Susan K. Rathe

A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma.

Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC50 of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

A microarray study of altered gene expression after cytarabine resistance in acute myeloid leukemia

A microarray study of altered gene expression after cytarabine resistance in acute myeloid leukemia

NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia.

Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

Synthesis and antileukemic activities of C1-C10-modified parthenolide analogues.

Parthenolide (PTL) is a sesquiterpene lactone natural product with anti-proliferative activity to cancer cells. Selective eradication of leukemic stem cells (LSCs) over healthy hematopoietic stem cells (HSCs) by PTL has been demonstrated in previous studies, which suggests PTL and related molecules may be useful for targeting LSCs. Eradication of LSCs is required for curative therapy. Chemical optimizations of PTL to improve potency and pharmacokinetic parameters have focused largely on the α-methylene-γ-butyrolactone, which is essential for activity. Conversely, we evaluated modifications to the C1-C10 olefin and benchmarked new inhibitors to PTL with respect to inhibitory potency across a panel of cancer cell lines, ability to target drug-resistant acute myeloid leukemia (AML) cells, efficacy for inhibiting clonal growth of AML cells, toxicity to healthy bone marrow cells, and efficiency for promoting intracellular reactive oxygen species (ROS) levels. Cyclopropane 4 was found to possess less toxicity to healthy bone marrow cells, enhanced potency for the induction of cellular ROS, and similar broad-spectrum anti-proliferative activity to cancer cells in comparison to PTL.

Deoxycytidine kinase is downregulated in Ara-C-resistant acute myeloid leukemia murine cell lines

Deoxycytidine kinase is downregulated in Ara-C-resistant acute myeloid leukemia murine cell lines

MMuFLR: missense mutation and frameshift location reporter

MOTIVATION Cancer researchers seeking immunotherapy targets in cancer cells need tools to locate highly expressed proteins unique to cancer cells. Missense mutation and frameshift location reporter (MMuFLR), a Galaxy-based workflow, analyzes next-generation sequencing paired read RNA-seq output to reliably identify small frameshift mutations and missense mutations in highly expressed protein-coding genes. MMuFLR ignores known SNPs, low quality reads and poly-A/T sequences. For each frameshift and missense mutation identified, MMuFLR provides the location and sequence of the amino acid substitutions in the novel protein candidates for direct input into epitope evaluation tools. AVAILABILITY http://toolshed.g2.bx.psu.edu/ CONTACT rath0096@umn.edu or johns198@umn.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma

Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype.

Comparative transcriptome analysis quantifies immune cell transcript levels, metastatic progression and survival in osteosarcoma

Overall survival of patients with osteosarcoma (OS) has improved little in the past three decades, and better models for study are needed. OS is common in large dog breeds and is genetically inducible in mice, making the disease ideal for comparative genomic analyses across species. Understanding the level of conservation of intertumor transcriptional variation across species and how it is associated with progression to metastasis will enable us to more efficiently develop effective strategies to manage OS and to improve therapy. In this study, transcriptional profiles of OS tumors and cell lines derived from humans (n = 49), mice (n = 103), and dogs (n = 34) were generated using RNA sequencing. Conserved intertumor transcriptional variation was present in tumor sets from all three species and comprised gene clusters associated with cell cycle and mitosis and with the presence or absence of immune cells. Further, we developed a novel gene cluster expression summary score (GCESS) to quantify intertumor transcriptional variation and demonstrated that these GCESS values associated with patient outcome. Human OS tumors with GCESS values suggesting decreased immune cell presence were associated with metastasis and poor survival. We validated these results in an independent human OS tumor cohort and in 15 different tumor data sets obtained from The Cancer Genome Atlas. Our results suggest that quantification of immune cell absence and tumor cell proliferation may better inform therapeutic decisions and improve overall survival for OS patients.Significance: This study offers new tools to quantify tumor heterogeneity in osteosarcoma, identifying potentially useful prognostic biomarkers for metastatic progression and survival in patients. Cancer Res; 78(2); 326-37. ©2017 AACR.