BRAF(V600E) mutation together with loss of Trp53 or pTEN drives the origination of hairy cell leukemia from B-lymphocytes.

Hairy cell leukemia (HCL) is a B-lymphoma induced by BRAF(V600E) mutation. However, introducing BRAF(V600E) in B-lymphocytes fails to induce hematological malignancy, suggesting that BRAF(V600E) needs concurrent mutations to drive HCL ontogeny. To resolve this issue, here we surveyed human HCL genomic sequencing data. Together with previous reports, we speculated that the tumor suppressor TP53, P27, or PTEN restrict the oncogenicity of BRAF(V600E) in B-lymphocytes, and therefore that their loss-of-function facilitates BRAF(V600E)-driven HCL ontogeny. Using genetically modified mouse models, we demonstrate that indeed BRAF(V600E)KI together with Trp53KO or pTENKO in B-lymphocytes induces chronic lymphoma with pathological features of human HCL. To further understand the cellular programs essential for HCL ontogeny, we profiled the gene expression of leukemic cells isolated from BRAF(V600E)KI and Trp53KO or pTENKO mice, and found that they had similar but different gene expression signatures that resemble that of M2 or M1 macrophages. In addition, we examined the expression signature of transcription factors/regulators required for germinal center reaction and memory B cell versus plasma cell differentiation in these leukemic cells and found that most transcription factors/regulators essential for these programs were severely inhibited, illustrating why hairy cells are arrested at a transitional stage between activated B cells and memory B cells. Together, our study has uncovered concurrent mutations required for HCL ontogeny, revealed the B cell origin of hairy cells and investigated the molecular basis underlying the unique pathological features of the disease, with important implications for HCL research and treatment.

The stability of R-spine defines RAF inhibitor resistance: A comprehensive analysis of oncogenic BRAF mutants with in-frame insertion of αC-ß4 loop.

Although targeting BRAF mutants with RAF inhibitors has achieved promising outcomes in cancer therapy, drug resistance remains a remarkable challenge, and underlying molecular mechanisms are not fully understood. Here, we characterized a previously unknown group of oncogenic BRAF mutants with in-frame insertions (LLRins506 or VLRins506) of αC-ß4 loop. Using structure modeling and molecular dynamics simulation, we found that these insertions formed a large hydrophobic network that stabilizes R-spine and thus triggers the catalytic activity of BRAF. Furthermore, these insertions disrupted BRAF dimer interface and impaired dimerization. Unlike BRAF(V600E), these BRAF mutants with low dimer affinity were strongly resistant to all RAF inhibitors in clinic or clinical trials, which arises from their stabilized R-spines. As predicted by molecular docking, the stabilized R-spines in other BRAF mutants also conferred drug resistance. Together, our data indicated that the stability of R-spine but not dimer affinity determines the RAF inhibitor resistance of oncogenic BRAF mutants.