Gfi1 is required for tumor maintenance

Our previous studies demonstrated that co-expression of Myc and Gfi1 drives transformation of neural progenitors into MB cells6. Although these results indicate that Gfi1 plays a role in tumor initiation, it is unknown whether it is required for continued tumor growth. To investigate this, we designed a conditional retroviral vector encoding Gfi1 flanked by loxp sites (Gfi1flox), which allows Gfi1 to be deleted by Cre recombinase (Fig. 1a). We isolated Prominin1 + neural progenitor cells9 from the cerebella of neonatal CAG-CreERTM mice (which express tamoxifen-inducible Cre protein in all cells10) and infected them with viruses encoding Myc-IRES-luciferase and Gfi1flox-IRES-GFP. Orthotopic transplantation of infected cells into the cerebella of adult mice resulted in tumor formation within 5 weeks (Fig. 1b). The latency and penetrance of Myc + Gfi1flox (MGflox) tumors was similar to that observed for Myc + Gfi1WT (MG) tumors6 (Supplementary Fig. 1).

Fig. 1 Gfi1 is required for tumor maintenance. a Constructs used to generate MGflox tumors. NSCs from a CAG-CreERTM mouse were transduced with viruses encoding Gfi1flox-IRES-GFP and MycT58A-IRES-Luciferase. b Bioluminescence imaging and whole mount fluorescence image of representative MGflox tumor. c Western blot for Gfi1 protein in MGflox tumor cells treated overnight with vehicle control (DMSO) or 5 µM 4-hydroxytamoxifen (4-OHT). d Bioluminescence imaging of mice transplanted with cells treated with vehicle or 4-OHT. X’s denote animals euthanized before they could be imaged. e Survival curves from a representative experiment (control n = 23, 4-OHT n = 24). p Value < 0.0001 was determined by Log-rank (Mantel–Cox) test. f Western blot of Gfi1 protein in resulting MG tumors from vehicle and 4-OHT treatment groups Full size image

Treatment of MGflox tumor cells with 4-hydroxytamoxifen (4-OHT) to activate CreERTM caused a marked reduction in Gfi1 protein expression compared to cells treated with vehicle (DMSO) (Fig. 1c). Importantly, when cells were retransplanted into the cerebella of naïve mice, those that had been treated with vehicle gave rise to tumors within 4–5 weeks, whereas those that had been exposed to 4-OHT did not generate tumors in most recipients (Fig. 1d, e). Of 24 mice that received cells treated with 4-OHT, we observed only 2 cases where tumors developed, and Western blotting showed that these tumors still expressed Gfi1 protein (Fig. 1f). These studies demonstrate that continued expression of Gfi1 is necessary for maintaining tumor growth.

Gfi1 recruits Lsd1

Given the importance of Gfi1 in MB initiation and maintenance, we sought to further understand the mechanisms by which Gfi1 promotes tumor growth. Studies in the hematopoietic system suggest that Gfi1/1b repress target genes via their interactions with cofactors, including Lsd111,12,13,14, the corepressor CoREST12,13,15, and the histone deacetylases HDAC1 and HDAC216,17,18. To determine whether these interactions are also involved in Gfi1-driven MB, we performed co-immunoprecipitation experiments on lysates from MG tumors. After immunoprecipitation of Gfi1, we detected interactions with Lsd1 and CoREST, but not HDAC1 or HDAC2, as determined by Western blotting (Fig. 2a; Supplementary Fig. 2). Immunoprecipitation of Lsd1 and CoREST yielded similar results, showing interactions with one another as well as with Gfi1 (Fig. 2b, c). Interestingly, the amount of Gfi1 protein detected after immunoprecipitation of Lsd1 or CoREST was similar to the amount detected after Gfi1 immunoprecipitation, suggesting that the majority of Gfi1 in the tumor cells complexes with Lsd1 and CoREST. In contrast, the amount of Lsd1 detected after immunoprecipitation of Gfi1 was only a small fraction of the total Lsd1, suggesting that Lsd1 also interacts with partners other than Gfi1. These data indicate that Gfi1 interacts with the epigenetic regulators Lsd1 and CoREST in Gfi1-driven MB.

Fig. 2 Gfi1 is associated with Lsd1. a–c Co-immunoprecipitations of a Gfi1, b Lsd1, and c CoREST were performed on MG tumor cells, and Gfi1, Lsd1, and CoREST protein levels were detected by Western blotting. Input represents 10% of total lysate before immunoprecipitation with experimental and isotype control antibodies Full size image

The SNAG domain is critical for Gfi1-driven tumorigenesis

Gfi1 family proteins contain two highly conserved domains: a SNAG (Snail/Gfi1) domain at the N-terminus and six C 2 H 2 -type zinc fingers at the C-terminus19. The ability of Gfi1/1b to recruit and interact with cofactors such as Lsd1 and CoREST has been attributed to the SNAG domain13,14,19 (Fig. 3a). To determine the importance of this domain in MB pathogenesis, we utilized a Gfi1 SNAG domain mutant with a proline to alanine change at amino acid 2 (Gfi1-P2A). This mutation has been shown previously to abrogate the function of the SNAG domain19,20; we confirmed this by co-immunoprecipitation, demonstrating that wild-type Gfi1 associates with Lsd1, but Gfi1-P2A does not (Supplementary Fig. 3). When overexpressed, the SNAG mutant still produced a full-length protein (Fig. 3b) and resulted in mRNA and protein levels comparable to those of wild-type Gfi1 (Fig. 3b, c). We co-infected neural progenitors with Myc and the Gfi1-P2A mutant, transplanted them into NSG mice, and monitored animals for tumor growth. By 4–5 weeks, mice transplanted with cells carrying WT Gfi1 had to be sacrificed (median survival = 27 days). In contrast, mice transplanted with cells expressing Gfi1-P2A were monitored for 7 months with no signs of tumor development (Fig. 3d, e). The stark difference in tumorigenic potential of the SNAG mutant strongly suggests that the ability of Gfi1 to recruit and interact with other proteins is essential for its oncogenic activity in MB.

Fig. 3 The SNAG domain is required for Gfi1-driven tumorigenesis. a Structure of Gfi1, illustrating the N-terminal SNAG domain and six C-terminal zinc fingers. Arrow denotes the proline to alanine point mutation of the SNAG mutant. b Western blot for Gfi1 protein levels in 293T cells transduced with empty vector, Gfi1-WT, or Gfi1-P2A. c qPCR for Gfi1 mRNA levels in NSCs transduced with empty vector, Gfi1-WT, or Gfi1-P2A. Data shown are from one representative experiment, where experiments were repeated in at least three biological replicates. Error bars represent 95% confidence intervals calculated using the sum of squares method. d Bioluminescence imaging of mice transplanted with NSCs that were co-infected with viruses encoding Myc and Gfi1-WT (MG) or Myc and Gfi1-P2A (MG-P2A) overnight. X’s denote animals euthanized before they could be imaged. e Survival curves comparing mice transplanted with MG or MG-P2A cells (MG n = 6, MG-P2A n = 6). p Value = 0.0005 was determined by Log-rank (Mantel–Cox) test Full size image

Genetic deletion of Lsd1 impairs growth of Gfi1-driven MB

As shown above, our data suggest that Gfi1 interacts with Lsd1 and that this interaction may be important for tumor growth. To determine whether Lsd1 is required for growth of MG tumors, we crossed CAG-CreERTM mice with Lsd1fl/fl mice21 to obtain CAG-CreERTM; Lsd1fl/fl mice (hereafter called Lsd1-inducible knockout, or Lsd1-iKO mice). We isolated neural progenitors from Lsd1-iKO pups and transduced them with Myc and Gfi1 to generate Lsd1-iKO MG tumors. We then treated tumor cells with 4-OHT overnight to delete Lsd1. Assaying these cells by Western blotting confirmed that treatment with 4-OHT significantly reduced the amount of Lsd1 protein when compared to treatment with vehicle (DMSO) (Fig. 4a). When tumor cells were implanted into mice, those that had been treated with vehicle all gave rise to tumors (median survival = 19 days). In contrast, only 8/26 of mice that received 4-OHT-treated cells developed tumors, and they did so with increased latency (Fig. 4b, c). Importantly, the tumors that did arise from 4-OHT-treated cells expressed substantial amounts of Lsd1 protein, suggesting that these tumors arose from cells that had escaped Lsd1 deletion (Fig. 4d).

Fig. 4 Genetic deletion of Lsd1 impairs growth of Gfi1-driven MB. a Western blot for Lsd1 protein in Lsd1-iKO tumor cells that were treated overnight with vehicle control (DMSO) or 5 µM 4-hydroxytamoxifen (4-OHT). b Bioluminescence imaging of mice transplanted with Lsd1-iKO cells treated with vehicle or 4-OHT. X’s denote animals euthanized before they could be imaged. c Survival curves from a representative experiment (control n = 10, 4-OHT n = 10). p Value < 0.0001 was determined by Log-rank (Mantel–Cox) test. d Western blot for Lsd1 protein in tumors from vehicle and 4-OHT treatment groups. e In vitro proliferation assay for Lsd1-iKO MG tumor cells treated with vehicle (DMSO), 1 or 5 µM 4-OHT. Proliferation was measured via 3H-thymidine incorporation at 48 h. Data are from a representative experiment and are plotted as the means of technical triplicate samples ± SEM. Experiments were repeated in at least three biological replicates. f Quantification of Ki67 staining in Lsd1-iKO MG cells after treatment with vehicle or 5 µM 4-OHT. p Value = 0.0047, t = 10.27, df = 2, one-tailed paired t test. g Quantification of active Caspase-3 staining in Lsd1-iKO MG cells after treatment with vehicle or 5 µM 4-OHT. p Value = 0.0286, t = 4.003, df = 2, one-tailed paired t test. f, g Staining and flow cytometric analysis for Ki67 and Caspase 3 were performed on fixed and permeabilized cells. Resulting values were normalized to vehicle control, and data shown represent the means of three biological replicates ± SEM Full size image

To understand the mechanisms by which loss of Lsd1 prevented tumor growth, we examined tumor cells for changes in proliferation and death after Lsd1 deletion. To measure proliferation, we treated Lsd1-iKO MG tumor cells with 1 or 5 μM 4-OHT for 48 h and performed 3-H Thymidine incorporation assays. Cells treated with both concentrations of 4-OHT showed markedly lower levels of incorporation compared to those treated with vehicle (DMSO), indicating that Lsd1 deletion impaired proliferation (Fig. 4e). Similarly, we observed a 63% decrease in the proportion of Ki67+ cells after treatment with 4-OHT (Fig. 4f, Supplementary Fig. 4a, b). We then looked for changes in cell death by staining cells for active Caspase 3, a marker for cells undergoing apoptosis. Tumor cells treated with 4-OHT exhibited 2.2-fold increase in active Caspase-3 expression, indicating that Lsd1 deletion also had a significant effect on tumor cell survival (Fig. 4g, Supplementary Fig. 4a, c). Staining with Annexin V and 7-AAD also showed a shift from live cells to dying and dead cells after 4-OHT treatment (Supplementary Fig. 4d, e).

To ensure that the effects of 4-OHT on tumor growth were caused by loss of Lsd1 and not by 4-OHT itself, we carried out parallel experiments using Lsd1fl/fl MG tumor cells without the CreERTM allele. Treatment of these tumor cells with 4-OHT did not activate CreERTM and consequently did not delete Lsd1 (Supplementary Fig. 5a). Mice receiving vehicle-treated or 4-OHT-treated tumor cells developed tumors with 100% penetrance, similar latencies, and no difference in Lsd1 protein levels (Supplementary Fig. 5b–d). Thus, the effects of 4-OHT on tumor growth depend on deletion of Lsd1. Collectively, these findings demonstrate that Lsd1 is required for growth of MG tumors.

Since Lsd1 is expressed in many cancer types and is involved in a wide range of biological processes22,23, we sought to determine whether the inhibitory effect of Lsd1 deletion was specific to MG tumors. We therefore repeated the deletion experiments described above using cells from Lsd1-iKO MP tumors, which are driven by overexpression of Myc and DNp53. As shown in Supplementary Fig. 6a–c, loss of Lsd1 had a much more modest effect on MP tumors than it did on MG tumors: the majority of animals receiving 4-OHT-treated Lsd1-iKO MP tumor cells still developed tumors. Moreover, in contrast to the persistent expression of Lsd1 in Lsd1-iKO MG tumors, 10/12 of the tumors arising from Lsd1-iKO cells exhibited reduced expression of Lsd1 protein (Supplementary Fig. 6c). These results suggested that Lsd1 is dispensable for the growth of MP but not MG tumors.

The p53 pathway remains functional in Gfi1-driven tumors

The studies described above show that Gfi1 depends on interactions with Lsd1 to promote tumor growth, but the signaling pathways and target genes affected by Gfi1 and its cofactors remain unknown. In the hematopoietic system, several groups have reported that Gfi1 can repress genes in the p53 pathway18,24,25. Furthermore, two previously established models of MB combine Myc overexpression with p53 loss of function4,5. Based on these observations, we wondered whether Gfi1 might contribute to MB growth by suppressing the p53 pathway. To assess the activity of the pathway, we treated cells with doxorubicin or with γ-irradiation to elicit p53-dependent DNA damage responses. After 4 h, samples were analyzed by Western blotting for p53 and its downstream effector p21. Mouse embryonic fibroblasts (MEFs), which express wild-type p53, showed increased expression of p53 and its target p21 in response to doxorubicin and γ-irradiation (Fig. 5a, b). Likewise, doxorubicin and γ-irradiation caused dose dependent increases in p53 and p21 in MG tumors (Fig. 5c, d). To confirm that p53 inactivation can desensitize MB cells to these treatments, we overexpressed DNp53 in MG tumors and then analyzed their response to doxorubicin and γ-irradiation. While DNp53-expressing MG tumor cells exhibited accumulation of p53 protein (because DNp53 prevents upregulation of the Mdm2 ubiquitin ligase that would otherwise promote p53 degradation), they no longer showed induction of p21 in response to DNA damage (Fig. 5e, f).

Fig. 5 The p53 pathway remains functional in Gfi1-driven tumors. a, b Western blots for p53 and p21 protein in wild-type mouse embryonic fibroblasts (MEFs) after 4-h treatment with a 0, 0.1, or 0.5 μM doxorubicin or b 0, 2, 4, or 8 Gy of γ-irradiation. c, d Western blots for p53 and p21 protein in MG tumor cells after 4-h treatment with c doxorubicin or d γ-irradiation. e, f Western blots for p53 and p21 protein in MG tumor cells expressing DNp53 after 4-h treatment with e doxorubicin or f γ-irradiation Full size image

Given that both Gfi1 and Lsd1 are necessary for MG tumors, it is also notable that others have demonstrated Lsd1 demethylation of p53 protein, which destabilizes it and prevents its association with coactivators25,26. We thus considered the possibility that MG tumors might have increased Lsd1 expression levels that could lead to reduced p53 activity. However, Lsd1 levels in MG tumors were not higher than those in NSCs, from which MG tumors are derived (Supplementary Fig. 7). Together, these results suggest that MG tumors have normal p53 function and that it is unlikely that the critical role of Gfi1 in MG tumorigenesis is to repress p53.

Neuronal differentiation genes are decreased in MG tumors

To identify other potential mechanisms by which Gfi1/Lsd1 promote tumor formation, we analyzed transcriptional profiles obtained from MG tumors (n = 7) and NSCs (n = 5). Our analysis identified a total of 2402 differentially expressed genes, of which 1170 were downregulated ((FDR)-adjusted p value < 10−2, log 2 FC < −1.5) and 1232 were upregulated ((FDR)-adjusted p value < 10−2, log 2 FC > 1.5) (Fig. 6a). Pathway analysis of differentially expressed genes revealed an enrichment of genes associated with neuronal fate commitment and differentiation, neuron migration, neuron projection guidance, and neuron apoptotic processes (Fig. 6b); regulators of these processes were expressed at significantly lower levels in MG tumors than in NSCs (Fig. 6c). Consistent with this, many of the most downregulated genes in MG tumors have functions in neuronal differentiation and migration: Ptf1a27, Sox1128, Zic329, Dcc30, Neurog231, Zic132, Barhl133, Neurod134, Nfib35, and Fbxo536 (Supplementary Data 1). These findings suggest that repression of neuronal commitment and differentiation may play a key role in the transformation of normal NSCs into MG tumors.

Fig. 6 Neuronal commitment and differentiation pathways are downregulated in MG tumors. a Volcano plot of of 21,304 genes whose expression was compared in MG tumors (n = 7) and NSCs (n = 5). Red indicates genes that were significantly upregulated in MG (log 2 FC > 1.5 and FDR-corrected p value < 10−2) and green indicates those that were significantly downregulated in MG (log 2 FC < −1.5 and FDR-corrected p value < 10−2). b Pathway analysis of differentially expressed genes. Each node represents a GO term. Node size reflects the enrichment significance of the term. Edges represent association strength between the terms calculated using chance corrected kappa statistics (a standard kappa score level threshold was used). c Heat maps comparing expression of genes involved in central nervous system neuron differentiation (left), neuron fate commitment (center), and neuron migration (right) in MG tumor cells and NSCs. d Fbxo5 expression from microarray analysis comparing NSCs to MG tumor cells (left) and qPCR validation of Fbxo5 mRNA expression in MG tumor samples compared to NSC samples (right). p values < 0.0001 were determined by one-sided Welch Two Sample t test. Whiskers indicate minimum and maximum expression values, bottom of boxes indicate first quartiles, midline indicates median expression values, and top of boxes indicate third quartiles. e Bioluminescence imaging of mice transplanted with cells infected with empty vector control (GFP) or Fbxo5. X’s denote animals euthanized before they could be imaged. f Survival curves comparing mice transplanted with cells infected with empty vector (GFP) or Fbxo5 (GFP n = 28, Fbxo5 n = 27). p Value < 0.0001 was determined by Log-rank (Mantel–Cox) test. g FBXO5 expression in human Group 3/Group 4 MB samples with or without GFI1/GFI1B activation (GFI1/GFI1B n = 17, WT n = 130). p Value was determined by one-sided Welch Two Sample t test. Whiskers indicate minimum and maximum expression values, bottom of boxes indicate first quartiles, midline indicates median expression values, and top of boxes indicate third quartiles Full size image

As an additional approach to elucidate the targets of Gfi1, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) in MG tumors and identified 10,840 significant peak regions bound by Gfi1 (Supplementary Data 2). Since we have demonstrated that Lsd1 is an essential cofactor of Gfi1 in MG tumors, we predicted that Gfi1/Lsd1 might co-occupy similar genomic regions and therefore carried out ChIP-seq using antibodies specific for Lsd1 as well. We identified 12,083 peaks where Lsd1 was bound and confirmed a high concordance between Lsd1 and Gfi1 peaks (Supplementary Fig. 8a, Supplementary Data 2): 9594 peaks were common to both Gfi1 and Lsd1 datasets, representing ~89% of all Gfi1 peaks and ~79% of all Lsd1 peaks (Supplementary Fig. 8b). The sizable overlap between Gfi1 and Lsd1 binding in the genome further substantiates that these proteins interact to co-regulate common downstream target genes and pathways.

To evaluate the functional relevance of predicted Gfi1 target genes, we tested a subset of the genes that were bound by Gfi1 and differentially expressed in MG tumors compared to NSCs. We focused specifically on genes that have been reported to regulate differentiation, which were downregulated in MG tumors (Fig. 6d, Supplementary Fig. 9a, c, e, g). We hypothesized that genes whose downregulation was critical for tumorigenesis might inhibit tumor growth if they were re-expressed in tumor cells. Therefore, we used retroviruses to overexpress these genes in tumor cells and examined the effects on tumor growth in vivo. While the majority of genes we tested did not affect tumor growth or latency (Supplementary Fig. 9b, d, f, h), overexpression of Fbxo5 caused a significant delay in tumor formation (Fig. 6e, f). Notably, analysis of human MB showed that FBXO5 levels were lower in GFI1/1B-activated Group 3/Group 4 tumors than in tumors without GFI1/1B-activation (Fig. 6g). Together these findings suggest that Fbxo5 may be a downstream effector of Gfi1 in MB.

Pharmacological inhibition of Lsd1 to treat Gfi1-driven MB

Based on our finding that the interaction between Gfi1 and Lsd1 is crucial for MG tumor growth, we sought to determine whether small molecule inhibitors of Lsd1 could serve as therapeutic agents for these tumors. We performed thymidine incorporation assays on cells treated with two different Lsd1 inhibitors: GSK-LSD1 and ORY-1001. Both compounds potently inhibited proliferation of MG tumor cells in vitro, with IC 50 s ranging from 0.05 to 0.1 nM (Fig. 7a, b). In contrast, the effect on MB tumor models not driven by Gfi1 was much less pronounced. As shown in Fig. 7a, b, the proliferation of MP tumor cells was inhibited only with much higher concentrations of these compounds (IC 50 = 440–3300 nM). Cells derived from Glt1-tTA:TRE-MYCN/Luc (GTML) and Math1-Cre;Ptch1fl/fl Luciferase (MPL) tumors, models of Group 3 and SHH MB respectively, were also relatively insensitive to Lsd1 inhibitors (Supplementary Fig. 10a, b). Finally, we tested the effects of GSK-LSD1 and ORY-1001 on normal granule neurons, and did not see adverse effects on viability (Fig. 7c). These data suggest that pharmacological inhibition of Lsd1 potently and selectively inhibits proliferation of Gfi1-activated tumor cells.

Fig. 7 Pharmacological inhibitors of Lsd1 inhibit growth of Gfi1-driven MB. a, b In vitro proliferation assays for MG and MP tumor cells treated with a GSK-LSD1 or b ORY-1001. Proliferation was measured via 3H-thymidine incorporation at 48hrs. c Cell viability of post-mitotic granule neurons treated with GSK-LSD1 (blue) or ORY-1001 (purple). Viability was measured via CellTiter-Glo Luminescent Assay at 48 h. Data shown in a–c are from representative experiments and are plotted as the means of technical triplicate samples ± SEM. All experiments were repeated in at least three biological replicates. d–h Mice implanted with subcutaneous MG tumors underwent surgical resection of tumors and were subsequently treated with vehicle (saline) or 10 mg/kg GSK-LSD1 in cycles of four days on and three days off (vehicle n = 21, GSK-LSD1 n = 22). d Representative images of two tumors before (left) and after (right) surgical resection. Tumors are outlined by white dotted lines. Tumor growth was monitored weekly by e bioluminescent imaging and f caliper measurements. Red arrowhead indicates time of surgical resection. Blue arrowhead indicates start of drug treatment. Data shown are from a representative experiment and are plotted as the means ± SEM. When tumors reached the maximum allowed diameter, mice were sacrificed and resulting tumors were g collected and h weighed. Compared to resection alone, resection and GSK-LSD1 treatment significantly reduced tumor burden in mice. p < 0.0001, t = 6.042, df = 41, one-tailed unpaired t test. Data shown in h are from three replicate experiments and plotted as the means ± SD Full size image

To test whether Lsd1 inhibition also impedes tumor growth in vivo, we implanted MG tumors into the flanks of NSG mice and treated them with vehicle (4% DMSO in saline) or 10 mg/kg GSK-LSD1. Tumor growth was monitored weekly by bioluminescent imaging and caliper measurements (Supplementary Fig. 11a, b). When tumors reached 2 cm in diameter, the experiment was terminated, and tumors were collected and weighed (Supplementary Fig. 11c, d). As shown in Supplementary Fig. 11a-d, treatment with GSK-LSD1 significantly slowed tumor growth and decreased the size of MG tumors in vivo. We also tested the effects of Lsd1 inhibitors on mice bearing intracranial MG tumors, but saw no effects on tumor growth or survival (Supplementary Fig. 12). The fact that we observed inhibition of MG tumors in the flank but not in the brain suggests that these compounds do not accumulate to sufficient levels within brain tumors to exert a therapeutic effect.

The experiments above tested the effects of Lsd1 inhibitors alone. Because standard therapy for MB includes surgery and radiation, we asked whether Lsd1 inhibitors could be combined with these modalities. To test the combination with surgical resection, we implanted MG tumors into the flanks of NSG mice, and when tumors reached a volume of 200–300 mm3, we performed surgery to remove the bulk of the tumor (Fig. 7d). After 1 week of recovery, mice were randomized into treatment groups based on tumor size, and treated with vehicle (saline) or 10 mg/kg GSK-LSD1. Tumor growth was monitored via bioluminescent imaging and caliper measurements, and endpoint tumors were collected and weighed. As shown in Fig. 7e–h, GSK-LSD1 was highly effective at suppressing tumor growth following surgical resection. GSK-LSD1 also potently inhibited tumor growth after ionizing radiation (Supplementary Fig. 11e–h). These results strongly support the notion that targeting Lsd1 with small molecule inhibitors could be an effective strategy for treating patients with Gfi1-driven MB.