Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis

The evolution of colorectal cancer suggests the involvement of many genes. We performed insertional mutagenesis with the Sleeping Beauty (SB) transposon system in mice carrying germline or somatic Apc mutation. Analysis of common insertion sites (CISs) isolated from 446 tumors revealed many hundreds of candidate cancer drivers. Comparison to human datasets suggested that 234 CIS genes are also deregulated in human colorectal cancers. 183 CIS genes are candidate Wnt targets, and 20 are shown to be novel modifiers of canonical Wnt signaling. We also identified gene mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included members of the FGF signaling cascade. Some 70 genes showed pairwise co-occurrence clustering into 38 sub-networks that may regulate tumor development

Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in PtenLoxP/LoxP;BrafCA/+ Mice

Current genetically-engineered mouse melanoma models are often based on Tyr::CreERT2-controlled MAPK pathway activation by the BRAFV600E mutation and PI3K pathway activation by loss of PTEN. The major drawback of these models is the occurrence of spontaneous tumors caused by leakiness of the Tyr::CreERT2 system, hampering long-term experiments. To address this problem, we investigated several approaches to optimally provide local delivery of Cre recombinase, including injection of lentiviral particles, DNA tattoo administration and particle-mediated gene transfer, to induce melanomas in PtenLoxP/LoxP;BrafCA/+ mice lacking the Tyr::CreERT2 allele. We found that dermal delivery of the Cre recombinase gene under the control of a non-specific CAG promoter induced the formation of melanomas, but also keratoacanthoma and squamous cell carcinomas. Delivery of Cre recombinase DNA under the control of melanocyte-specific promoters in PtenLoxP/LoxP;BrafCA/+ mice resulted in sole melanoma induction. The growth rate and histological features of the induced tumors were similar to 4-hydroxytamoxifen-induced tumors in Tyr::CreERT2;PtenLoxP/LoxP;BrafCA/+ mice, while the onset of spontaneous tumors was prevented completely. These novel induction methods will allow long-term experiments in mouse models of skin malignancies

GFAP-Cre-Mediated Transgenic Activation of Bmi1 Results in Pituitary Tumors

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16(INK4A)/Rb rather than on regulation of p19(ARF)/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse mode

Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma

Immunotherapy of advanced melanoma with CTLA-4 or PD-1/PD-L1 checkpoint blockade induces in a proportion of patients long durable responses. In contrast, targeting the MAPK-pathway by selective BRAF and MEK inhibitors induces high response rates, but most patients relapse. Combining targeted therapy with immunotherapy is proposed to improve the long-term outcomes of patients. Preclinical data endorsing this hypothesis are accumulating. Inhibition of the PI3K-Akt-mTOR pathway may be a promising treatment option to overcome resistance to MAPK inhibition and for additional combination with immunotherapy. We therefore evaluated to which extent dual targeting of the MAPK and PI3K-Akt-mTOR pathways affects tumor immune infiltrates and whether it synergizes with PD-1 checkpoint blockade in a BRAFV600E/PTEN-/--driven melanoma mouse model. Short-term dual BRAF + MEK inhibition enhanced tumor immune infiltration and improved tumor control when combined with PD-1 blockade in a CD8+ T cell dependent manner. Additional PI3K inhibition did not impair tumor control or immune cell infiltration and functionality. Analysis of on-treatment samples from melanoma patients treated with BRAF or BRAF + MEK inhibitors indicates that inhibitor-mediated T cell infiltration occurred in all patients early after treatment initiation but was less frequent found in on-treatment biopsies beyond day 15. Our findings provide a rationale for clinical testing of short-term BRAF + MEK inhibition in combination with immune checkpoint blockade, currently implemented at our institutes. Additional PI3K inhibition could be an option for BRAF + MEK inhibitor resistant patients that receive targeted therapy in combination with immune checkpoint blockade

IHC analysis of pituitary hormones in Gcre; Bm1^LSL induced tumors.

<p>n.d., not done, n.o., not observed.</p

Mendelian distribution of transgenic allele before and after birth.

<p>Mendelian distribution of transgenic allele before and after birth.</p

Reported over expression of Bmi1 in astroglial and neural tumors.

<p>Reported over expression of Bmi1 in astroglial and neural tumors.</p

Transgenic expression of Bmi1 is sufficient to induce intermediate and anterior lobe pituitary tumors but does not induce medulloblastoma or glioma.

<p>(A) Kaplan Meier survival curves of mice carrying tumors because of GCre induced transgenic expression of Bmi1 complemented with loss of Rb. These data show that Bmi1 transgenic mice develop pituitary tumors after about one year. Pituitary tumors are also observed in Rb^Lox/Lox and Bmi1^LSL;Rb^Lox/Lox transgenic mice. (B) Histograms showing the relative frequency and penetrance of tumors generated by the individual genotypic groups. All genotypes shown are GCre positive. Total cohort size: GCre;Bmi1^LSL n = 14, GCre;Rb^Lox/Lox n = 20, GCre;Rb^Lox/Lox;Bmi1^LSL n = 12, GCre;p53^Lox/Lox;Rb^Lox/Lox n = 6, GCre;p53^Lox/Lox;Rb^Lox/Lox; Bmi1^LSL n = 4, WT mice did not develop tumors, n = 7. (C) IHC results showing transgenic Bm1 expression in tumors raised on a GCre;Bmi1^LSL background (5/5), no expression in tumors raised on a Gcre;Rb^lox/lox background (0/5) while some of the GCre;Bmi1^LSL; Rb^lox/lox mice were positive (2/5, 40%). These results are summarized in a histogram (D). Bar is 50 µm.</p

Histology of a representative example of a typical pituitary tumor as found in this study.

<p>(A) Average location of pituitary tumors observed in the GCre;Bmi1^LSL mice showing localization to the pituitary gland (n = 6). Localization is based on Allen’s mouse brain database, (B) H&E staining of a coronal section of the brain showing a typical pituitary tumor. Immunohistochemistry shows expression analysis of (C) Chromogranin A, (D) adrenocorticotropic hormone ACTH, (E) Growth Hormone hGH, (F) Prolactin PRL, (G) Thyroid Stimulating Hormone TSH, (H) cytokeratin 8/18 CAM5.2 and (I) CD56/NCAM. Of these markers, ACTH is clearly positive in this tumor. Electron microscopy (EM) shows secretory vesicles (indicated by the arrowheads) in Bmi1 transgenic tumors (J, K) in a similar way as observed in human tumors (M, N). Ki67 is clearly positive in this tumor (L). Immunohistochemistry of human pituitary adenomas show over expression of BMI1, which is visualized by a nuclear signal (O). Bars, 100 µm unless otherwise noted.</p