Patient-derived xenografts and in vitro model show rationale for imatinib mesylate repurposing in HEY1-NCoA2-driven mesenchymal chondrosarcoma.

Mesenchymal chondrosarcoma (MCS) is a high-grade malignancy that represents 2-9% of chondrosarcomas and mostly affects children and young adults. HEY1-NCoA2 gene fusion is considered to be a driver of tumorigenesis and it has been identified in 80% of MCS tumors. The shortage of MCS samples and biological models creates a challenge for the development of effective therapeutic strategies to improve the low survival rate of MCS patients. Previous molecular studies using immunohistochemical staining of patient samples suggest that activation of PDGFR signaling could be involved in MCS tumorigenesis. This work presents the development of two independent in vitro and in vivo models of HEY1-NCoA2-driven MCS and their application in a drug repurposing strategy. The in vitro model was characterized by RNA sequencing at the single-cell level and successfully recapitulated relevant MCS features. Imatinib, as well as specific inhibitors of ABL and PDGFR, demonstrated a highly selective cytotoxic effect targeting the HEY1-NCoA2 fusion-driven cellular model. In addition, patient-derived xenograft (PDX) models of MCS harboring the HEY1-NCoA2 fusion were developed from a primary tumor and its distant metastasis. In concordance with in vitro observations, imatinib was able to significantly reduce tumor growth in MCS-PDX models. The conclusions of this study serve as preclinical results to revisit the clinical efficacy of imatinib in the treatment of HEY1-NCoA2-driven MCS.

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization.

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.

An epigenetic switch regulates the ontogeny of AXL-positive/EGFR-TKi-resistant cells by modulating miR-335 expression.

Despite current advancements in research and therapeutics, lung cancer remains the leading cause of cancer-related mortality worldwide. This is mainly due to the resistance that patients develop against chemotherapeutic agents over the course of treatment. In the context of non-small cell lung cancers (NSCLC) harboring EGFR-oncogenic mutations, augmented levels of AXL and GAS6 have been found to drive resistance to EGFR tyrosine kinase inhibitors such as Erlotinib and Osimertinib in certain tumors with mesenchymal-like features. By studying the ontogeny of AXL-positive cells, we have identified a novel non-genetic mechanism of drug resistance based on cell-state transition. We demonstrate that AXL-positive cells are already present as a subpopulation of cancer cells in Erlotinib-naïve tumors and tumor-derived cell lines and that the expression of AXL is regulated through a stochastic mechanism centered on the epigenetic regulation of miR-335. The existence of a cell-intrinsic program through which AXL-positive/Erlotinib-resistant cells emerge infers the need of treating tumors harboring EGFR-oncogenic mutations upfront with combinatorial treatments targeting both AXL-negative and AXL-positive cancer cells.

The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein.

Phosphatase and tensin homologue (PTEN) protein levels are critical for tumor suppression. However, the search for a recurrent cancer-associated gene alteration that causes PTEN degradation has remained futile. In this study, we show that Importin-11 (Ipo11) is a transport receptor for PTEN that is required to physically separate PTEN from elements of the PTEN degradation machinery. Mechanistically, we find that the E2 ubiquitin-conjugating enzyme and IPO11 cargo, UBE2E1, is a limiting factor for PTEN degradation. Using in vitro and in vivo gene-targeting methods, we show that Ipo11 loss results in degradation of Pten, lung adenocarcinoma, and neoplasia in mouse prostate with aberrantly high levels of Ube2e1 in the cytoplasm. These findings explain the correlation between loss of IPO11 and PTEN protein in human lung tumors. Furthermore, we find that IPO11 status predicts disease recurrence and progression to metastasis in patients choosing radical prostatectomy. Thus, our data introduce the IPO11 gene as a tumor-suppressor locus, which is of special importance in cancers that still retain at least one intact PTEN allele.

Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.

The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes.

TGF-ß reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24- cancer cells.

Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24- cell surface marker profile. Here, we report that human CD44+/CD24- cancer cells are genetically highly unstable because of intrinsic defects in their DNA-repair capabilities. In fact, in CD44+/CD24- cells, constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are crucial in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24- state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24- cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness.

TP53 exon-6 truncating mutations produce separation of function isoforms with pro-tumorigenic functions.

TP53 truncating mutations are common in human tumors and are thought to give rise to p53-null alleles. Here, we show that TP53 exon-6 truncating mutations occur at higher than expected frequencies and produce proteins that lack canonical p53 tumor suppressor activities but promote cancer cell proliferation, survival, and metastasis. Functionally and molecularly, these p53 mutants resemble the naturally occurring alternative p53 splice variant, p53-psi. Accordingly, these mutants can localize to the mitochondria where they promote tumor phenotypes by binding and activating the mitochondria inner pore permeability regulator, Cyclophilin D (CypD). Together, our studies reveal that TP53 exon-6 truncating mutations, contrary to current beliefs, act beyond p53 loss to promote tumorigenesis, and could inform the development of strategies to target cancers driven by these prevalent mutations.

Effectors and potential targets selectively upregulated in human KRAS-mutant lung adenocarcinomas.

Genetic and proteomic analysis of human tumor samples can provide an important compliment to information obtained from model systems. Here we examined protein and gene expression from the Cancer Genome and Proteome Atlases (TCGA and TCPA) to characterize proteins and protein-coding genes that are selectively upregulated in KRAS-mutant lung adenocarcinomas. Phosphoprotein activation of several MAPK signaling components was considerably stronger in KRAS-mutants than any other group of tumors, even those with activating mutations in receptor tyrosine kinases (RTKs) and BRAF. Co-occurring mutations in KRAS-mutants were associated with differential activation of PDK1 and PKC-alpha. Genes showing strong activation in RNA-seq data included negative regulators of RTK/RAF/MAPK signaling along with potential oncogenic effectors including activators of Rac and Rho proteins and the receptor protein-tyrosine phosphatase genes PTPRM and PTPRE. These results corroborate RAF/MAPK signaling as an important therapeutic target in KRAS-mutant lung adenocarcinomas and pinpoint new potential targets.

The Evolution of TP53 Mutations: From Loss-of-Function to Separation-of-Function Mutants.

As the most mutated gene in cancer, it is no surprise that TP53 has been the center of cancer biology discourse since its discovery in the late 1970s. Although early demonstrations of p53's role in the modulation of cell proliferation and survival solidified its classification as a tumor suppressor and transcription factor, our conceptualization of p53 is ever-evolving. Here, we present novel evidence of the role of alternative splicing isoforms, truncating/separation-of-function mutations, and hotspot silent mutations in the regulation of p53's activities.

MYC Drives Pten/Trp53-Deficient Proliferation and Metastasis due to IL6 Secretion and AKT Suppression via PHLPP2.

UNLABELLED: We have recently recapitulated metastasis of human PTEN/TP53-mutant prostate cancer in the mouse using the RapidCaP system. Surprisingly, we found that this metastasis is driven by MYC, and not AKT, activation. Here, we show that cell-cell communication by IL6 drives the AKT-MYC switch through activation of the AKT-suppressing phosphatase PHLPP2, when PTEN and p53 are lost together, but not separately. IL6 then communicates a downstream program of STAT3-mediated MYC activation, which drives cell proliferation. Similarly, in tissues, peak proliferation in Pten/Trp53-mutant primary and metastatic prostate cancer does not correlate with activated AKT, but with STAT3/MYC activation instead. Mechanistically, MYC strongly activates the AKT phosphatase PHLPP2 in primary cells and prostate cancer metastasis. We show genetically that Phlpp2 is essential for dictating the proliferation of MYC-mediated AKT suppression. Collectively, our data reveal competition between two proto-oncogenes, MYC and AKT, which ensnarls the Phlpp2 gene to facilitate MYC-driven prostate cancer metastasis after loss of Pten and Trp53. SIGNIFICANCE: Our data identify IL6 detection as a potential causal biomarker for MYC-driven metastasis after loss of PTEN and p53. Second, our finding that MYC then must supersede AKT to drive cell proliferation points to MYC inhibition as a critical part of PI3K pathway therapy in lethal prostate cancer.

p53Ψ is a transcriptionally inactive p53 isoform able to reprogram cells toward a metastatic-like state.

Although much is known about the underlying mechanisms of p53 activity and regulation, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation involving alternative splicing of the TP53 gene. We found that the use of an alternative 3' splice site in intron 6 generates a unique p53 isoform, dubbed p53Ψ. At the molecular level, p53Ψ is unable to bind to DNA and does not transactivate canonical p53 target genes. However, like certain p53 gain-of-function mutants, p53Ψ attenuates the expression of E-cadherin, induces expression of markers of the epithelial-mesenchymal transition, and enhances the motility and invasive capacity of cells through a unique mechanism involving the regulation of cyclophilin D activity, a component of the mitochondrial inner pore permeability. Hence, we propose that p53Ψ encodes a separation-of-function isoform that, although lacking canonical p53 tumor suppressor/transcriptional activities, is able to induce a prometastatic program in a transcriptionally independent manner.

TAp73 is essential for germ cell adhesion and maturation in testis.

A core evolutionary function of the p53 family is to protect the genomic integrity of gametes. However, the role of p73 in the male germ line is unknown. Here, we reveal that TAp73 unexpectedly functions as an adhesion and maturation factor of the seminiferous epithelium orchestrating spermiogenesis. TAp73 knockout (TAp73KO) and p73KO mice, but not ΔNp73KO mice, display a "near-empty seminiferous tubule" phenotype due to massive premature loss of immature germ cells. The cellular basis of this phenotype is defective cell-cell adhesions of developing germ cells to Sertoli nurse cells, with likely secondary degeneration of Sertoli cells, including the blood-testis barrier, which leads to disruption of the adhesive integrity and maturation of the germ epithelium. At the molecular level, TAp73, which is produced in germ cells, controls a coordinated transcriptional program of adhesion- and migration-related proteins including peptidase inhibitors, proteases, receptors, and integrins required for germ-Sertoli cell adhesion and dynamic junctional restructuring. Thus, we propose the testis as a unique organ with strict division of labor among all family members: p63 and p53 safeguard germ line fidelity, whereas TAp73 ensures fertility by enabling sperm maturation.

Metabolic alterations in lung cancer-associated fibroblasts correlated with increased glycolytic metabolism of the tumor.

Cancer cells undergo a metabolic reprogramming but little is known about metabolic alterations of other cells within tumors. We use mass spectrometry-based profiling and a metabolic pathway-based systems analysis to compare 21 primary human lung cancer-associated fibroblast lines (CAF) to "normal" fibroblast lines (NF) generated from adjacent nonneoplastic lung tissue. CAFs are protumorigenic, although the mechanisms by which CAFs support tumors have not been elucidated. We have identified several pathways whose metabolite abundance globally distinguished CAFs from NFs, suggesting that metabolic alterations are not limited to cancer cells. In addition, we found metabolic differences between CAFs from high and low glycolytic tumors that might reflect distinct roles of CAFs related to the tumor's glycolytic capacity. One such change was an increase of dipeptides in CAFs. Dipeptides primarily arise from the breakdown of proteins. We found in CAFs an increase in basal macroautophagy which likely accounts for the increase in dipeptides. Furthermore, we show a difference between CAFs and NFs in the induction of autophagy promoted by reduced glucose. In sum, our data suggest that increased autophagy may account for metabolic differences between CAFs and NFs and may play additional as yet undetermined roles in lung cancer.

A rapid and scalable system for studying gene function in mice using conditional RNA interference.

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:

TGF-beta IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer.

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-beta-mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-beta-dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-beta are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.

Increased prevalence of EGFR-mutant lung cancer in women and in East Asian populations: analysis of estrogen-related polymorphisms.

PURPOSE: Somatic mutations in the epidermal growth factor receptor (EGFR) gene occur in a subset of non-small-cell lung cancer (NSCLC) and are highly predictive of the clinical response to selective EGFR kinase inhibitors. The prevalence of EGFR-mutant NSCLC is appreciably higher in females than in males and in East Asian than in Caucasian populations. We hypothesized that these disparate frequencies may be attributable to underlying genetic modifiers. Given the coincident differences in sex and ethnic origin, we tested allozymatic variants of enzymes involved in estrogen biosynthesis and metabolism, encoded by polymorphic alleles known to differ in frequency between Caucasian and Asian populations, as modifying alleles. EXPERIMENTAL DESIGN: We genotyped nine polymorphisms in the CYP1A1, CYP17A1, CYP19, HSD17B1, COMT, GSTM1, and GSTT1 genes, in a series of 100 Japanese NSCLCs, selected for equal representation of EGFR wild-type (wt) and EGFR-mutant cases, as well as male and female cases. Associations between polymorphic variants and the EGFR genotype and sex of NSCLC cases were examined using Fisher's exact test of significance. RESULTS: Only CYP1A1 2C showed a difference in allele frequency that approached statistical significance. Heterozygotes were underrepresented among EGFR-mutant cases compared with EGFR-wt cases (27% versus 47%, P = 0.08), with a concurrent trend toward overrepresentation of CYP1A1 2C(Ile/Ile) homozygotes among EGFR-mutant cases as compared with EGFR-wt cases (69% versus 51%, P = 0.13). CONCLUSION: Within the power of this study, our findings suggest that the selected polymorphic variants in the estrogen biosynthesis and metabolism pathways are unlikely to be major genetic modifiers of the prevalence of EGFR-mutant NSCLC.

DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma.

Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.

Dialogue between RhoA/ROCK and members of the Par complex in cell polarity.

Cell polarity is essential for many biological processes and is regulated by conserved protein complexes, including the Par complex, Rho GTPases, and their regulators. In this issue of Developmental Cell, studies by Nakayama et al. and Zhang and Macara examine how interplay between Rho GTPases and the Par complex control polarized cell migration and dendritic spine morphogenesis in alternate ways.