Tumor Heterogeneity-Based Sscr-Score Predicts Prognosis and Reveals Mechanism of SORL1 in Restraining CLL Progression

Introduction

Complex heterogeneity is a hallmark of chronic lymphocytic leukemia (CLL) that challenges oncological research and optimal treatment regimens. Although genetic markers with prognostic values have been developed for risk assessment in CLL, the existing prognostic models are far from satisfaction. Single-cell RNA sequencing (scRNA-seq) technologies have made it possible to illustrate the genetic heterogeneity and cell differentiation status at the single-cell level. A prognostic model associated with CLL cell differentiation status was established to refine the risk assessment of CLL patients.

Methods

Primary CLL cells from 90 patients and CD19+ B cells from 20 healthy donors were collected with informed consents according to the Declaration of Helsinki. Expression levels of SORL1 mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Cell viability was measured by the CCK-8 assay. Cell cycle and apoptosis were analyzed by PI and Annexin V-PE/7AAD staining. Fatty acid oxidation rate was assessed with Fatty Acid Oxidation Complete Assay Kit. Red oil O staining was performed to evaluate the lipid deposition in CLL cells. Single cell sequencing data were analyzed by the Seurat package and Monocle package in R4.0.5.

Results

The scRNA-seq profile revealed remarkable intra- and inter-tumoral heterogeneity of CLL. According to the differentiation status, this study reconstructed the evolutionary history of CLL cells, which reflected the molecular classification and risk subgroups of CLL. At the key point of cell differentiation, CLL cells were divided into two subsets with progressive and indolent genetic signatures respectively. Genes differentially expressed between aggressive and indolent subsets were identified. Through univariate and multivariate analysis, four genes (SORL1, SGK1, CYBB, RPL22L1) with significant prognostic values were selected to construct a risk model named SSCR-Score. The risk score was calculated and validated in two independent validation cohorts (Fig 1A).

SSCR-Score has revealed the positive prognostic value of SORL1 in CLL. Increased expression of SORL1 was associated with prolonged overall survival and improved time to first treatment in CLL. Among patients with ZAP70 (-) or IGHV mutated status, the expression levels of SORL1 were up-regulated. Although increased expression of SORL1 was associated with favorable prognosis, the biological mechanism of SORL1 still remained ill-defined. This study demonstrated that overexpression of SORL1 restrained cell proliferation, induced cell apoptosis and G2/M cell cycle arrest in CLL cells. In accordance with the RNA sequencing profile, overexpression of SORL1 suppressed abnormal lipid reposition and fatty acid oxidation in CLL cells. On a molecular level, SORL1 down-regulated the expression of lipid-metabolism related genes, including lipoprotein lipase (LPL), carnitine palmitoyltransferase 1A (CPT1A) and carnitine palmitoyltransferase 2 (CPT2).

To illustrate the SORL1-mediated regulation of LPL, immunofluorescence staining was performed. Results showed that SORL1 accelerated the degradation of LPL. After the inhibition of lysosome function, accelerated degradation of LPL was not observed in CLL cells overexpressing SORL1. Rescue experiments substantiated that SORL1 impeded progression of CLL through downregulating the activity of LPL which promoted lipid metabolism. Immuno-coprecipitation experiment showed that anti-SORL1 antibody precipitated LPL protein. Additionally, 6-Shohaol induced overexpression of SORL1 in CLL cells. It exhibited potent anti-CLL efficacy by suppressing cell proliferation, promoting cell apoptosis and G2/M cell cycle arrest in CLL cells (Fig 1B).

Conclusion

CLL presented remarkable heterogeneity which reflected different biological subclasses and risk groups. Through identifying genes differentially expressed between aggressive and indolent cell subsets, a novel prognostic model named SSCR-Score was developed to optimize risk stratification of CLL patients. Moreover, SORL1 restrained progression of CLL by inhibiting lipid metabolism regulated by LPL in CLL cells. SORL1 may serve as a potential strategy for the development of novel therapeutic target in CLL.

No relevant conflicts of interest to declare.