rare nature of WT-GISTs many aspects of these malignancies are still unknown and very few animal models have been developed for studying these rare tumors. In close contact with MyPart clinicians we have designed and generated a new mouse model of SDH-deficient GISTs, one of the most common pediatric WT-GISTs subtype which is currently not responding to standard therapies and has no suitable animal models. By CRISPR/Cas9 editing technology we generate a conditional knock-out mouse for SdhB (SdhB cKO). After validation of the effectiveness of the conditional targeting, we sought for a Cre-driver models able to target ICCs. We used a c-Kit-CreERT mouse model to allow for temporal control of Cre expression through tamoxifen administration. Since BRAF activating mutation (V600E) is also one of the most frequent mutation found in WT-GISTs, we also included the conditional BRAFV600E mutation in our study to investigate its role in GISTs tumorigenesis and to evaluate any possible synergistic effect with the loss of Sdh activity. Although the process was laborious and lengthy, we generated four experimental groups with approximately 12 animals in each group, including a Ctrl group (c-Kit-CreERT), an SdhB group (c-Kit-CreERT; SdhBlox/lox), a BRAF group (c-Kit-CreERT; BRAFV600E) and an SdhB-BRAF group (c-Kit-CreERT; SdhBlox/lox; BRAFV600E). All the animals were injected intraperitoneally with a single dose of tamoxifen (75 mg/kg) at post-natal day 21. Surprisingly, all animals in the BRAF and SdhB-BRAF group showed signs of distress around 3 weeks post tamoxifen induction and they were sacrificed and subjected to pathological evaluation. Necropsy revealed an abnormal stomach shape that appeared to be bloated and distended in all the mice analyzed. Histological evaluation showed ICCs hyperplasia in the stomach leading occasionally to GIST-like tumors. Importantly, the most severe manifestation of hyperplasia and/or tumor-like lesions were occurring in the pyloric region of the stomach, coinciding with the area where, according to the MyPart clinicians, most human GIST develop. No hyperplasia was found in the stomach of Ctrl and SdhB mice while a minimal ICC hyperplasia was found in the cecum area in some SdhB mice sugegsting that single SdhB deletion in ICCs is not sufficient to initiate the hyperplastic and invasive transformation of ICCs. However, the development of hyperplasia in the stomach of both BRAF and SdhB-BRAF groups strongly suggests that the BRAF activating mutation (V600E) is acting as a potent neoplastic driver in ICCs as observed in human GISTs. Interestingly, the grade of hyperplasia was more severe in the absence of SdhB since SdhB-BRAF mice were about 20% more prone to display severe ICC hyperplasia or GIST-like tumors compared to the mice with only the BRAF mutation. Therefore, we further investigated whether Sdh inactivation can contribute to the worsening of the hyperplastic/neoplastic condition triggered by the BRAF activation. Inactivation of the succinate dehydrogenase enzyme (SDH) leads to accumulation of its substrate 'succinate', and elevated intracellular succinate levels are potent inhibitors of the TET family of enzymes. Therefore, we are currently testing whether in the lesions with the SDH2 deletion there is a reduction in the global DNA de-methylation caused by TET inhibition. Preliminary experiments appear to show that this is the case. If this is confirmed, it would suggest that SdhB-BRAF derived malignancies found in this mouse model may have a global DNA-hypermethylation phenotype similarly to what has been described in human SDH-deficient tumors. A second effect caused by the accumulation of succinate is the inhibition of prolyl-hydrolases (PHDs) which results in the stabilization of the hypoxia-inducible factors HIF1a and/or HIF2b. Since it has been reported that in SdhB-deficient cells HIF2a is induced and stabilized but not HIF1a (Morin, Goncalves et al. 2020), we are testing the levels of HIF2a in the GIST-like tumors of BRAF and SdhB-BRAF mice. This could be important because if we find that HIF2a is induced in SdhB-deficient invasive cells, it identifies a new, and to date only target for SDH-deficient GISTs. Importantly, the FDA has recently approved (2021) Belzutifan (WeliregTM), as an HIF2a specific inhibitor for the treatment of Von Hippel-Lindau (VHL) disease-associated tumors and this inhibitor could be tested in our mouse model. Because of the severity of the phenotype caused by induction of c-Kit-CreERT with the injection of 75 mg/Kg of tamoxifen we have also initiated the generation of a new cohort of mice with a 10 times lower dose of tamoxifen (7.5 mg/kg) to reduce the severity of the phenotype and study the tumors over a longer period of time. From the initial group of mice generated with this protocol it appears that indeed we can prolong the survival of the animals since out of seventeen SdhB-BRAF mice induced with the lower tamoxifen dose, nine showed signs of distress at about 50-60 days post-induction. Interestingly, in the BRAF group, only two animals out of fifteen showed similar distress around the same age (60-65 days post-induction). Thus, it appears that SdhB-BRAF mutant mice have a more severe phenotype than the BRAF group. Overall, this project promise to generate a new mouse model or rare WT-GIST. Importantly it showed the power of grouping clinicians with investigators with a wide variety of expertise as found in the MCGP (e.g. genome editing, cancer biology, epigenetics, and metabolism).