Cell cycle checkpoint proficiency of endometrial cancer cells [bulk RNA-Seq]

Endometrial cancers (EC) are the most common gynecologic malignancy in the US. Most ECs harbor limited targetable somatic alterations, and are often grouped by histology (endometrioid, serous, or clear cell), mismatch repair, or TP53 status, none of which perfectly predict therapeutic response. A better mechanistic understanding of the key functional defects in ECs and more therapies with which to engage those targets in advanced stage EC are needed. Here we utilize functional, transcriptomic and genomic assays on a panel of EC cell lines and patient-derived organoids across EC histologic and genomic subtypes to characterize the TP53 and RB1 cell cycle regulatory proficiency and therapeutic vulnerabilities in this disease. We were surprised to find that TP53 genomic and functional status has little predictive capacity for EC therapeutic response. Rather, RB regulatory status correlated better with response to G1/S targeted therapies. Overall design: For cell lines, doubling times were calculated. For HEC1B and ARK1 models, cells were treated for one doubling time (approximately 26 hours for ARK1 and 33.5 hours for HEC1B) with either 0.25µM Abemaciclib methanesulfonate (referred to as "Abemaciclib" henceforth) or media containing DMSO vehicle (used for analysis as batch 1). For organoids, a subset of the organoids were treated with media containing either 0.25µM Abemaciclib or media containing DMSO vehicle for 24 hours (used for analysis as batch 2). For additional cell lines and organoids to be used only for baseline transcriptomic comparisons, the cells were treated with media containing DMSO at 1:100,000 for 24 hours (used for analysis as batch 3). After treatments, cell lines and organoids were harvested and stored at -80°C until RNA was prepared. RNA was prepared using Qiagen's RNEasy Mini kit (Qiagen Cat #74104) with on-column DNASE digest (Qiagen Cat. #79254) according to the manufacturer's protocol. RNA was snap frozen and sent to GENEWIZ (South Plainfield, NJ) for Standard RNA sequencing. Each individual sample was sequenced in duplicate to provide technical replicates for validation. The following library preparation and sequencing methods in quotation marks are provided verbatim by GENEWIZ and may represent a standard pipeline used there. "RNA samples were quantified using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was checked using an Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using the manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First-strand and second-strand cDNA were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on a flow cell. After clustering, the flow cell was loaded on the Illumina instrument according to the manufacturer's instructions. The samples were sequenced using a 2x150bp Paired-End (PE) configuration, targeting 30 M reads/sample. The control software conducted image analysis and base calling. Raw sequence data (.bcl files) generated by the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.20 software. One mismatch was allowed for index sequence identification."