SUMMARY Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, whereas prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

SUMMARY Monitoring cancer and aging in vivo remains experimentally challenging. Here, we describe a luciferase knockin mouse (p16LUC), which faithfully reports expression of p16INK4a, a tumor suppressor and aging biomarker. Lifelong assessment of luminescence in p16+/LUC mice revealed an exponential increase with aging, which was highly variable in a cohort of contemporaneously housed, syngeneic mice. Expression of p16INK4a with aging did not predict cancer development, suggesting that the accumulation of senescent cells is not a principal determinant of cancer-related death. In 14 of 14 tested tumor models, expression of p16LUC was focally activated by early neoplastic events, enabling visualization of tumors with sensitivity exceeding other imaging modalities. Activation of p16INK4a was noted in the emerging neoplasm and surrounding stromal cells. This work suggests that p16INK4a activation is a characteristic of all emerging cancers, making the p16LUC allele a sensitive, unbiased reporter of neoplastic transformation.

The Collaborative Cross Consortium reports here on the development of a unique genetic resource population. The Collaborative Cross (CC) is a multiparental recombinant inbred panel derived from eight laboratory mouse inbred strains. Breeding of the CC lines was initiated at multiple international sites using mice from The Jackson Laboratory. Currently, this innovative project is breeding independent CC lines at the University of North Carolina (UNC), at Tel Aviv University (TAU), and at Geniad in Western Australia (GND). These institutions aim to make publicly available the completed CC lines and their genotypes and sequence information. We genotyped, and report here, results from 458 extant lines from UNC, TAU, and GND using a custom genotyping array with 7500 SNPs designed to be maximally informative in the CC and used a novel algorithm to infer inherited haplotypes directly from hybridization intensity patterns. We identified lines with breeding errors and cousin lines generated by splitting incipient lines into two or more cousin lines at early generations of inbreeding. We then characterized the genome architecture of 350 genetically independent CC lines. Results showed that founder haplotypes are inherited at the expected frequency, although we also consistently observed highly significant transmission ratio distortion at specific loci across all three populations. On chromosome 2, there is significant overrepresentation of WSB/EiJ alleles, and on chromosome X, there is a large deficit of CC lines with CAST/EiJ alleles. Linkage disequilibrium decays as expected and we saw no evidence of gametic disequilibrium in the CC population as a whole or in random subsets of the population. Gametic equilibrium in the CC population is in marked contrast to the gametic disequilibrium present in a large panel of classical inbred strains. Finally, we discuss access to the CC population and to the associated raw data describing the genetic structure of individual lines. Integration of rich phenotypic and genomic data over time and across a wide variety of fields will be vital to delivering on one of the key attributes of the CC, a common genetic reference platform for identifying causative variants and genetic networks determining traits in mammals.

The claudin-low subtype is a recently identified rare molecular subtype of human breast cancer that expresses low levels of tight and adherens junction genes and shows high expression of epithelial-to-mesenchymal transition (EMT) genes. These tumors are enriched in gene expression signatures derived from human tumorinitiating cells (TICs) and human mammary stem cells. Through crossspecies analysis, we discovered mouse mammary tumors that have similar gene expression characteristics as human claudin-low tumors and were also enriched for the human TIC signature. Such claudinlow tumors were similarly rare but came from a number of distinct mouse models, including the p53 null transplant model. Here we present a molecular characterization of 50 p53 null mammary tumors compared with other mouse models and human breast tumor subtypes. Similar to human tumors, the murine p53 null tumors fell into multiple molecular subtypes, including two basallike, a luminal, a claudin-low, and a subtype unique to this model. The claudin-low tumors also showed high gene expression of EMT inducers, low expression of the miR-200 family, and low to absent expression of both claudin 3 and E-cadherin. These murine subtypes also contained distinct genomic DNA copy number changes, some of which are similarly altered in their cognate human subtype counterpart. Finally, limiting dilution transplantation revealed that p53 null claudin-low tumors are highly enriched for TICs compared with the more common adenocarcinomas arising in the same model, thus providing a unique preclinical mouse model to investigate the therapeutic response of TICs. genetically engineered mouse model | gene profiling | array comparative genomic hybridization B reast cancer (BC) is the second leading cause of cancerrelated deaths among women in the United States (1). The large compendium of underlying genetic alterations and the resulting histological and molecular subtypes illustrate the heterogeneous nature of this disease. Both this intertumor heterogeneity and the cellular heterogeneity found within a breast tumor (intratumor heterogeneity) are major obstacles to effective treatments. One common feature of BC (and most cancers) is the loss of the tumor suppressor p53 function. p53 has been shown to be mutated in ≈40% of BCs, associated with poor clinical outcomes, and a higher frequency of mutations occurs in more-aggressive molecular subtypes, including the basal-like subtype of human BC (2).Mice homozygous for p53 loss have been shown to develop lymphomas and sarcomas with a short latency (3, 4). When crossed into the BALB/c background, mammary tumors were observed in p53 +/− mice (5). To circumvent the appearance of other tumor types that occurred with short latency, the model was further modified (6); namely, 6-wk-old p53 −/− glands were removed and transplanted into 3-wk-old wild-type BALB/c recipients. These mice develop mammary tumors stochastically with an average latency of approximately 12 mo. Interestingly, the p53 null epithelium initially forms normal du...

Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumor formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumors with OCCC-like histopathology, culminating in hemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting tumor cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signaling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumor growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodeling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signaling. We propose that ARID1A protects against inflammation-driven tumorigenesis.