Identify the experimental technique. Describe the experimental treatment (drug o
ID: 264595 • Letter: I
Question
Identify the experimental technique. Describe the experimental treatment (drug or genetic approach to introduce a change in the experimental condition) and control(s).
Describe the data the authors reported in the panels. Summarize the authors’ interpretation of the data.
Panels
Technique Used and Methodology
Results and Interpretation
2 a, h
2 b, i
Using DNp63 (ref. 15) and DNp73 conditional knockout mice (Extended Data Fig. 1a, b), we generated DNp631/2 and DNp732/2 mice (Extended Data Fig. 1c–f). To ask whether the DN isoforms of p63 and p73 act as oncogenesin vivo by interacting with p53,DNp631/2;p532/2 and DNp732/2;p532/2 mice were aged for the development of thymic lymphomas,whichformin nearly all p532/2 mice16.Wefound a remarkable diminution in the number and size of thymic lymphomas in DNp631/2;p532/2 andDNp732/2;p532/2 mice, leading to an extended lifespan (Extended Data Fig. 2a–c) and suggesting that theDN isoforms of p63 and p73 restrain a tumour suppressive program that can compensate for p53 function. We found that TAp63 and TAp73 were upregulated in thymic lymphomasfromDNp631/2;p532/2 andDNp732/2;p532/2 mice (Extended Data Fig. 2d, e) along with an upregulation of apoptosis (Extended Data Fig. 2f–j) and senescence (Extended Data Fig. 2k–o).We also examined thymocytes from 4-week-old mice after treatment with 10 Gy gamma irradiation, a dose that is known to elicit p53-dependent apoptosis9,17. Indeed,TAp63 andTAp73 are higherinDNp631/2;p532/2 andDNp732/2; p532/2 thymocytes, which was further exacerbated after gamma irradiation (Extended Data Fig. 3a–c)with an increase in apoptosis (Extended Data Fig. 3d–h) and senescence (Extended Data Fig. 3i–m). To determine whether TAp63 or TAp73 compensatefor p53function in tumoursin vivo, we acutely removedDNp63 orDNp73 byintratumoral infection with adenovirus-Cre-mCherry (Extended Data Fig. 4a–d and Fig. 1a–f) inDNp63fl/fl;p532/2 andDNp73fl/fl;p532/2 at 10 weeks of age. Tumours were 2.3–5.8 mm3 in size at the time of infection and monitored weekly bymagnetic resonance imaging (MRI; Fig. 1a–i).Mice deficient for either DNp63 or DNp73 and p53 showed marked decreases in tumour burden (Fig. 1h, i). The reduction ofDNp63 andDNp73 expression resulted in increased expression of TAp63 and TAp73 (Fig. 1j–m and Extended Data 4d) and increased apoptosis (Extended Data Fig. 4e–h) and senescence (Extended Data Fig. 4i–k).DNp63D/D ;p532/2 and DNp73D/D ;p532/2 mice also had anincreased lifespan (Fig. 1n).Wefound differences in CD4/CD8-positive cells in young mice (4 weeks) (Extended Data Fig. 4l–p), indicating that changes in T-cell development may lead to a lower tumour incidence in double-mutant mice. Indeed, we found that p532/2 thymic lymphomas are composed primarily of CD4/CD8 double-positive thymocytes while theDNp63D/D ;p532/2 and DNp73D/D ; p532/2 lymphomas contain very few CD4/CD8 double-positive thymocytes (Extended Data Fig. 4q–t). Lastly, we asked whether thymic stromal cells contribute to the apoptosis in the regressing lymphomas. We sorted CD45-positive cells to select for T lymphocytes in p532/2, DNp63fl/fl;p532/2 and DNp73fl/fl;p532/2 mice and infected them with adenovirus-Cre (Extended Data Fig. 4u).DNp63D/D ;p532/2 andDNp73D/D ; p532/2 thymocytes underwent apoptosis independent of the presence of the stromal cells (Extended Data Fig. 4v). These data indicate that inhibition of the DN isoforms of p63 and p73 serves to upregulate TAp63 and TAp73 to compensate for loss of p53 in tumour suppression. We found that the DN isoforms of p63 and p73 bind to the promoters of the TA isoforms of p63 and p73, suggesting that theDN isoforms of p63 and p73 can transcriptionally repress TAp63 and TAp73 transcription (Extended Data Fig. 5a–i). We also found that the increase in apoptosis and cellular senescence was dependent on TAp63 and TAp73 (Extended Data Fig. 5j–q). We performed RNA sequencing of lymphomas after infection with Ad-mCherry (DNp63fl/fl;p532/2 and DNp73fl/fl;p532/2) and Ad-CremCherry (DNp63D/D ;p532/2 and DNp73D/D ;p532/2) and found that thymic lymphomas from mice deficient for p53 and DNp63 clustered with thosefrommice deficientfor p53 andDNp73 (Extended Data Fig. 6a). Ingenuity pathway analysis (IPA) (Fig. 1q) revealed genes involved in metabolism including TP53-inducible glycolysis and apoptosis regulator (TIGAR) 18, and glutaminase 2 (GLS2) 19,20. While we found that TIGAR and GLS2 were upregulated in either DNp63D/D ;p532/2 or DNp73D/D ;p532/2 thymic lymphomas, we identified a novel gene, islet amyloid polypeptide (IAPP) or amylin, which was upregulated by over fivefold in both double-mutant thymic lymphomas. IAPP limits glucose uptake, resulting in increased intracellular glucose-6-phosphate (G-6-P)21 levels and decreased glycolysis21.We validatedIAPP,TIGARand GLS2 expression in thymic lymphomas derived fromDNp63D/D ;p532/2 and DNp73D/D ;p532/2 mice and found that IAPP is expressed at levels over twofold higher in double-mutant mice (Fig. 1p and Extended Data Fig. 6b–d). IAPP and GLS2 expression depend on TAp63 and TAp73 (Fig. 1q and Extended Data Fig. 6d). To determine whether TAp63 or TAp73 transcriptionally regulate IAPP, we performed chromatin immunoprecipitation in mouse embryonic fibroblasts (MEFs; Extended Data Fig. 6e–g) and thymocytes (Fig. 1r, s).Wefound that TAp63 and TAp73 bind to sites located in the promoter (site 1), 1,756 nucleotides upstream of the transcriptional start site, and intron 2 (site 2) of IAPP, 706 nucleotides downstream of the transcriptional start site (Extended Data Fig. 6e–g). Because a greater binding affinity of TAp63 and TAp73 was detected in the promoter region (site 1) ofIAPP, we cloned this site into a luciferase reporter gene and also mutated this site (Extended Data Fig. 6h–k). Only the luciferase reporter gene containing wild-type IAPP promoter site 1 was transactivated by TAp63 and TAp73 whereas the mutant version was not. Taken together, these data indicate that IAPP is a transcriptional target gene of TAp63 and TAp73 (Fig. 1t). Expression of IAPP in p532/2MEFs resulted in lowlevels of glycolysis comparable to that in DNp632/2;p532/2 and DNp732/2;p532/2 MEFs (Extended Data Fig. 6l–m and Fig. 1u). Conversely, when we knocked downIAPPinDNp632/2;p532/2 andDNp732/2;p532/2MEFs, the levels of glycolysis were similar to that of p532/2 MEFs (Fig. 1u) indicating that IAPP inhibits glycolysis.In vivo, we detected massive tumour regression in DNp63fl/fl;p532/2 or DNp73fl/fl;p532/2 thymic lymphomas treated with IAPP (Extended Data Fig. 7a and Fig. 2a, b, h, i, o, p), P , 0.05. Conversely, in DNp63D/D;p532/2 and DNp73D/D;p532/2 thymic lymphomas treated with Ad-shIAPP-mCherry the tumours continued to grow comparable to that of p532/2 thymic lymphomas (Fig. 2a–k, o–r), P . 0.05 at 13 weeks. Additionally, p532/2 mice treated with Ad-IAPP had an extended tumour-free survival period compared to p532/2 mice orDNp63D/D ;p532/2 andDNp73D/D ;p532/2 mice treated intratumorally with Ad-shIAPP-mCherry (Extended Data Fig. 7a, b), indicating that IAPP is a tumour suppressor gene and is causally involved in the in vivo effects seen upon inactivation of DNp63 or DNp73. Given that pramlintide, a synthetic analogue of amylin, is used to treat type I and type II diabetes22, we treated thymic lymphomas in DNp63fl/fl;p532/2 andDNp73fl/fl;p532/2 mice. Indeed, three-weekly intratumoral injections resulted in rapid tumour regression (Fig. 2e, l, s), P , 0.005 at 13 weeks.
Figure 2 | IAPP is causally involved in tumorigenesis suppression in p53-deficient thymic lymphomas. a–n, Thymic lymphomas were infected with adenovirus (Ad)-mCherry (a, h), Ad-IAPP-mCherry (1IAPP) (b, i), Ad-shIAPP-mCherry (c, d, j, k), or treated with pramlintide intratumorally (IT) (e, l) or intravenously (IV) (f, m), or with 2DG (g, n). Yellow dashed lines indicate tumour. Volume of tumour shown. UN-D, undetectable. o–u, Quantification of the indicated thymic lymphomas, n 5 5 mice per group. Significance indicated by the asterisks, P , 0.005. v, Quantification of in vivo pyruvate to lactate conversion using dynamic magnetic resonance spectroscopy as a measurement of glycolysis, n 5 3 mice, P , 0.005. w, Immunohistochemistry for reactive oxygen species (ROS) or cleaved caspase 3. Positive nuclei are brown.
Panels
Technique Used and Methodology
Results and Interpretation
2 a, h
2 b, i
Explanation / Answer
Identify the experimental technique. Describe the experimental treatment (drug o