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The ABL2 kinase regulates an HSF1-dependent transcriptional program required for lung adenocarcinoma brain metastasis

Significance Among all cancer types, lung cancer patients exhibit the highest prevalence of brain metastasis, often associated with cognitive impairment, seizures, decline in quality of life, and decreased survival. Limited therapeutic options are currently available to treat brain metastasis. A comprehensive understanding of the signaling pathways and transcriptional networks required for survival and growth of brain-metastatic cancer cells is needed to develop effective strategies to treat this disease. Here, we report that the Heat Shock Transcription Factor 1 (HSF1) is upregulated in brain-metastatic lung cancer cells and is required for brain metastasis in mice. Importantly, we show that the HSF1-dependent expression of E2F target genes implicated in cell cycle progression and survival is decreased by blood–brain barrier-penetrant ABL allosteric inhibitors.


RNA-sequencing analysis
For differential expression analysis of RNA-seq data comparing PC9 parental to PC9-BrM3 cells, total RNA was collected from equally sub-confluent cell cultures using the Libraries were sequenced on an Illumina HiSeq 2000 sequencing system using 50-bp single-end reads by the Duke University Genome Sequencing facility. RNA-seq fastq data files were processed using the TrimGalore toolkit followed by read quality assessment with the Fast-QC analysis tool. Reads were mapped to the GRCh37 version of the human genome and transcriptome using the HISAT2 RNA-seq alignment tool (1). Reads were used for subsequent analysis if they mapped to a single genomic location, and gene counts were compiled using FeatureCounts (2). Normalization and differential expression were carried out using the DESeq2 Bioconductor package (3) in the R statistical programming environment. Datasets containing differential expression analysis corresponding to each RNA-seq experiment are provided as Supplementary File 1 and Supplementary File 2.
RNA sequencing raw data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE149246. 3

Gene set enrichment analysis
Following analysis of RNA-seq differential expression data comparing PC9 parental to PC9-BrM3 cells, data were sorted by statistical rank and imported into the GSEA software tool from the Broad Institute (4). The pre-ranked gene list was processed under default settings and size filters for analysis across all signatures contained within the following

Generation of HSF1-regulated E2F target gene panel
Following differential expression analysis of RNA-seq data from PC9-BrM3 values for each gene in the setting of brain metastasis. As a final filtering step, the top 20 genes from the BPE+HS gene list with reads present in the PC9-BrM3 RNA-sequencing data were included to form a representative HSF1 heat shock target gene panel.

Real-time quantitative PCR
RNA was isolated using the RNeasy RNA isolation kit (Qiagen), and cDNA synthesis was performed using oligo(dT) primers and M-MLV reverse transcriptase (Invitrogen).
Samples for RT-qPCR analysis were loaded in triplicate wells with iTaq Universal SYBR

Co-immunoprecipitation assays
Cells were lysed with immunoprecipitation buffer (50 µM Tris-HCl pH 8.0, 150 mM NaCl, 1.0% NP-40, 1x protease inhibitor cocktail) and lysates were incubated for 30 min on ice prior to clearing by centrifugation at 15,000 rpm for 10 min. Supernatants were incubated with antibodies overnight followed by incubation with protein A/G PLUS-agarose beads (Santa Cruz) for 3 h. Following incubation, beads were washed three times with immunoprecipitation buffer. Bound proteins were eluted with 4x Laemmli Sample Buffer (Bio-Rad) and visualized by SDS-PAGE on nitrocellulose membranes followed by immunoblot analysis. Total expression for each protein was visualized by running equal concentrations of whole protein lysate input on an SDS-PAGE gel prior to transfer onto nitrocellulose membranes and subsequent immunoblot analysis.

Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR)
ChIP-qPCR analysis was performed as previously described (6). Briefly, cells were crosslinked with formaldehyde (1% final concentration) on ice for 5 min with gentle shaking.
Formaldehyde was quenched by adding 125 M glycine in PBS for 5 min on ice with gentle shaking. Cells were then lysed in cell lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) containing protease and phosphatase inhibitors 7 and transferred to a 15 mL conical tube for 15 min on ice. 1 mL of IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) was added, and lysates were then sonicated ten times for 30 s each on ice using a microprobe on setting 5. Lysates were cleared by spinning down cellular debris at 13,000 RPM at 4°C for 30 min. Samples containing antibody against HSF1 (Enzo) or IgG control were rotated overnight at 4°C, after which Dynabeads Protein G were added to all samples and rotated at 4°C for 43h.
Beads were washed twice with IP buffer, twice with IP buffer + 0.5 M NaCl, and twice with TE Buffer (10 mM Tris, pH 7.5, 1 mM EDTA). Proteins were then eluted by adding TE buffer + 1% SDS and incubating at 65°C for 10 min, and beads were spun down at 14,000 rpm for 10 min. Samples were then incubated overnight at 65°C to reverse crosslinks, followed by treatment with proteinase K for 1.5 h at 37°C and purification with a PCR cleanup kit (Qiagen) prior to analysis via qPCR. Primer sequences are listed in Supplementary Table S1.

Nuclear/Cytosolic Subcellular Fractionation Immunoprecipitation
Cells plated onto 15 cm dishes were harvested by trypsinization and pelleted by spinning at 1,000 RPM for 4 min. The cell pellet was washed with 5 mL PBS, pelleted by centrifugation, and then resuspended in 1 mL PBS, transferred to a 1.5 mL tube, and repelleted. The cell pellet was resuspended in 500 µL cytosolic lysis buffer (20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2) and incubated for 15 min on ice. 25 µL of 10 % NP-40 was added to the cell homogenate and the sample was vortexed for 10 s followed by centrifugation at 3,000 RPM for 10 min at 4°C. The supernatant was then removed and designated as the cytosolic fraction. The remaining nuclear pellet was resuspended in 8 nuclear lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40) and incubated on ice for 30 min with vortexing at 10 min intervals. The sample was spun down at 14,000 RPM at 4°C for 30 min. The supernatant was transferred to a new 1.5 mL tube and designated the nuclear fraction. 1000 µg of each fraction were used for subsequent immunoprecipitation following the method described above.

FACS analysis
Subconfluent monolayers of PC9-BrM3 cells transduced with inducible shRNAs against non-target control (shNTC) or HSF1 (shHSF1 clone 7481) were treated with 500 ng/mL doxycycline for 48 h prior to harvest by trypsinization, fixation by EtOH, and staining with propidium iodide using the Propidium Iodide Flow Cytometry Kit (Abcam #ab139418) following the manufacturer-supplied protocol. Fixed and stained cells were analyzed by FACS on a BD FACSCanto IIs analyzer. Post-FACS cell cycle analysis was performed using FlowJo 10 software.

Kaplan-Meier Analysis and Mutual Exclusivity Analysis
Lung adenocarcinoma patient microarray data were analyzed using the KMplot analysis tool (kmplot.com) (7). Patient groups were stratified by tertile (n=720 total patients across all cohorts). Affimetrix identifiers for each gene were as follows: 228361_at (E2F1), (MCM7). Survival data were plotted in GraphPad Prism 8 software. Mutual exclusivity analysis of HSF1-E2F target genes was performed with CBioPortal using the TCGA 9 Provisional lung adenocarcinoma dataset (n=515 patients) (8,9). A user-defined list of the 15 gene HSF1-E2F target panel was used, and patients with mRNA expression data (RNAseq, z-score threshold ± 2.0) were included. Heatmaps of patient cohorts were generated using default clustering settings on CBioPortal, and mutual exclusivity analysis heatmaps were plotted using GraphPad Prism 8 software. confocal microscope, and analysis was performed using ImageJ software (10).

DNA plasmids
Sequences for stable shRNAs targeting the ABL kinases were as follows: In vitro GST-pulldown assay 12 GST-ABL2 fragments were added dissolved at a final concentration of 1 µM in binding buffer (25 mM Tris HCl, pH 7.5, 100 mM NaCl, 0.1%, 1 mM DTT, and 5% glycerol). A

Promoter analysis of predicted degenerate HSF1 binding sites at E2F target genes
To identify predicted degenerate HSEs located near promoter regions of canonical E2F targets identified in the 15-gene HSF1-regulated E2F target gene panel, we utilized GAAnnTTC, AAnnTTCnnGA, and TTCnnGAA. Degenerate HSE sequences were then used to search the promoter regions of genes from the E2F target panel.