Reproducibility Project: Cancer Biology Replication Study

Abstract

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by replicating selected results from a substantial number of high-profile papers in the field of cancer biology published between 2010 and 2012. This Registered report describes the proposed replication plan of key experiments from ‘BET bromodomain inhibition as a therapeutic strategy to target c-Myc’ by Delmore and colleagues, published in Cell in 2011 (Delmore et al., 2011). The key experiments that will be replicated are those reported in Figures 3B and 7C-E. Delmore and colleagues demonstrated that treatment with JQ1, a small molecular inhibitor targeting BET bromodomains, resulted in the transcriptional down-regulation of the c-Myc oncogene in vitro (Figure 3B; Delmore et al., 2011). To assess the therapeutic efficacy of JQ1 in vivo, mice bearing multiple myeloma (MM) lesions were treated with JQ1 before evaluation for tumor burden and overall survival. JQ1 treatment significantly reduced disease burden and increased survival time (Figure 7C-E; Delmore et al., 2011). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Introduction

c-Myc is a DNA binding transcription factor involved in the regulation of cell proliferation, differentiation, and apoptosis (McKeown and Bradner, 2014). Abnormal expression of c-Myc is frequently observed in a range of malignancies including breast, colon and cervical cancer, small cell lung carcinoma, osteosarcomas, glioblastomas and myeloid leukemias (Meyer and Penn, 2008; Conacci-Sorrell et al., 2014). While c-Myc expression is required for tumor initiation and maintenance, c-Myc inactivation leads to tumor regression (Felsher and Bishop, 1999; Flores et al., 2004; Soucek et al., 2008; Gabay et al., 2014). Therefore, c-Myc represents an enticing target for pharmacological inhibition.

Multiple myeloma (MM) is an incurable disease characterized by the unrestricted proliferation of terminally differentiated plasma cells (Anderson et al., 2011). The primary tumor-initiating events include genetic translocation and hyperploidy, while secondary events, such as oncogenic c-Myc activation and overexpression, drive MM progression (Bergsagel and Kuehl, 2005; Morgan et al., 2012). Furthermore, studies by Chng and colleagues determined that c-Myc activation was prevalent in more than 60% of patient-derived MM cells (Chng et al., 2011). Despite therapeutic advances and increases in survival, patients eventually succumb to treatment-refractory disease (Anderson, 2011; Kumar et al., 2012).

Therapeutic strategies targeting c-Myc are complicated by the fact that c-Myc lacks a clear ligand-binding domain (Darnell, 2002). However, it is possible that c-Myc could be disrupted by other means, such as through disruption of chromatin-dependent signaling. Bromodomain and extraterminal (BET) proteins are transcriptional regulators that epigenetically control the expression of genes involved in cell cycle, growth and inflammation (Darnell, 2002; Wu and Chiang, 2007; LeRoy et al., 2008; Dey et al., 2009; Nicodeme et al., 2010). BETs therefore provide potential therapeutic targets for modulating gene expression programs associated with various human diseases. Specifically, bromodomain protein 4 (BRD4), a member of the BET subfamily that associates with acetylated chromatin to promote transcription, was reported to interact with the positive transcription elongation factor complex b (P-TEFb) (Dey et al., 2009; Filippakopoulos et al., 2010). Recruitment of P-TEFb by c-Myc was also reported, providing the rationale for Delmore and colleagues to explore targeting BET proteins to inhibit c-Myc transcriptional activity (Bisgrove et al., 2007). Importantly, BRD4 expression was found to positively correlate with MM disease progression (Delmore et al., 2011). To interrogate this relationship, they used JQ1, a small molecule inhibitor of the BET family of bromodomain-containing proteins, which has the highest affinity with BRD4 and competitively inhibits BET proteins from binding to chromatin (Filippakopoulos et al., 2010). While the (+)-JQ1 enantiomer potently inhibits BET proteins, the (−)-JQ1 enantiomer is structurally incapable of inhibiting BET bromodomains supporting an on-target mechanism of action (Filippakopoulos et al., 2010). Further support for the relationship between c-Myc and BET proteins was reported by Mertz and colleagues, who used gene expression profiling of cells treated with the active and inactive forms of the JQ1 inhibitor to identify MYC as a highly down-regulated gene following BET bromodomain inhibition (Mertz et al., 2011).

As an alternative approach to direct c-Myc-targeting, Delmore and colleagues tested whether the BET inhibitor, JQ1, could effect c-Myc-specific gene silencing in MM (Delmore et al., 2011). In Figure 3B, Delmore and colleagues assessed the ability of JQ1 to downregulate MYC transcription in the MM cell line MM.1S. In this experiment, MM.1S cells were treated with JQ1 for up to 8 hours and the relative expression of MYC was compared to untreated control cells. JQ1 treatment resulted in a significant reduction in MYC transcripts as determined by qRT-PCR. This key experiment shows that JQ1 was effective at silencing MYC gene transcription and will be replicated in Protocol 1. Importantly, Loven and colleagues also recently corroborated these results through the demonstration that JQ1 treatment in MM.1S cells significantly decreases MYC mRNA levels (Loven et al., 2013). In addition to MM cell lines, JQ1 has proven to potently inhibit MYC in Merkel cell carcinoma cells (MCC-3 and 5), primary effusion lymphoma cells (PELs) and B cell acute lymphoblastic lymphomas (B-ALL) cells at the transcript level, as well as in diffuse large B cell lymphoma (DLBCL) cells at the protein expression level (Ott et al., 2012; Shao et al., 2014; Tolani et al., 2014; Trabucco et al., 2015). However, JQ1-resistant cells have also been described. Specifically, JQ1 did not alter MYC transcription in embryonic stem cells (ESCs) or in non-small cell lung carcinoma (NSCLC) harboring alteration in KRAS (Shimamura et al., 2013; Horne et al., 2014). In lung adenocarcinoma cells (LACs), JQ1 was found to inhibit cell growth independent of MYC down regulation (Lockwood et al., 2012).

In Figure 7C, 7D and 7E, the efficacy of JQ1 treatment was tested in mice harboring bioluminescent MM lesions. In these experiments, tumor burden was measured by whole-body bioluminescent imaging. Delmore and colleagues showed that JQ1 treatment significantly decreased disease burden and increased survival time compared to vehicle-treated control animals (Delmore et al., 2011). Similar findings recapitulating the suppressive effect of JQ1 on solid tumor growth have been reported in MCC, DLBCL and PEL xenograft models (Ott et al., 2012; Tolani et al., 2014; Trabucco et al., 2015), and reduced leukemic burden in a B-ALL xenograft model with corresponding improvements in survival (Ott et al., 2012). These experiments will be replicated in Protocol 2.

Materials and methods

Protocol 1: evaluation of MYC expression in JQ1-treated MM.1S cells

This experiment analyzes the expression of endogenous MYC during pharmacological inhibition of BET bromodomains with JQ1. This is a replication of the data presented in Figure 3B and assesses the levels of MYC by quantitative RT-PCR.

Sampling

• Each experiment has 9 conditions:
  • qRT-PCR of MYC (and GAPDH) 0 hr after (+)-JQ1 treatment.
  • qRT-PCR of MYC (and GAPDH) 1 hr after (+)-JQ1 treatment.
  • qRT-PCR of MYC (and GAPDH) 8 hr after (+)-JQ1 treatment.
  • qRT-PCR of MYC (and GAPDH) 0 hr after (−)-JQ1 treatment [additional].
  • qRT-PCR of MYC (and GAPDH) 1 hr after (−)-JQ1 treatment [additional].
  • qRT-PCR of MYC (and GAPDH) 8 hr after (−)-JQ1 treatment [additional].
  • qRT-PCR of MYC (and GAPDH) 0 hr after vehicle treatment [additional].
  • qRT-PCR of MYC (and GAPDH) 1 hr after vehicle treatment [additional].
  • qRT-PCR of MYC (and GAPDH) 8 hr after vehicle treatment [additional].
• Experiment will be performed five times with each run using three technical replicates, for a total power of ≥91%.
  • See ‘Power calculations’ section for details.

Materials and reagents

Procedure

Notes

• All cells will be sent for mycoplasma testing and STR profiling.
• Cells maintained in RPMI 1640 with 2 mM L-glutamine supplemented with 10% FBS, 100 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in a humidified atmosphere at 5% CO ₂.

1. Seed 8 × 10 ⁵ MM.1S-LucNeo cells into three 35-mm tissue culture dishes.
2. The next day treat the dishes of cells with 2 ml of media with a final concentration of 500 nM (+)-JQ1, 500 nM (−)-JQ1, or an equivalent volume of DMSO.
   • Make 10 mM stock of (+)-JQ1 and (−)-JQ1 by diluting in DMSO.
3. Isolate RNA from dishes at the following time points after treatment using TRI Reagent following manufacturer's instructions.
   • 0 hr (immediately).
   • 1 hr.
   • 8 hr.
4. Reverse transcribe total RNA to cDNA with reverse transcription kit following manufacturer's instructions.
   • Record RNA concentration and purity.
   • Use 1 µg of RNA per 50 µl reaction.
   • Use random hexamers for first-strand synthesis.
5. Perform qPCR to assess MYC expression levels using a real-time PCR system with a real-time PCR kit following manufacturer's instructions. Perform triplicate technical replicates for each biological replicate.
   • Use 5 µl of undiluted cDNA mixture per 50 µl reaction.
   • Use TaqMan probes for MYC (Hs00905030_m1) and Gapdh (Hs02758991_g1).
6. Analyze and compute ΔΔC _T values.
   • The first qRT-PCR assay will be analyzed to ensure conditions are appropriate for proper quantitation. If it is determined that conditions need to be adjusted, such as input volume, the conditions will be adjusted and the reaction will be repeated. Once optimized, the conditions will be used for all subsequent reactions.
     • All details and data associated with this process will be recorded.
7. Repeat steps 1–6 independently four additional times.

Deliverables