Abstract B10: HGF enhances tumor cell survival in canertinib-treated medulloblastoma cell lines

Abstract Acute myelogeneous leukemia (AML) is a heterogeneous disease and includes a subset of neoplasms that harbor activating mutations of the fms-like tyrosine kinase-3 (FLT3) gene. Mutated FLT3 has recently been shown to activate downstream oncogenic pathways including the PI3K/AKT pathway (Scheijen, et al. Oncogene. 23:3338–3349, 2004; Choudhary, et al. Blood. 106:265–273, 2005). It is known that activated AKT mediates its effects, at least in part, through activation of mammalian target of rapamycin (mTOR). However, the potential role of PI3K/AKT/mTOR signaling pathway in tumor cell survival in AML remains largely unknown. We hypothesized that the PI3K/AKT signaling pathway is activated in AML and contributes to tumor cell survival through activation (phosphorylation) of mTOR and its downstream effectors 4EBP1, p70S6K, ribosomal protein S6 (rpS6), and eIF-4E. We used 3 AML cell lines, including MV4-11 and MOLM-13, that are homozygous and heterozygous for mutated FLT3, respectively, as well as U937 (wild-type FLT3). All 3 cell lines expressed activated (serine 473-phosphorylated) AKT (Ser473pAKT), and phosphorylated 4EBP1, p70S6K and rpS6 shown by Western Blot analysis. Treatment of AML cell lines with LY294002, an inhibitor of PI3K, resulted in a dose-dependent decrease of phosphorylation of AKT, mTOR, 4EBP1, p70S6K, and rpS6. This was associated with decreased cell viability as assessed by trypan-blue exclusion assay. Cell death following inhibition of the PI3K/AKT pathway was predominantly attributed to apoptosis as shown by increased annexin V staining assessed by flow cytometry. These changes were associated with downregulation of the anti-apoptotic proteins cFLIP, Mcl-1, and Bcl-XL that are involved in the extrinsic and intrinsic apoptosis. Cell cycle analysis using flow cytometry also showed that inhibition of PI3K resulted in decreased S-phase and increased G1-phase fraction. These cell cycle changes were associated with increased levels of the cyclin-dependent kinase inhibitor p27 and underphosphorylated Rb in a dose-dependent manner. Similar biologic effects, although to a lesser degree, were found after treatment of AML cells with rapamycin, an inhibitor of mTOR. In addition, expression of activated AKT, mTOR, 4EBP1, p70S6K and rpS6 was assessed in AML tumors (n=19) using tissue microarrays of bone marrow samples and immunohistochemical methods. These included tumors with (n=14) and without (n=5) FLT3 mutations. Using a 10% cutoff to define positivity, 13/19 (68%) expressed Ser473pAKT, 16/18 (89%) mTOR, 15/19 (79%) p4E-BP1, 18/19 (95%) p-p70S6K, and 15/18 (83%) p-rpS6. However, no association between expression of activated AKT, or mTOR signaling proteins and FLT3 mutational status was observed. Our study provides first evidence that the AKT/mTOR signaling pathway is activated in AML cell lines and tumors regardless of FLT3 mutational status. The AKT/mTOR signaling pathway may contribute to cell cycle progression and tumor cell survival in AML. Inhibition of this oncogenic pathway represents a potential target for therapy in patients with AML.

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PD.188 Radiation induced release of angiogenic growth factors by HNSCC cell lines increases in vitro tumor cell survival

Oral Oncology Supplement ◽

10.1016/s1744-7895(05)80308-6 ◽

2005 ◽

Vol 1 (1) ◽

pp. 124

Author(s):

J. Brieger ◽

P. Schroeder ◽

W.J. Mann

Keyword(s):

Growth Factors ◽

Tumor Cell ◽

Cell Survival ◽

Cell Lines ◽

Angiogenic Growth Factors ◽

Tumor Cell Survival ◽

Radiation Induced ◽

Hnscc Cell

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Functional Genomic Landscape of Genes with Recurrent Mutations in Multiple Myeloma

Blood ◽

10.1182/blood-2018-189 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 189-189 ◽

Cited By ~ 1

Author(s):

Ricardo De Matos Simoes ◽

Ryosuke Shirasaki ◽

Huihui Tang ◽

Michal Sheffer ◽

Olga Dashevsky ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Cell ◽

Cell Survival ◽

Cell Lines ◽

Research Funding ◽

Functional Genomic ◽

Loss Of Function ◽

Functional Studies ◽

Tumor Cell Survival ◽

Genome Scale

Abstract During the last two decades, cell lines and patient-derived samples from multiple myeloma (MM) have been extensively profiled for alterations in their genome with the anticipation that those genes with the most recurrent lesions could represent attractive novel therapeutic targets or markers for aggressive disease. Yet for many of these genes, their functional significance for MM cells has not been formally evaluated. With the advent of new CRISPR/Cas9-based functional genomics platforms, it is possible to generate in genome- or subgenome-scale direct quantitative information on the impact that perturbation of these genes exerts on tumor cell survival, proliferation or other phenotypes. We therefore examined the landscape of our CRISPR-based functional genomic data for these recurrently dysregulated genes We specifically curated information from the MMRF CoMMpass study and multiple other publicly available studies, to identify genes which are recurrently identified to harbor nonsynonymous mutation (SNV or indel), DNA copy number loss or gain, or participation in chromosomal translocations. We then examined the patterns of results for these genes in our genome scale CRISPR-based gene-editing studies for loss-of-function in n=18 MM cell lines. We identified a subset of genes (e.g. FAM46C, CDKN2C, RASA2) which are considered targets for recurrent loss-of-function events and indeed exhibit, for large fractions of the cell lines tested enrichment, of their sgRNAs in CRISPR knock-out studies, consistent with a role of these genes as suppressors of tumor cell survival or proliferation. CRISPR KO of TP53 leads to increased survival/proliferation of only a small minority (2/18 of cell lines tested thus far), which reflects the fact that the overwhelming majority of MM cell lines already harbor LOF events for this gene. Interestingly, a substantial number of genes which have been considered to harbor recurrent LOF events in MM patient samples (e.g. NF1, NF2, CYLD) do not exhibit sgRNA enrichment in CRISPR KO screens in the MM cell lines tested so far. In addition, several other recurrently mutated genes for which their loss- or gain-of-function status had not been previously evaluated with extensive functional studies in MM (e.g. SP140, LTB, EGR1, ATM, PARK2, PRKD2, RAPGEF5, DOCK5, TGDS, TNFAIP8) exhibit in the majority of cell lines tested in in CRISPR knockout studies no significant enrichment or depletion of their sgRNAs. In contrast, PTPN11, CREBBP, EP300, KMT2B, KMT2C, SETD2, SF3B1 and UBR5, are notable examples of recurrently mutated genes which represent dependencies for large fractions of MM cell lines in vitro. These results highlight the value of interpreting results from next generation sequencing studies in the context of information provided by the genome scale by use of functional genomic characterization of available cell line models. We envision that, similar sub-genome scale assays were performed at the level of patient derived samples will also provide direct information about the relevance of some of these genes. In addition, functional studies conducted with context of tumor-microenvironemtn compartment interactions and tumor interface will be needed to evaluate several genes identified in the study. Disclosures Licht: Celgene: Research Funding. Mitsiades:Takeda: Other: employment of a relative; Janssen/ Johnson & Johnson: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding; TEVA: Research Funding.

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Serum and Glucocorticoid Inducible Kinase 1-Sensitive Survival, Proliferation and Migration of Rhabdomyosarcoma Cells

Cellular Physiology and Biochemistry ◽

10.1159/000481842 ◽

2017 ◽

Vol 43 (3) ◽

pp. 1301-1308 ◽

Cited By ~ 2

Author(s):

Evi Schmid ◽

Matias Julian Stagno ◽

Jing Yan ◽

Sabine Schleicher ◽

Willi Yu ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cell ◽

Cell Survival ◽

Cell Lines ◽

Therapy Resistance ◽

Tumor Cell Survival ◽

Rhabdomyosarcoma Cell ◽

And Migration ◽

Tumor Types ◽

Proliferation And Migration

Background/Aims: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, may show an intrinsic refractoriness to standard chemotherapy in advanced tumor stages, which is associated with poor prognosis. Cellular mechanisms conferring tumor cell survival and therapy resistance in many tumor types include the serum & glucocorticoid inducible kinase (SGK) 1 pathway, a kinase expressed ubiquitously with particularly strong expression in skeletal muscle and some tumor types. The present study explored whether SGK1 is expressed in rhabdomyosarcoma and, if so, whether this kinase impacts on tumor cell survival, proliferation and migration. Multiple in vitro techniques were used to study the role of SGK1 in rhabdomyosarcoma. Methods: The Gene Chip® Human Genome U133 Plus 2.0 Array were employed to examine SGK1 transcriptional activity in healthy muscle and rhabdomyosarcoma tissue. SGK1 transcript levels were quantified in rhabdomyosarcoma cell lines RD (embryonal subtype) and RH30 (alveolar subtype) by RT-PCR, cell viability was measured using MTT assays. Clonal cell growth was assessed via colony forming assays and migration experiments were performed in a transwell system. Results: SGK1 is expressed in embryonal and alveolar rhabdomyosarcoma tissue samples and in RD and RH30 rhabdomyosarcoma cell lines. Administration of EMD638683 – an inhibitor specific for SGK1 - decreased viability of RD and RH30 cells, enhanced the effects of the cytotoxic drug doxorubicin leading to reduced migration and decreased cell proliferation. Conclusions: SGK1 is expressed in rhabdomyosarcoma cells where it contributes to survival, therapy resistance, cell proliferation and migration. Thus, SGK1 inhibitors may be considered a therapeutic option for the treatment of therapy-resistant rhabdomyosarcoma.

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