Abstract 1252: Role of purine and pyrimidine metabolism in CLL B cell survival and drug resistance mediated via interaction with bone marrow microenvironment
Authors: Sinha Sutapa , Wang Zhiquan , Han Weiguo , Rabe Kari G. , Slager Susan L. , McCabe Chantal E. , O'Brien Daniel R. , Parikh Sameer A. , Braggio Esteban , Kay Neil E. 
Published: April 2023

Multiple studies including ours have shown that the interaction of chronic lymphocytic leukemia (CLL) B cells with the bone marrow (BM) microenvironment promotes cell survival and protects these cells from the cytotoxic effects of therapy. A detailed understanding of this interaction will help better understand CLL leukemic cell survival and drug resistance mechanisms. We performed RNA-seq in paired CLL B cells isolated from untreated blood and BM (n=6) and in untreated CLL B cells (n=4) cultured alone or co-cultured with bone marrow stromal cells (BMSCs) derived from either normal individuals (n=2) or CLL patients (n=2). We found upregulation of 232 and 917 genes in CLL B cells from BM and in co-cultured CLL B cells compared to the CLL B cells from the same patient’s blood and CLL B cells cultured alone respectively (p&lt;0.05, fold change &gt;1.5). However we found only 13 genes overlapped between BM CLL B cells and co-cultured CLL B cells. To explore the functional importance of these 13 genes, we analyzed the expression of these genes in CLL blood B cells from an additional 169 untreated patients by RNA-seq and found a positive relationship between 5 of these 13 genes (MKI67, PNP, C16orf54, MOB3A, CDK2AP2) with CLL patient’s clinical outcome including overall survival (OS), progression free survival (PFS), and time to first treatment (TTFT) [univariate Cox regressions analysis (UCRA) p&lt;0.05]. Out of 5 genes, purine nucleotide phosphorylase (PNP) which is an enzyme in the purine salvage pathway, showed the significant association for patient’s OS, PFS, and TTFT using UCRA [hazard ratio (HR) {95% CI}: 2.8 {1.8-4.3}, 2.7 {1.9-3.8}, 2.9 {2.0-4.2}, respectively, p&lt;.0001], suggesting purine metabolism plays an important role in BM induced CLL B cell survival. We also observed an increase in PNP protein levels in CLL B cells co-cultured with BMSCs or from BM using Western blot analysis. Untargeted metabolomic profiling (LC-MS+GC-MS) showed an increased level of purine salvage pathway metabolites adenosine, inosine, and hypoxanthine in co-cultured CLL B cells. Consistent with this altered nucleotide metabolism data, we also found increased pyrimidine pathway enzyme thymidylate synthase (TYMS) at RNA and protein levels in co-cultured CLL B cells and in BM CLL B cells. We further analyzed the RNA-seq data generated from the 169 CLL patients and it also showed a positive association of TYMS gene with their OS, PFS and TTFT using UCRA [HR (95% CI): 1.35 (1.0-1.8), 1.3 (1.0-1.6), 1.63 (1.3-2.1) respectively, p&lt;0.05]. In conclusion, these results indicate that active purine/pyrimidine metabolism in CLL B cells may be augmented by the BM environment to support CLL B cell survival. Future studies to understand the exact mechanism of PNP and TYMS mediated CLL B cell survival for both untreated and treated CLL B cells will uncover unique approaches for therapy.

Citation Format: Sutapa Sinha, Zhiquan Wang, Weiguo Han, Kari G. Rabe, Susan L. Slager, Chantal E. McCabe, Daniel R. O'Brien, Sameer A. Parikh, Esteban Braggio, Neil E. Kay. Role of purine and pyrimidine metabolism in CLL B cell survival and drug resistance mediated via interaction with bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1252.