The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To ...identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562R, a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562R cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34+ cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34+ cells from normal cord blood or from a patient harboring the BCR-ABL1T315I mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.
•A function-first shRNA library screen identifies pathways involved in BCR-ABL1 kinase-independent TKI resistance.•RAN or XPO1 inhibition impairs survival of progenitors from newly diagnosed or TKI-resistant CML patients.
Chronic inflammation is associated with increased cancer risk. Furthermore, the transcription factor NF-κB, a central regulator of inflammatory responses, is constitutively active in most tumors. To ...determine whether active NF-κB inherently contributes to malignant transformation, we isolated a set of NF-κB-activating genetic elements and tested their oncogenic potential in rodent cell transformation models. Genetic elements with desired properties were isolated using biologically active selectable peptide technology, which involves functional screening of lentiviral libraries encoding 20 or 50 amino acid-long polypeptides supplemented with endoplasmic reticulum-targeting and oligomerization domains. Twelve NF-κB-activating selectable peptides (NASPs) representing specific fragments of six proteins, none of which was previously associated with NF-κB activation, were isolated from libraries of 200,000 peptides derived from 500 human extracellular proteins. Using selective knockdown of distinct components of the NF-κB pathway, we showed that the isolated NASPs act either via or upstream of TNF receptor-associated factor 6. Transduction of NASPs into mouse and rat embryo fibroblasts did not, in itself, alter their growth. However, when coexpressed with oncogenic Ras (H-Ras(V12)), NASPs allowed rodent fibroblasts to overcome H-Ras(V12)-mediated p53-dependent senescence and acquire a transformed tumorigenic phenotype. Consistent with their ability to cooperate with oncogenic Ras in cell transformation, NASP expression reduced the transactivation activity of p53. This system provides an in vitro model of NF-κB-driven carcinogenesis and suggests that the known carcinogenic effects of inflammation may be at least partially due to NF-κB-mediated abrogation of oncogene-induced senescence.
Bluetongue is an insect-transmitted disease of sheep and wild ruminants that is caused by bluetongue virus (BTV). Cattle are asymptomatic reservoir hosts of BTV. Infection of lung microvascular ...endothelial cells (ECs) is central to the pathogenesis of BTV infection of both sheep and cattle, but it is uncertain as to why sheep are highly susceptible to BTV-induced microvascular injury, whereas cattle are not. Thus, to better characterize the pathogenesis of bluetongue, the transcription of genes encoding a variety of vasoactive and inflammatory mediators was quantitated in primary ovine lung microvascular ECs (OLmVECs) exposed to BTV and/or inflammatory mediators. BTV infection of OLmVECs increased the transcription of genes encoding interleukin- (IL) 1 and IL-8, but less so IL-6, cyclooxygenase-2, and inducible nitric oxide synthase. In contrast, we previously have shown that transcription of genes encoding all of these same mediators is markedly increased in BTV-infected bovine lung microvascular ECs and that BTV-infected bovine ECs produce substantially greater quantities of prostacyclin than do sheep ECs. Thus, sheep and cattle were experimentally infected with BTV to further investigate the role of EC-derived vasoactive mediators in the pathogenesis of bluetongue. The ratio of thromboxane to prostacyclin increased during BTV infection of both sheep and cattle, but was significantly greater in sheep (P = 0.001). Increases in the ratio of thromboxane to prostacyclin, indicative of enhanced coagulation, coincided with the occurrence of clinical manifestations of bluetongue in BTV-infected sheep. The data suggest that inherent species-specific differences in the production and activities of EC-derived mediators contribute to the sensitivity of sheep to BTV-induced microvascular injury.
Abstract CD8+ cells from healthy, asymptomatic HIV-1-infected individuals can inhibit HIV-1 replication in naturally or acutely infected CD4+ cells in the absence of cell killing. This CD8+ cell ...noncytotoxic anti-HIV response (CNAR) is mediated by a soluble CD8+ cell antiviral factor (CAF). CNAR/CAF inhibits HIV-1 replication by blocking viral RNA transcription. HIV transcription is regulated by a variety of cis -acting DNA sequence elements within the proviral long terminal repeat (LTR). We hypothesized that one of the HIV-1 LTR proviral DNA sequence elements that binds host cell transcriptional factors is involved in this antiviral activity. To assess this possibility, we constructed full-length infectious HIV-1 molecular clones with mutations in the LTR elements NFAT, AP-1, IL-2 homology region, and the downstream ISRE. We also tested full-length infectious molecular clones that had deletions of either the NF-κB or Sp1 sites of the LTR or lacked functional Tat and TAR elements. Viruses generated from these molecular clones were used to acutely infect CD4+ cells that subsequently were either co-cultured with CD8+ cells from individuals that exhibited strong CNAR or cultured with CAF-containing fluids. The replication of all of the mutant HIV-1 viruses tested was substantially reduced in the presence of CNAR/CAF. These findings suggest that other regions in the viral LTR or other host cell processes are involved in the transcriptional block elicited by CNAR/CAF.
Oncogenic MYC activation promotes proliferation in Burkitt lymphoma, but also induces cell-cycle arrest and apoptosis mediated by p53, a tumor suppressor that is mutated in 40% of Burkitt lymphoma ...cases. To identify molecular dependencies in Burkitt lymphoma, we performed RNAi-based, loss-of-function screening in eight Burkitt lymphoma cell lines and integrated non-Burkitt lymphoma RNAi screens and genetic data. We identified 76 genes essential to Burkitt lymphoma, including genes associated with hematopoietic cell differentiation (
) or B-cell development and activation (
) and found a number of context-specific dependencies including oncogene addiction in cell lines with
/
or
mutation. The strongest genotype-phenotype association was seen for
. MDM4, a negative regulator of
, was essential in
wild-type (TP53wt) Burkitt lymphoma cell lines.
knockdown activated p53, induced cell-cycle arrest, and decreased tumor growth in a xenograft model in a p53-dependent manner. Small molecule inhibition of the MDM4-p53 interaction was effective only in TP53wt Burkitt lymphoma cell lines. Moreover, primary TP53wt Burkitt lymphoma samples frequently acquired gains of chromosome 1q, which includes the
locus, and showed elevated MDM4 mRNA levels. 1q gain was associated with TP53wt across 789 cancer cell lines and
was essential in the TP53wt-context in 216 cell lines representing 19 cancer entities from the Achilles Project. Our findings highlight the critical role of p53 as a tumor suppressor in Burkitt lymphoma and identify MDM4 as a functional target of 1q gain in a wide range of cancers that is therapeutically targetable. SIGNIFICANCE: Targeting MDM4 to alleviate degradation of p53 can be exploited therapeutically across Burkitt lymphoma and other cancers with wild-type p53 harboring 1q gain, the most frequent copy number alteration in cancer.
Abstract
RNAi loss-of-function screening with pooled shRNA expression libraries has proven to be a useful tool to identify essential cancer genes and drug resistance mechanisms in numerous cell lines ...in vitro. The effects of knocking down large numbers of genes on cancer cell growth and viability can be measured simultaneously using this approach. Now there is considerable interest to adapt RNAi viability screens to ex vivo xenograft mouse models. However, small take rates, growth rate variance of cells, and differing cell microenvironments in these more complex tumor models impose significant challenges to this sort of assay. We have developed a novel approach based on the use of pooled shRNA libraries with clonal barcodes that enables proliferation measurements of clonal populations produced by the individual shRNA-expressing founder cells that produce the tumor. This RNAi clonal analysis provides a basis to separate, across large cell populations, the external parameters that strongly influence cell growth rates from shRNA-induced growth inhibition in these systems. We will present in vivo RNAi screening validation data for several cancer models.
Citation Format: Paul Diehl, Donato Tedesco, Kyle Bonneau, Mikhail Makhanov, Debbie Deng, Peiqing Sun, Alex Chenchik. Moving beyond in vitro models and addressing the challenges of pooled RNAi screens in mouse xenografts. 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 1721. doi:10.1158/1538-7445.AM2015-1721
Abstract
RNAi loss-of-function screening with pooled shRNA expression libraries has proven to be a useful tool to identify essential cancer genes and drug resistance mechanisms in numerous cell lines ...in vitro. The effects of knocking down large numbers of genes on cancer cell growth and viability can be measured simultaneously using this approach. Now there is considerable interest to adapt RNAi viability screens to ex vivo xenograft mouse models. However, small take rates, growth rate variance of cells, and differing cell microenvironments in these more complex tumor models impose significant challenges to this sort of assay. We have developed a novel approach based on the use of pooled shRNA libraries with clonal barcodes that enables proliferation measurements of clonal populations produced by the individual shRNA-expressing founder cells that produce the tumor. This RNAi clonal analysis provides a basis to separate, across large cell populations, the external parameters that strongly influence cell growth rates from shRNA-induced growth inhibition in these systems. We will present in vivo RNAi screening validation data for several cancer models.
Citation Format: Donato Tedesco, Kyle Bonneau, Mikhail Makhanov, Debbie Deng, Paul Diehl, Peiqing Sun, Alex Chenchik. Moving beyond in vitro models and addressing the challenges of pooled RNAi screens in mouse xenografts. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4266. doi:10.1158/1538-7445.AM2014-4266
Chronic inflammation is associated with increased cancer risk. Furthermore, the transcription factor NF-...B, a central regulator of inflammatory responses, is constitutively active in most tumors. ...To determine whether active NF-...B inherently contributes to malignant transformation, we isolated a set of NF-...B-activating genetic elements and tested their oncogenic potential in rodent cell transformation models. Genetic elements with desired properties were isolated using biologically active selectable peptide technology, which involves functional screening of lentiviral libraries encoding 20 or 50 amino acid-long polypeptides supplemented with endoplasmic reticulum-targeting and oligomerization domains. Twelve NF-...B-activating selectable peptides (NASPs) representing specific fragments of six proteins, none of which was previously associated with NF-...B activation, were isolated from libraries of 200,000 peptides derived from 500 human extracellular proteins. Using selective knockdown of distinct components of the NF-...B pathway, we showed that the isolated NASPs act either via or upstream of TNF receptor-associated factor 6. Transduction of NASPs into mouse and rat embryo fibroblasts did not, in itself, alter their growth. However, when coexpressed with oncogenic Ras (H-RasV12), NASPs allowed rodent fibroblasts to overcome H-RasV12-mediated p53-dependent senescence and acquire a transformed tumorigenic phenotype. Consistent with their ability to cooperate with oncogenic Ras in cell transformation, NASP expression reduced the transactivation activity of p53. This system provides an in vitro model of NF-...B-driven carcinogenesis and suggests that the known carcinogenic effects of inflammation may be at least partially due to NF-...B-mediated abrogation of oncogene-induced senescence. (ProQuest: ... denotes formulae/symbols omitted.)
Abstract
We present results of clonal analysis studies aimed at characterizing the heterogeneity of in vivo cancer cell growth in xenograft models. Using a high-throughput sequencing method, we ...followed the fate of thousands of individually bar-coded cancer cells grown in vitro, or in vivo upon sub-cutaneous implantation in immunocompromised mice. By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we found that xenograft growth is marked by a phenomenon of clonal dominance, in which a small subset of cancer cell sub-clones account for the bulk of the resulting tumor mass. This phenomenon has important implications for the field of in vivo shRNA screening. Subsequently, we developed a new proprietary clonal RNAi screening platform specifically designed to circumscribe the clonal dominance effect in xenograft model viability screens. We used this technology to perform xenograft “drop-out” screens in a panel of tumorigenic cell lines, including human breast, ovarian and colon carcinomas. Preliminary data will be presented. These results demonstrate that complex pooled shRNA libraries provide a highly efficient, flexible, and unique tool for in vivo screens aimed the discovery of potential cancer therapy targets.
Citation Format: Donato Tedesco, Kyle Bonneau, Mikhail Makhanov, Debbie Deng, Paul Diehl, Peiqing Sun, Alex Chenchik. Pooled RNAi screens in xenograft mouse models. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3172. doi:10.1158/1538-7445.AM2013-3172
To determine the variability of the NS3/NS3A gene of field strains of BTV contained in Culicoides sonorensis collected from a single site in California (CA), the NS3/NS3A gene was directly amplified ...and sequenced from 22 pools of C. sonorensis and compared with those of previously characterized field isolates from CA, as well as to viruses that caused recent outbreaks of bluetongue disease in ruminants in CA. Phylogenetic analysis established that the NS3/NS3A gene of strains of BTV contained in C. sonorensis collected from the site exists as a heterogeneous population. The two most divergent nucleotide sequences of the NS3/NS3A genes of these viruses differed by 2.5% (18 nucleotides). Comparison with the NS3/NS3A gene sequences from viruses that caused recent instances of bluetongue disease in ruminants in CA indicated that BTV strains from different geographic regions can exhibit a higher degree of genetic heterogeneity (up to 6.6%; 0-48 nucleotide differences) than those contained in C. sonorensis collected from a single site.
PDF
