Deletion of the p27(Kip1) gene restores normal development in cyclin D1-deficient mice

D-type cyclins (cyclins D1, D2, and D3) are key components of cell cycle machinery in mammalian cells. These proteins are believed to drive cell cycle progression by associating with their kinase partners, cyclin-dependent kinases, and by directing phosphorylation of critical cellular substrates. In addition, D-cyclins play a kinase-independent role by sequestering cell cycle inhibitors p27(Kip1) and p21(Cip1). In the past, we and others generated cyclin D1-deficient mice and have shown that these mice display developmental abnormalities, hypoplastic retinas, and pregnancy-insensitive mammary glands. To test the significance of cyclin D1–p27(Kip1) interaction within a living mouse, we crossed cyclin D1-deficient mice with mice lacking p27(Kip1), and we generated double-mutant cyclin D1(−/−)p27(−/−) animals. Here we report that ablation of p27(Kip1) restores essentially normal development in cyclin D1-deficient mice. Our results provide genetic evidence that p27(Kip1) functions downstream of cyclin D1

Development of mice expressing a single D-type cyclin

D-cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery. To directly test the ability of each D-cyclin to drive development of various lineages, we generated mice expressing only cyclin D1, or only cyclin D2, or only cyclin D3. We found that these “single-cyclin” embryos develop normally until late gestation. Our analyses revealed that in single-cyclin embryos, the tissue-specific expression pattern of D-cyclins was lost. Instead, mutant embryos ubiquitously expressed the remaining D-cyclin. These findings suggest that the functions of the three D-cyclins are largely exchangeable at this stage. Later in life, single-cyclin mice displayed focused abnormalities, resulting in premature mortality. “Cyclin D1-only” mice developed severe megaloblastic anemia, “cyclin D2-only” mice presented neurological abnormalities, and “cyclin D3-only” mice lacked normal cerebella. Analyses of the affected tissues revealed that these compartments failed to sufficiently up-regulate the remaining, intact D-cyclin. In particular, we found that in cerebellar granule neuron precursors, the N-myc transcription factor communicates with the cell cycle machinery via cyclins D1 and D2, but not D3, explaining the inability of D3-only mice to up-regulate cyclin D3 in this compartment. Hence, the requirement for a particular cyclin in a given tissue is likely caused by specific transcription factors, rather than by unique properties of cyclins

Preclinical activity of selinexor, an inhibitor of XPO1, in sarcoma

Selinexor is an orally bioavailable selective inhibitor of nuclear export that has been demonstrated to have preclinical activity in various cancer types and that is currently in Phase I and II clinical trials for advanced cancers. In this study, we evaluated the effects of selinexor in several preclinical models of various sarcoma subtypes. The efficacy of selinexor was investigated in vitro and in vivo using 17 cell lines and 9 sarcoma xenograft models including gastrointestinal stromal tumor (GIST), liposarcoma (LPS), leiomyosarcoma, rhabdomyosarcoma, undifferentiated sarcomas, and alveolar soft part sarcoma (ASPS). Most sarcoma cell lines were sensitive to selinexor with IC50s ranging from 28.8 nM to 218.2 nM (median: 66.1 nM). Selinexor suppressed sarcoma tumor xenograft growth, including models of ASPS that were resistant in vitro. In GIST cells with KIT mutations, selinexor induced G1- arrest without attenuation of phosphorylation of KIT, AKT, or MAPK, in contrast to imatinib. In LPS cell lines with MDM2 and CDK4 amplification, selinexor induced G1-arrest and apoptosis irrespective of p53 expression or mutation and irrespective of RB expression. Selinexor increased p53 and p21 expression at the protein but not RNA level, indicating a post-transcriptional effect. These results indicate that selinexor has potent in vitro and in vivo activity against a wide variety of sarcoma models by inducing G1-arrest independent of known molecular mechanisms in GIST and LPS. These studies further justify the exploration of selinexor in clinical trials targeting various sarcoma subtypes

MAX inactivation is an early event in GIST development that regulates p16 and cell proliferation

KIT, PDGFRA, NF1 and SDH mutations are alternate initiating events, fostering hyperplasia in gastrointestinal stromal tumours (GISTs), and additional genetic alterations are required for progression to malignancy. The most frequent secondary alteration, demonstrated in ∼70% of GISTs, is chromosome 14q deletion. Here we report hemizygous or homozygous inactivating mutations of the chromosome 14q MAX gene in 16 of 76 GISTs (21%). We find MAX mutations in 17% and 50% of sporadic and NF1-syndromic GISTs, respectively, and we find loss of MAX protein expression in 48% and 90% of sporadic and NF1-syndromic GISTs, respectively, and in three of eight micro-GISTs, which are early GISTs. MAX genomic inactivation is associated with p16 silencing in the absence of p16 coding sequence deletion and MAX induction restores p16 expression and inhibits GIST proliferation. Hence, MAX inactivation is a common event in GIST progression, fostering cell cycle activity in early GISTs

MAX inactivation is an early event in GIST development that regulates p16 and cell proliferation

KIT, PDGFRA, NF1 and SDH mutations are alternate initiating events, fostering hyperplasia in gastrointestinal stromal tumours (GISTs), and additional genetic alterations are required for progression to malignancy. The most frequent secondary alteration, demonstrated in ∼70% of GISTs, is chromosome 14q deletion. Here we report hemizygous or homozygous inactivating mutations of the chromosome 14q MAX gene in 16 of 76 GISTs (21%). We find MAX mutations in 17% and 50% of sporadic and NF1-syndromic GISTs, respectively, and we find loss of MAX protein expression in 48% and 90% of sporadic and NF1-syndromic GISTs, respectively, and in three of eight micro-GISTs, which are early GISTs. MAX genomic inactivation is associated with p16 silencing in the absence of p16 coding sequence deletion and MAX induction restores p16 expression and inhibits GIST proliferation. Hence, MAX inactivation is a common event in GIST progression, fostering cell cycle activity in early GISTs.status: publishe

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;Kras(G12D/+);Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5(+) stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.</p