Rap Guanine Nucleotide Exchange Factor RAPGEF5 Is a Novel Tumor Suppressor in 7/del7q Myeloid Neoplasia

Introduction

The loss of chromosome 7 or 7q arm, (-7/del7q), remains one of the most common cytogenetic abnormalities in myeloid malignancies, identified in 15% of MDS, 20-25% of secondary AML, and 10-15% of primary AML patients (Ogawa et al. Blood, 2019). Despite the relatively high frequency of this cytogenetic abnormality, the current prognosis remains dismal, with no existing options for targeted therapeutics, while comprehensive oncogenic mechanisms remain elusive.

Past attempts at identifying and characterizing pathogenic genes disrupted during the -7/del7q deletion have focused largely on identification of lost tumor suppressor genes (TSGs) within commonly deleted regions of chromosome 7 (McNerney et al. Blood, 2013). More recent studies have focused on systematic genome-wide proliferation screens utilizing CRISPR, gene-trap, as well as cDNA libraries to identify and characterize large collections of potentially targetable tumor suppressor (Baeten et al. Leukemia, 2022). A comprehensive understanding of pathogenesis remains elusive, however, due to lack of correlation between observed clinical and biological outcomes and the large number of potential false positive hits that are not relevant in the -7/del7q contexts.

Here, we utilized a novel iterative computational analysis coupled with experimental validation to identify targets and characterized a novel TSG in the context of a large and previously unexploited clinical dataset. Our approach identified and characterized Rap guanine nucleotide exchange factor 5 (RAPGEF5), which serves as a Ras/Rap1 regulator, acts as a novel tumor suppressor gene in the -7/del7q context.

Methods and Results

To determine potential TSG targets in a patient-focused context, a large in-house patient dataset was combined with public genomic datasets and systematically analyzed using novel computational algorithms to generate a unique list of potential TSGs. This rigorous statistical dataset was generated utilizing RNAseq data from 29 -7/del7q patients as well as whole exome sequencing and whole genome sequencing data from 47 -7/del7q patients. From this list of putative TSG targets, the most significant hits, including RAPGEF5, were selected for in vitro characterization.

To assess the relative protein levels of RAPGEF5 in AML cell lines, we performed a Western Blot utilizing 2 cell models including one del7q (KG1) and one -7 model (F36P) as well as a chromosome 7 diploid AML cell model (THP-1). The -7/del7q cell lines demonstrated lower levels of RAPGEF5 when compared to the diploid cell line. The relative decrease in RAPGEF5 protein levels support the in-silico findings.

To further evaluate the effects of RAPGEF5 loss on cell proliferation in vitro, we conducted a high efficiency CRISPR knockout (CRISPRko) utilizing an individualized sgRNA targeting RAPGEF5. The cell line KG1 was selected for the CRISPRko for its chromosome 7 defect and for the lack of expression data present in DepoMap, ensuring all subsequently generated findings will be novel. RAPGEF5 knockout efficiency was 95.1% at the time of assay. We performed a cell proliferation assay and interestingly, this knockout population demonstrated a significant increase in proliferation rate (18.0%, p=0.0091) when compared to the wild-type population. This finding is noteworthy considering that KG1 is a highly proliferative AML cell model. These findings suggest that RAPGEF5 acts in a tumor suppressive manner and is lost in the -7/del7q event.

Finally, we observed a change in the variable allele frequency (VAF) of the initial KG1 RAPGEF5 CRISPRko population from 81.0% to 95.1% in 21 days of culture. This suggests that the loss of RAPGEF5 as a tumor suppressor provides an advantage over the KG1 WT population.

Conclusion

The above findings comprise the first report of RAPGEF5 acting as a tumor suppressor in MN. Our initial in vitro characterization of RAPGEF5 demonstrates its potential role as a tumor suppressor in the -7/del7q lesion and offers a novel therapeutic target.

Maciejewski:Novartis: Consultancy, Honoraria, Speakers Bureau; Alexion: Consultancy, Honoraria.