Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12 with expression of FKBP12 in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRAS loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

Osteosarcoma is the most common primary bone tumor, yet there have been no substantial advances in treatment or survival in three decades. We examined 59 tumor/normal pairs by whole-exome, whole-genome, and RNA-sequencing. Only the TP53 gene was mutated at significant frequency across all samples. The mean nonsilent somatic mutation rate was 1.2 mutations per megabase, and there was a median of 230 somatic rearrangements per tumor. Complex chains of rearrangements and localized hypermutation were detected in almost all cases. Given the intertumor heterogeneity, the extent of genomic instability, and the difficulty in acquiring a large sample size in a rare tumor, we used several methods to identify genomic events contributing to osteosarcoma survival. Pathway analysis, a heuristic analytic algorithm, a comparative oncology approach, and an shRNA screen converged on the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway as a central vulnerability for therapeutic exploitation in osteosarcoma. Osteosarcoma cell lines are responsive to pharmacologic and genetic inhibition of the PI3K/mTOR pathway both in vitro and in vivo., the most common primary bone tumor, is an aggressive cancer that affects children, adolescents, and young adults. In contrast to the improvements in 5-year overall survival for childhood cancers from 58% to 82% in the past three decades, the overall survival for pediatric OS has remained static over that same time period at 60% (1, 2).Predisposition to OS is associated with germline syndromes, including hereditary retinoblastoma and Li-Fraumeni syndrome (3, 4). OS is also seen in syndromes with mutations in RECQ helicases and SQSTM1 (5, 6). However, most cases of OS develop sporadically and are characterized by complex genomics. The first genome-wide association study conducted in OS only identified two susceptibility loci implicating one gene, GRM4, a glutamate receptor (7).Linkage with hereditary retinoblastoma and Li Fraumeni led to the recognition of recurrent somatic alterations in TP53, RB1, and genes interacting with TP53 and RB1 in OS (8, 9). Candidate-gene approaches demonstrated recurrent somatic mutations, deletions, and rearrangement affecting TP53 (9). Additional mechanisms of p53 inactivation described in OS are MDM2 and COPS3 amplification (8, 9). RB1 mutations are present in 6% and deletions or structural alterations are seen in 40% of cases (10,11). CDKN2A is deleted in 10-20% of OS (9, 12). Multiple other cancer-associated genes have been reported to be altered in OS [reviewed in Kansara and Thomas (5)]. Many of these studies SignificanceWe present, to our knowledge, the first comprehensive nextgeneration sequencing of osteosarcoma in combination with a functional genomic screen in a genetically defined mouse model of osteosarcoma. Our data provide a strong rationale for targeting the phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway in osteosarcoma and a foundation for rational clinical trial design. These findings present an immed...

Osteosarcoma is the most common primary malignant tumor of bone. Analysis of familial cancer syndromes and sporadic cases has strongly implicated both p53 and pRb in its pathogenesis; however, the relative contribution of these mutations to the initiation of osteosarcoma is unclear. We describe here the generation and characterization of a genetically engineered mouse model in which all animals develop short latency malignant osteosarcoma. The genetically engineered mouse model is based on osteoblast-restricted deletion of p53 and pRb. Osteosarcoma development is dependent on loss of p53 and potentiated by loss of pRb, revealing a dominance of p53 mutation in the development of osteosarcoma. The model reproduces many of the defining features of human osteosarcoma including cytogenetic complexity and comparable gene expression signatures, histology, and metastatic behavior. Using a novel in silico methodology termed cytogenetic region enrichment analysis, we demonstrate high conservation of gene expression changes between murine osteosarcoma and known cytogentically rearranged loci from human osteosarcoma. Due to the strong similarity between murine osteosarcoma and human osteosarcoma in this model, this should provide a valuable platform for addressing the molecular genetics of osteosarcoma and for developing novel therapeutic strategies.[Keywords: Cancer; mouse model; osteocarcinoma] Supplemental material is available at http://www.genesdev.org.

Highlights d A selective, orally bioavailable Menin-MLL inhibitor, VTP50469, is developed d Displacement of Menin from chromatin leads to loss of MLL from specific loci d Treatment with VTP50469 leads to suppression of a subset of MLL fusion target genes d Treatment with VTP50469 improves survival in PDX models of MLL-r ALL