Xiaoyu Lin

siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments.

'Seed' analysis of off-target siRNAs reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-XL inhibitor ABT-737.

ABT-737 is a subnanomolar inhibitor of the antiapoptotic proteins Bcl-2, Bcl-XL and Bcl-w. Although ABT-737 triggers extensive cell death in many small-cell lung carcinoma (SCLC) cell lines, some of the SCLC cell lines and the majority of the cancer cell lines derived from other solid tumors were found to be resistant to ABT-737. To better understand the mechanism of resistance to ABT-737, we screened a short interfering RNA library consisting of short interfering RNA against 4000 ‘druggable’ targets in an SCLC-derived cell line, NCI-H196. By comparing the knockdowns with phenotypes, all of the three top ‘hits’ from the screen were found to result from off-target gene silencing. Interestingly, the three off-target siRNAs were found to knock down an antiapoptotic Bcl-2 family protein Mcl-1 owing to the complementation between their seed regions with the 3′ untranslated region (3′ UTR) of Mcl-1. Furthermore, reducing the level of Mcl-1 using siRNAs or the small-molecule compounds Bay43-9006 and Seliciclib was sufficient to overcome the resistance to ABT-737 in the resistant SCLC cell line and cancer cell lines derived from other solid tumors. These results provide further evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and the combination of Mcl-1 downregulating agents with ABT-737 could be potent therapeutic regimens for patient with ABT-737-resistant SCLC and many other types of solid tumors.

Evaluating Hypoxia-Inducible Factor-1α as a Cancer Therapeutic Target via Inducible RNA Interference In vivo

Validating potential targets is an important step in the drug discovery process. In this study, we tested the feasibility of using inducible RNA interference (RNAi) in vivo to obtain an unbiased evaluation on the efficacy of inhibiting hypoxia-inducible factor-1alpha (HIF-1alpha) in established tumors. We showed that HIF-1alpha inhibition resulted in transient tumor stasis or tumor regression, and inhibiting HIF-1alpha in early-stage tumors was found to be more efficacious than inhibiting HIF-1alpha in more established tumors. A differential requirement of HIF-1alpha for tumor growth was also observed among different tumor types. Examination of tumors resistant to HIF-1alpha inhibition suggested that the resistance might result from a less hypoxic tumor environment and the level of HIF-1alpha expression in tumors may be a useful marker for predicting tumor response to HIF-1 inhibition. This study shows that inducible RNAi is a versatile tool for evaluating cancer targets in vivo. In addition to broad implications on in vivo validation of cancer targets, results from this study will also be instructive for practical applications of HIF-1-based cancer therapeutics.

The Bcl-2/Bcl-X(L)/Bcl-w inhibitor, navitoclax, enhances the activity of chemotherapeutic agents in vitro and in vivo.

The ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of prosurvival proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small-molecule inhibitor of Bcl-2, Bcl-XL, and Bcl-w. While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-XL for survival, the expression of Mcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here, we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeutic agents in vitro and enhanced efficacy of both docetaxel and erlotinib in xenograft models. The ability of navitoclax to synergize with docetaxel or erlotinib corresponded to an altered sensitivity of the mitochondria toward navitoclax, which was associated with the downmodulation of Mcl-1 and/or upregulation of Bim. These data provide a rationale to interrogate these combinations clinically. Mol Cancer Ther; 10(12); 2340–9. ©2011 AACR.

A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development and progression by regulating genes that are vital for proliferation, glycolysis, angiogenesis, and metastasis. To identify strategies of targeting the HIF-1 pathway, we screened a siRNA library against the entire druggable genome and a small-molecule library consisting of 691,200 compounds using a HIF-1 reporter cell line. Although the siRNA library screen failed to reveal any druggable targets, the small-molecule library screen identified a class of alkyliminophenylacetate compounds that inhibit hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations. These compounds were found to inhibit hypoxia but not deferoxamine-induced HIF-1α protein stabilization. Further analysis indicated that the alkyliminophenylacetate compounds likely inhibit the HIF-1 pathway through blocking the hypoxia-induced mitochondrial reactive oxygen species (ROS) production. Strikingly, all of the nonalkyliminophenylacetate HIF-1 inhibitors identified from the small-molecule library screen were also found to target mitochondria like the alkyliminophenylacetate compounds. The exclusive enrichment of mitochondria inhibitors from a library of >600,000 diverse compounds by using the HIF-1 reporter assay highlights the essential role of mitochondria in HIF-1 regulation. These results also suggest that targeting mitochondrial ROS production might be a highly effective way of blocking HIF-1 activity in tumors.

Development of a tightly regulated U6 promoter for shRNA expression.

Short hairpin RNAs (shRNAs) have been used to achieve stable target knockdown in a variety of biological systems. Here, we report the development of a tightly regulated tetracycline‐responsive human U6 promoter for shRNA expression. By engineering two copies of the tet operators flanking the TATA box of the human U6 promoter, we created a U6 promoter derivative (2O2) that exhibited much lower basal transcriptional activity compared with recently reported inducible pol III dependent promoters. As a consequence of its tighter regulation, the 2O2 system greatly improved the success rate in making inducible knockdown cell lines.

Hypoxia-Inducible Factor-1 Inhibition in Combination with Temozolomide Treatment Exhibits Robust Antitumor Efficacy In vivo

Purpose: Inhibiting hypoxia-inducible factor-1 (HIF-1) represents a unique mechanism for cancer therapy. It is conceived that HIF-1 inhibitors may synergize with many classes of cancer therapeutic agents, such as angiogenesis inhibitors and cytotoxic drugs, to achieve a more robust tumor response. However, these hypotheses have not been rigorously tested in tumor models in vivo. The present study was carried out to evaluate the antitumor efficacy of combining HIF-1 inhibition with angiogenesis inhibitors or cytotoxic agents. Experimental Design: Using a D54MG-derived tumor model that allows knockdown of HIF-1α on doxycycline treatment, we examined the tumor responses to chemotherapeutic agents, including the angiogenesis inhibitor ABT-869 and cytotoxic agents 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide, in the presence or absence of an intact HIF-1 pathway. Results: Surprisingly, inhibiting HIF-1 in tumors treated with the angiogenesis inhibitor ABT-869 did not produce much added benefit compared with ABT-869 treatment alone, suggesting that the combination of an angiogenesis inhibitor with a HIF-1 inhibitor may not be a robust therapeutic regimen. In contrast, the cytotoxic drug temozolomide, when used in combination with HIF-1α knockdown, exhibited a superadditive and likely synergistic therapeutic effect compared with the monotherapy of either treatment alone in the D54MG glioma model. Conclusions: Our results show that the DNA alkylating agent temozolomide exhibits robust antitumor efficacy when used in combination with HIF-1 inhibition in D54MG-derived tumors, suggesting that the combination of temozolomide with HIF-1 inhibitors might be an effective regimen for cancer therapy. In addition, our results also show that the RNA interference–based inducible knockdown model can be a valuable platform for further evaluation of the combination treatment of other cancer therapeutics with HIF-1 inhibition.

Bcl-XL represents a druggable molecular vulnerability during aurora B inhibitor-mediated polyploidization.

Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This “addiction” can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.

Exploitation of Castration-Resistant Prostate Cancer Transcription Factor Dependencies by the Novel BET Inhibitor ABBV-075

Competitive inhibitors of acetyl-lysine binding to the bromodomains of the BET (bromodomain and extra terminal) family are being developed for the treatment of solid and hematologic malignancies. The function of BET family member BRD4 at enhancers/superenhancers has been shown to sustain signal-dependent or pathogenic gene expression programs. Here, the hypothesis was tested that the transcription factor drivers of castration-resistant prostate cancer (CRPC) clinical progression, including the androgen receptor (AR), are critically dependent on BRD4 and thus represent a sensitive solid tumor indication for the BET inhibitor ABBV-075. DHT-stimulated transcription of AR target genes was inhibited by ABBV-075 without significant effect on AR protein expression. Furthermore, ABBV-075 disrupted DHT-stimulated recruitment of BET family member BRD4 to gene-regulatory regions cooccupied by AR, including the well-established PSA and TMPRSS2 enhancers. Persistent BET inhibition disrupted the composition and function of AR-occupied enhancers as measured by a reduction in AR and H3K27Ac ChIP signal and inhibition of enhancer RNA transcription. ABBV-075 displayed potent antiproliferative activity in multiple models of resistance to second-generation antiandrogens and inhibited the activity of the AR splice variant AR-V7 and ligand-binding domain gain-of-function mutations, F877L and L702H. ABBV-075 was also a potent inhibitor of MYC and the TMPRSS2-ETS fusion protein, important parallel transcription factor drivers of CRPC. Implications: The ability of BET family inhibitor ABBV-075 to inhibit transcription activation downstream of the initiating events of transcription factors like AR and TMPRSS2:ETS fusion proteins provides a promising therapeutic option for CRPC patients who have developed resistance to second-generation antiandrogens. Mol Cancer Res; 15(1); 35–44. ©2016 AACR.

A robust in vivo positive-readout system for monitoring siRNA delivery to xenograft tumors

Delivering small interfering RNA (siRNA) to tumors is the major technical hurdle that prevents the advancement of siRNA-based cancer therapy. One of the difficulties associated with the development of clinically relevant delivery systems is the lack of reliable tools for monitoring siRNA delivery to tumors in vivo. We describe here a novel, positive-readout system where siRNA-mediated target knockdown elicits a rapid and robust increase of reporter activity. Using the positive-readout system, we created (1) β-galactosidase-based tumor models that allow the detection of target knockdown in 1%-2% of tumor cells and can distinguish between tumor areas where effective target knockdown occurs versus tumor areas that are not accessible to delivery, and (2) luciferase-based tumor models that allow the quantitative assessment of a large number of delivery systems. Using these positive-readout models, we screened a number of literature-described siRNA delivery systems and identified lipid nanoparticles as a promising delivery platform for siRNA-based cancer therapy.