Single‐cell rna‐seq of classic hairy cell leukemia reveals disease drivers linked to intrinsic treatment resistance and identifies dusp1 as potential new therapeutic target

Introduction: Standard treatment with purine analogs such as cladribine (2-CdA) achieves excellent remissions in classic hairy cell leukemia (HCL), but up to 25% of patients relapse early and there is a non-diminishing risk of late relapse in complete responders. Even when targeting the recurrent genetic driver mutation BRAF V600E, HCL cells frequently persist in the bone marrow suggesting cooperating biologic alterations facilitating disease survival and persistence. Methods: Single-cell RNA sequencing (scRNA-Seq) was performed in primary HCL cells derived from three long-term (>10 years progression-free survival, PFS) and three short-term responders after 2-CdA treatment (SR, <3 years PFS) and from up to three time points (HCL diagnosis, first, and second relapse). Functional studies were performed using the BRAF D594E-mutated HAIR-M cell line after validating BRAF D594E as an activating mutation with similar downstream signaling and inhibitory potential with BRAF inhibitors as compared to BRAF V600E in primary HCL cells. Results: scRNA-Seq revealed distinct HCL cell clusters characterized by marked overexpression of DUSP1, FOS and JUND in all six patients that were outgrowing at first and second relapse in all three SR. In these DUSP1hi/FOShi/JUNDhi HCL cells, PROGENy cancer pathway analysis demonstrated induced activation of MAPK pathways and gene ontology analysis found negative regulation of the pro-apoptotic p38-MAPK-cascade as potential underlying biologic alteration. BRAF D594E-mutated HAIR-M cells were selected as substitute for primary HCL cells in cell culture due to comparable changes upon exposure to BRAF inhibitors in BRAF kinase activity conformation as assessed by the modular kinase conformation biosensor platform (KinCon) and similar reduction in ERK phosphorylation levels in HEK293T cells transiently overexpressing either flag-tagged BRAF D594E or BRAF V600E. In HAIR-M, we confirm stable expression levels of DUSP1, FOS and JUND, which were highly increased when co-cultured with HS5 stromal cells. Whereas increased expression of FOS and JUND was reversible upon exposure to BRAF inhibitors, expression of DUSP1, a direct target of BRAF-MEK-ERK signaling, and phosphorylation of p38 remained unaffected. As DUSP1 preferentially dephosphorylates/inactivates p38, we studied exposure of the DUSP1-inhibitor BCI in co-culture and found re-phosphorylated p38. Conclusion: DUSP1hi/FOShi/JUNDhi HCL cells may represent a distinct subset of HCL cells highly dependent on environmental cues with an inherently more resistant phenotype to treatment with 2-CdA or BRAF-inhibitors. Inhibition of DUSP1 may represent a novel therapeutic approach by effectively truncating HCL cells from extracellular pro-survival stimuli. Finally, our results suggest that BRAF D594E-mutated HAIR-M cells may serve as a representative disease model for functional studies. Keywords: bioinformatics, computational and systems biology, indolent non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.