Prediction of potent shRNAs with a sequential classification algorithm

Pelossof, Raphael; Fairchild, Lauren; Huang, Chun‐Hao; Widmer, Christian; Sreedharan, Vipin T.; Sinha, Nishi; Lai, Dan-Yu; Guan, Yuanzhe; Premsrirut, Prem K.; Tschaharganeh, Darjus F.; Hoffmann, Thomas; Thapar, Vishal; Qiu, Xiang; Garippa, Ralph; Rätsch, Gunnar; Zuber, Johannes; Lowe, Scott W.; Leslie, Christina; Fellmann, Christof

doi:10.1038/nbt.3807

Cited by 139 publications

(119 citation statements)

References 40 publications

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“…Small-interfering RNAs (siRNAs) were designed using RNAi Sensor assay technology against RAS pathway genes (Fellmann et al, 2011; Pelossof et al, 2017; Yuan et al, 2014) (Table S1, Figure S1A-B). The selectivity and potency of these siRNAs allow for simultaneous knockdown of multiple functional paralogs (i.e.…”

Section: Resultsmentioning

confidence: 99%

Differential Effector Engagement by Oncogenic KRAS

et al. 2018

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KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.

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Section: Resultsmentioning

confidence: 99%

Differential Effector Engagement by Oncogenic KRAS

et al. 2018

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“…shRNA cloning. shRNAs were designed with the splashRNA software 32 and cloned into the backbone by Gibson assembly as previously described 33 . All designed shRNAs, lentiviral backbones and primers used for cloning are listed in Supplementary Table 3…”

Section: Methodsmentioning

confidence: 99%

LSD1 inhibitors induce neuronal differentiation of Merkel cell carcinoma by disrupting the LSD1-CoREST complex and activating TGFβ signaling

Leiendecker

Jung

Neumann

et al. 2020

Preprint

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Merkel cell carcinoma (MCC) is a highly aggressive, neuroendocrine skin cancer that is either associated with the clonal integration of the Merkel cell polyomavirus or with chronic sun exposure 1,2 . Immunotherapy is initially effective in many patients with metastatic MCC, but the response is rarely durable 3,4 . MCC lacks actionable mutations that could be utilized for targeted therapies, but epigenetic regulators, which govern cell fate, provide unexplored therapeutic entry points. Here, we performed a pharmacological screen in MCC cells, targeting epigenetic regulators. We discovered that the lysine-specific histone demethylase 1A (LSD1/KDM1A) is required for MCC growth in vitro and in vivo. HMG20B (BRAF35), a poorly characterized subunit of the LSD1-CoREST complex, is also essential for MCC proliferation. LSD1 inhibition in MCC disrupts the LSD1-CoREST complex, directly induces the expression of key regulators of the neuronal lineage and of members of the TGFβ pathway, and activates a gene expression signature corresponding to normal Merkel cells. Our results provide a rationale for evaluating LSD1 inhibitors, which are currently being tested in patients with leukemia and solid tumors, in MCC.

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“…In order to confirm the specificity of TERT regulation by GABPB1L in TERTp mutant cells, we made use of a doxycycline-inducible, microRNA-embedded short hairpin RNA (shRNA) expression system with a green fluorescent protein (GFP) marker reporting shRNA induction [33][34][35] . We used a machine learning algorithm 33 to design four shRNAs each targeting either GABPB1L or GABPB1S specifically. Doxycycline-mediated induction of GABPB1L and GABPB1S shRNA expression led to reductions in the respective mRNA and protein levels in a TERTp mutant cell line ( Fig.…”

Section: Gabpb1l-containing Transcription Factor Complexes Bind the Mmentioning

confidence: 99%

“…MicroRNA-embedded miR-E shRNA sequences were predicted using SplashRNA 33 and cloned into LT3GEPIR 35 , an all-in-one doxycycline-inducible miR-E shRNA expression vector. All shRNA sequences are shown (Table S1), and were cloned as previously described 65 .…”

Section: Design Of Shrnas For Reversible Target Inhibitionmentioning

confidence: 99%

Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage

Amen¹,

Fellmann²,

Soczek³

et al. 2020

Preprint

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Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose expression is associated with TERT reactivation and telomere maintenance. Here, using biochemical and cell biology approaches, we show direct evidence that a specific GABP complex containing the subunit protein GABPB1L forms predominantly at the mutant TERT promoter, leading to TERT re-expression. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, are immediately dependent on GABPB1L for proliferation in cell culture and post-tumor establishment in vivo. Notably, when combined with frontline temozolomide (TMZ) chemotherapy, GABPB1L knockdown and the associated TERT reduction lead to an impaired DNA damage response that results in profoundly reduced growth of intracranial GBM tumors.Together, these findings provide new insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of TERT suppression in GBM maintenance, and establish GABPB1L inhibition, alone or in combination with chemotherapy, as a therapeutic strategy for TERTp mutant GBM.

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Prediction of potent shRNAs with a sequential classification algorithm

Cited by 139 publications

References 40 publications

Differential Effector Engagement by Oncogenic KRAS

Differential Effector Engagement by Oncogenic KRAS

LSD1 inhibitors induce neuronal differentiation of Merkel cell carcinoma by disrupting the LSD1-CoREST complex and activating TGFβ signaling

Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage

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