Figure legends for supplementary figures.

<p>Supplementary Figure 1: Locations and types of chromatin regulator mutations found in human lung adenocarcinomas. Supplementary Figure 2: Identification of high efficiency Brg1, Arid1a and Setd2 sgRNAs using a fluorescent reporter-based sensor assay. Supplementary Figure 3: Examples of tumors with mixed expression of Brg1, Arid1a and H3K36me3. Supplementary Figure 4: Proliferative and apoptotic rates of KrasG12D/+ tumors targeted with sgRNAs against GFP, Brg1, Arid1a, or Setd2. Supplementary Figure 5: Identification of chromatin regulator expression status and measurement of tumor cell purity in RNA-Seq samples. Supplementary Figure 6: Ingenuity Pathway Analysis of top 10 enriched canonical pathways in Arid1a and Setd2 deficient tumors. Supplementary Figure 7: Proliferative and apoptotic rates of KrasG12D/+;p53-/- tumors targeted with sgRNAs against GFP, Brg1, Arid1a, or Setd2. Supplementary Figure 8: Analysis of markers of tumor cell identity in early and late KrasG12D/+;p53-/- tumors. Supplementary Figure 9: Representative images of tumors from KrasG12D/+;p53flox/flox mice, and the distinct morphology of Setd2 deficient tumors. Supplementary Figure 10: Analysis of multinucleate giant cells, aberrant mitoses and DNA damage in Setd2 deficient tumors.</p>

Supplementary Methods, Tables 1-3, Figures 1-6 from Targeting Hyaluronidase for Cancer Therapy: Antitumor Activity of Sulfated Hyaluronic Acid in Prostate Cancer Cells

<p>Supplementary Table 2: Ingenuity Pathway Analysis results for Setd2 vs. GFP tumors.</p>

Four Supplementary Figures and Seven Supplementary Tables

<p>Supplementary Table 2: Ingenuity Pathway Analysis results for Setd2 vs. GFP tumors.</p>

<p>Supplementary Table 3: Gene ontology analysis for genes deregulated by both Arid1a and Setd2 loss.</p>

<p>Supplementary Table 1: Ingenuity Pathway Analysis results for Arid1a vs. GFP tumors.</p>

The 5-year survival rate of patients with metastatic renal cell carcinoma (mRCC) is <12% due to treatment failure. Therapeutic strategies that overcome resistance to modestly effective drugs for mRCC, such as sorafenib (SF), could improve outcome in mRCC patients. SF is terminally biotransformed by UDP-glucuronosyltransferase-1A9 (A9) mediated glucuronidation, which inactivates SF. In a clinical-cohort and the TCGA-dataset, A9 transcript and/or protein levels were highly elevated in RCC specimens and predicted metastasis and overall-survival. This suggested that elevated A9 levels even in primary tumors of patients who eventually develop mRCC could be a mechanism for SF failure. 4-methylumbelliferone (MU), a choleretic and antispasmodic drug, downregulated A9 and inhibited SF-glucuronidation in RCC cells. Low-dose SF and MU combinations inhibited growth, motility, invasion and downregulated an invasive signature in RCC cells, patient-derived tumor explants and/or endothelial-RCC cell co-cultures; however, both agents individually were ineffective. A9 overexpression made RCC cells resistant to the combination, while its downregulation sensitized them to SF treatment alone. The combination inhibited kidney tumor growth, angiogenesis and distant metastasis, with no detectable toxicity; A9-overexpressing tumors were resistant to treatment. With effective primary tumor control and abrogation of metastasis in preclinical models, the low-dose SF and MU combinations could be an effective treatment option for mRCC patients. Broadly, our study highlights how targeting specific mechanisms that cause the failure of "old" modestly effective FDA-approved drugs could improve treatment response with minimal alteration in toxicity profile.

Abstract Purpose: Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer progression. Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in bladder cancer. Experimental Design: In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder. Results: HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In bladder cancer tissues, V1 levels associated with metastasis and cancer-specific survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically. Conclusions: Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant bladder cancer and potential predictor of outcome in patients with bladder cancer.

Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1–3. Although RB was the first tumour suppressor to be identified, the molecular and cellular basis that underlies selection for persistent RB loss in cancer remains unclear4–6. Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and are currently under evaluation for the treatment of lung adenocarcinoma7–9. Whether RB pathway reactivation will have therapeutic effects and whether targeting CDK4 and CDK6 is sufficient to reactivate RB pathway activity in lung cancer remains unknown. Here we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in mice. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for amplification of the MAPK signal during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to inhibition of CDK4 and CDK6. Second, RB inactivation deregulates the expression of cell-state-determining factors, facilitates lineage infidelity and accelerates the acquisition of metastatic competency. By contrast, reactivation of RB reprograms advanced tumours towards a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs that they control and critical determinants of successful cancer therapy. Loss of RB promotes both malignant progression and the development of metastatic disease; however, whereas reactivation of the RB pathway can revert metastatic tumour cell states to non-metastatic cell states, malignant cell proliferation is supported by MAPK–CDK2-dependent suppression of RB.

Mutations in the Rb tumor suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma. Additionally, recent clinical successes with cyclin dependent kinase inhibitors have reinvigorated interest in reactivating the Retinoblastoma (Rb) pathway to treat lung adenocarcinoma and other tumor types. Remarkably though, Rb’s role in suppressing lung adenocarcinoma remains unclear, and whether Rb pathway reactivation would be efficacious in this disease remains unknown. To model Rb pathway reactivation as treatment strategy in lung adenocarcinoma and to shed light on its role in this disease, we established an Rb XTR allele that enables Cre-dependent inactivation of Rb in developing tumors, and allows Flp recombinase-inducible reactivation of Rb after tumors are established. In the Kras Lox-Stop-Lox-G12D/+ ;p53 flox/flox ( KP ) mouse model of lung adenocarcinoma, we show that Rb inactivation facilitates the bypass of two molecularly distinct barriers to tumor progression and dramatically accelerates malignant conversion and the development of metastatic disease. Although, in the presence of Rb, malignant conversion requires amplification of the Raf/Mek/Erk (MAPK) signaling pathway beyond that normally activated by the Kras oncogene, we find that this requirement is abrogated when Rb is inactivated. Mechanistically, we identified Cdk2 as an important effector downstream of amplified MAPK signaling and that this activity suppresses Rb’s ability to limit the adenoma-to-carcinoma transition. Importantly, inactivation of Cdk2 reduces cell proliferation in Rb wild-type cells and confers sensitivity to Cdk4/6 inhibition in both human and mouse lung adenocarcinoma cell lines were intrinsically resistant. Acquiring metastatic competency in Rb wild-type tumors is causally linked to epigenetic changes resulting in loss of lung lineage cell fate-determining transcription factors and concomitant derepression of factors normally restricted to embryonic cell types. However, inactivation of Rb uncouples the onset of metastatic competency from the loss of lung lineage factors, facilitates the early derepression of prometastatic factors, and significantly enhances metastatic proclivity. Finally, we demonstrate that reactivation of Rb in metastatic disease settings reprograms these tumors toward a less aggressive cell state and improves overall survival. Our study highlights an unappreciated role for Rb in regulating metastasis-promoting programs, and the potential of Rb restorative therapies to treat lung adenocarcinoma. Further, we suggest that a renewed investment in the development of specific Cdk2 inhibitors may be necessary for Rb pathway reactivation in certain cancer types. This abstract is also being presented as Poster A27. Citation Format: Travis Yates, Caroline Kim-Kiselak, Walter Wang, David M. Feldser. Modeling Rb loss and pathway reactivation in lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR01.

Mutations in the Rb tumor suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma. Additionally, recent clinical successes with cyclin dependent kinase inhibitors have reinvigorated interest in reactivating the Retinoblastoma (Rb) pathway to treat lung adenocarcinoma and other tumor types. Remarkably though, Rb’s role in suppressing lung adenocarcinoma remains unclear, and whether Rb pathway reactivation would be efficacious in this disease remains unknown. To model Rb pathway reactivation as treatment strategy in lung adenocarcinoma and to shed light on its role in this disease, we established an Rb XTR allele that enables Cre-dependent inactivation of Rb in developing tumors, and allows Flp recombinase-inducible reactivation of Rb after tumors are established. In the Kras Lox-Stop-Lox-G12D/+ ;p53 flox/flox ( KP ) mouse model of lung adenocarcinoma, we show that Rb inactivation facilitates the bypass of two molecularly distinct barriers to tumor progression and dramatically accelerates malignant conversion and the development of metastatic disease. Although, in the presence of Rb, malignant conversion requires amplification of the Raf/Mek/Erk (MAPK) signaling pathway beyond that normally activated by the Kras oncogene, we find that this requirement is abrogated when Rb is inactivated. Mechanistically, we identified Cdk2 as an important effector downstream of amplified MAPK signaling and that this activity suppresses Rb’s ability to limit the adenoma-to-carcinoma transition. Importantly, inactivation of Cdk2 reduces cell proliferation in Rb wild-type cells and confers sensitivity to Cdk4/6 inhibition in both human and mouse lung adenocarcinoma cell lines were intrinsically resistant. Acquiring metastatic competency in Rb wild-type tumors is causally linked to epigenetic changes resulting in loss of lung lineage cell fate-determining transcription factors and concomitant derepression of factors normally restricted to embryonic cell types. However, inactivation of Rb uncouples the onset of metastatic competency from the loss of lung lineage factors, facilitates the early derepression of prometastatic factors, and significantly enhances metastatic proclivity. Finally, we demonstrate that reactivation of Rb in metastatic disease settings reprograms these tumors toward a less aggressive cell state and improves overall survival. Our study highlights an unappreciated role for Rb in regulating metastasis-promoting programs, and the potential of Rb restorative therapies to treat lung adenocarcinoma. Further, we suggest that a renewed investment in the development of specific Cdk2 inhibitors may be necessary for Rb pathway reactivation in certain cancer types. This abstract is also being presented as Poster A27. Citation Format: Travis Yates, Caroline Kim-Kiselak, Walter Wang, David M. Feldser. Modeling Rb loss and pathway reactivation in lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR01.

Background: Aberrantly expressed miRNAs promote renal cell carcinoma (RCC) growth and metastasis and are potentially useful biomarkers for metastatic disease. However, a consensus clinically significant miRNA signature has not been identified. To identify an miRNA signature for predicting clinical outcome in RCC patients, we used a four-pronged interconnected approach. Methods: Differentially expressed miRNAs were identified and analyzed in 113 specimens (normal kidney: 59; tumor: 54). miRNA profiling was performed in matched normal and tumor specimens from 8 patients and extended to 32 specimens. Seven aberrantly expressed miRNAs were analyzed by qPCR, and their levels were correlated with RCC subtypes and clinical outcome. miRNA signature was confirmed in The Cancer Genome Atlas RCC dataset (n = 241). Results: Discovery phase identified miR-21, miR-142-3p, miR-142-5p, miR-150, and miR-155 as significantly upregulated (2–4-fold) and miR-192 and miR-194 as downregulated (3–60-fold) in RCC; miR-155 distinguished small tumors (<4 cm) from benign oncocytomas. In univariate and multivariate analyses, miRNA combinations (miR-21+194; miR-21+142-5p+194) significantly predicted metastasis and/or disease-specific mortality; miR-21+142-5p+194 (for metastasis): P = 0.0017; OR, 0.53; 95% confidence interval (CI), 0.75–0.33; 86.7% sensitivity; 82% specificity. In the TCGA dataset, combined biomarkers associated with metastasis and overall survival (miR-21+142-5p+194: P < 0.0001; OR, 0.37; 95% CI, 0.58–0.23). Conclusions: The interconnected discovery–validation approach identified a three-miRNA signature as a potential predictor of disease outcome in RCC patients. Impact: With 10% survival at 5 years, metastatic disease presents poor prognosis for RCC patients. The three-miRNA signature discovered and validated may potentially at an early stage detect and predict metastasis, to allow early intervention for improving patient prognosis. Cancer Epidemiol Biomarkers Prev; 27(4); 464–72. ©2018 AACR.

Abstract Chromatin-modifying genes are frequently mutated in human lung adenocarcinoma, but the functional impact of these mutations on disease initiation and progression is not well understood. Using a CRISPR-based approach, we systematically inactivated three of the most commonly mutated chromatin regulatory genes in two KrasG12D-driven mouse models of lung adenocarcinoma to characterize the impact of their loss. Targeted inactivation of SWI/SNF nucleosome–remodeling complex members Smarca4 (Brg1) or Arid1a had complex effects on lung adenocarcinoma initiation and progression. Loss of either Brg1 or Arid1a were selected against in early-stage tumors, but Brg1 loss continued to limit disease progression over time, whereas loss of Arid1a eventually promoted development of higher grade lesions. In contrast to these stage-specific effects, loss of the histone methyltransferase Setd2 had robust tumor-promoting consequences. Despite disparate impacts of Setd2 and Arid1a loss on tumor development, each resulted in a gene expression profile with significant overlap. Setd2 inactivation and subsequent loss of H3K36me3 led to the swift expansion and accelerated progression of both early- and late-stage tumors. However, Setd2 loss per se was insufficient to overcome a p53-regulated barrier to malignant progression, nor establish the prometastatic cellular states that stochastically evolve during lung adenocarcinoma progression. Our study uncovers differential and context-dependent effects of SWI/SNF complex member loss, identifies Setd2 as a potent tumor suppressor in lung adenocarcinoma, and establishes model systems to facilitate further study of chromatin deregulation in lung cancer. Cancer Res; 77(7); 1719–29. ©2017 AACR.

Tumor cell-derived hyaluronidase HYAL-1 degrades hyaluronic acid (HA) into angiogenic fragments (AGF: 10-12 disaccharides). AGF support tumor growth and progression. Urine and tissue HAase/HYAL-1 levels are sensitive markers for high-grade bladder cancer (BCa) and its metastasis. In preclinical models of BCa, we evaluated whether o-sulfated AGF (sHA-F) inhibits HAase activity and has antitumor activity. At IC50 for HAase activity inhibition (5-20 μg/ml [0.4-1.7 μM]), sHA-F significantly inhibited proliferation, motility and invasion of HYAL-1 expressing BCa cells (253J-Lung, HT1376, UMUC-3), P<0.001. sHA-F did not affect the growth of HYAL-1 non-expressing BCa (5637, RT4, T24, TCCSUP) and normal urothelial (Urotsa, SV-HUC1) cells. sHA-F treatment induced apoptosis by death receptor pathway. sHA-F downregulated transcript and/or protein levels of HA receptors (CD44, RHAMM), p-AKT, β-catenin, pβ-Catenin(S552), Snail and Twist but increased levels of pβ-Catenin(T41/S45), pGSK-3α/β(S21/S9) and E-cadherin. sHA-F also inhibited CD44/Phosphoinositide 3-kinase (PI-3K) complex formation and PI-3K activity. AGF addition or myristoylated-AKT overexpression attenuated sHA-F effects. Contrarily, HYAL-1 expression sensitized RT4 cells to sHA-F treatment. In the 253J-L and HT1376 xenograft models, sHA-F treatment significantly inhibited tumor growth (P<0.001), plausibly by inhibiting angiogenesis and HA receptor-PI-3K/AKT signaling. This study delineates that sHA-F targets tumor-associated HA-HAase system and could be potentially useful in BCa treatment.

Background: Molecular markers of clinical outcome may aid in designing targeted treatments for bladder cancer. However, only a few bladder cancer biomarkers have been examined as therapeutic targets. Methods: Data from The Cancer Genome Atlas (TCGA) and bladder specimens were evaluated to determine the biomarker potential of the hyaluronic acid (HA) family of molecules – HA synthases, HA receptors and hyaluronidase. The therapeutic efficacy of 4-methylumbelliferone (4MU), a HA synthesis inhibitor, was evaluated in vitro and in xenograft models. Results: In clinical specimens and TCGA data sets, HA synthases and hyaluronidase-1 levels significantly predicted metastasis and poor survival. 4-Methylumbelliferone inhibited proliferation and motility/invasion and induced apoptosis in bladder cancer cells. Oral administration of 4MU both prevented and inhibited tumour growth, without dose-related toxicity. Effects of 4MU were mediated through the inhibition of CD44/RHAMM and phosphatidylinositol 3-kinase/AKT axis, and of epithelial–mesenchymal transition determinants. These were attenuated by HA, suggesting that 4MU targets oncogenic HA signalling. In tumour specimens and the TCGA data set, HA family expression correlated positively with β -catenin, Twist and Snail expression, but negatively with E-cadherin expression. Conclusions: This study demonstrates that the HA family can be exploited for developing a biomarker-driven, targeted treatment for bladder cancer, and 4MU, a non-toxic oral HA synthesis inhibitor, is one such candidate.

Abstract INTRODUCTION AND OBJECTIVE: Tumor cell-derived hyaluronidase HYAL1, which degrades hyaluronic acid (HA) into angiogenic fragments, promotes tumor growth and metastasis. HYAL-1 is an independent prognostic marker for predicting metastasis. Small molecular mass sulfated hyaluronic acid (sHA) derivatives (sHA8k) inhibit HYAL1 activity through a mixed inhibition mechanism. Antitumor activity of sHA8k and mechanism of action was evaluated in BCa models.. METHODS: Effect of sHA8k (0-40 ug/ml) on cell proliferation and apoptosis was examined in BCa cells (253J-Lung, HT1376, UMUC-3, T24, RT4) by cell counting and Cell Death ELISA kit. Matrigel invasion and Boyden chamber assays were used to test the effect of sHA8k on invasive activity. Effect of sHA on signaling, apoptosis cascade, HA receptor (CD44, RHAMM), EMT markers (β-catenin, E-cadherin, Snail, Twist) levels, was evaluated by Q-pCR and immunoblotting assays. Angiogenic fragment addition and mAkt transfection were performed to elucidate mechanism of action. Athymic mice bearing 253J-Lung xenografts were treated with sHA (25 and 50 mg/kg) by i.p. injection. RESULTS: sHA8k inhibited proliferation, motility and invasion in BCa cells that expressed HYAL-1. At IC50 for HAase activity inhibition (∼ 20-μg/ml), sHA induced > 3-fold apoptosis and inhibited invasive activity of BCa cells. sHA8k induced caspase-3, -8, -9 activation, up-regulation of Fas, Fas-L, FADD, DR4, DR5 and E-cadherin. sHA8k downregulated CD44, RHAMM, bcl-2, phospho(p)-Akt, pGSK3β, pβcatenin(ser552), snail and Twist expression. Effect of sHA8k were attenuated by angiogenic HA fragments or overexpression of m-Akt and downregulation of CD44 and RHAMM mimicked sHA8k effects. sHA significantly inhibited 253J-Lung xenograft growth. The majority of the animals did not form palpable tumors at 50-mg/kg dose. No weight loss or serum and organ toxicity was observed in sHA treated animals. Biochemical analysis of tumors also showed the same alterations in Akt and EMT pathways as observed cell culture. CONCLUSION: This is the first study that shows sHA8k, a small molecular mass HYAL-1 inhibitor has potent antitumor activity. Support: R01 CA 72821-14 (VBL) Citation Format: Andre R. Jordan, Juan Chipollini, Luis Lopez, Travis Yates, Vinata Lokeshwar. Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2647. doi:10.1158/1538-7445.AM2015-2647

334 Background: Stroma-Derived Factor (SDF)-1 is a ligand for chemokine receptors CXCR4 and CXCR7. The six known SDF-1 isoforms are generated by alternative mRNA splicing. While SDF-1 expression has been detected in various malignancies, only a few studies have reported differential expression of SDF-1 isoforms and its clinical significance. In this study we evaluated the expression three SDF-1 isoforms (α,β,γ) in bladder cancer (BCa). Methods: Using quantitative PCR, mRNA levels of SDF-1α, SDF-1β and SDF-1γ were measured in bladder tissues (normal: 25; BCa: 44) and urine specimens (n=210; normal: 28; benign conditions: 74; BCa: 57, history of BCa (HxBCa): 35, Hx other Ca: 8; other Ca: 8) from consecutive patients. The transcript levels were normalized to β-actin. Correlation between SDF1-isoform transcript levels and clinical outcome (metastasis and disease specific mortality (DSM)) was evaluated in univariate and multivariate analyses. Results: Among SDF-1 isoforms, only SDF-1β mRNA was significantly overexpressed by 2.5-6-fold in BCa tissues when compared to normal bladder tissues. While SDF-1α was expressed in bladder tissues, SDF-1γ expression was undetectable. In univariate analysis, pathologic stage, lymph node positivity for tumor, SDF-1α and SDF-1β transcript levels significantly associated with metastasis and DSM. However, in multivariate analysis, stage (P=0.005) and SDF-1β levels (P=0.017) were independent predictors of metastasis and DSM. In exfoliated urothelial cells, only SDF-1β mRNA levels were differentially expressed and having 91.2% sensitivity and 73.8% specificity for detecting BCa. In patients with HxBCa, elevated SDF-1β levels indicated 4.3-fold increased risk (chi-square: 15.1; P=0.0001; relative risk = 4.3) for developing recurrence within 6-months. Conclusions: SDF-1 isoforms are differentially expressed in bladder tissues and exfoliated urothelial cells. SDF-1β mRNA levels in BCa tissues predict poor prognosis. Further, SDF-1β mRNA levels in exfoliated cells detect BCa with high sensitivity and are potential predictors of future recurrence. Support: Grant NCI/NIH R01 CA-72821-14; Women’s Cancer Association – University of Miami.

Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. Here we report the development and characterization of a synthetic gene switch that, when targeted in the mouse germline, enables conditional inactivation, reports gene expression and allows inducible restoration of the targeted gene. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems, to study the impact of p53 or Rb inactivation and restoration. We term this allele system XTR, to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR) and Restored (R).