Gain-of-function mutation in IRS1 and NF1/2 loss result in de novo vemurafenib resistance in cHCL. (A) Serial flow cytometric analyses of cHCL cells (CD103⁺/CD25⁺) in the peripheral blood of a patient with cHCL before vemurafenib initiation and during the first month of treatment (top), as well as a different patient before vemurafenib initiation until time of death (bottom). Mutations (B) and CN alterations (C) detected in pretreatment cHCL sample from the vemurafenib-resistant patient. (D) Western blot analysis of human (hIRS1) and mouse IRS1 (mIRS1) complementary DNA constructs in wild-type (WT) or mutant forms on AKT and ERK phosphorylation related to empty vector. (E) Cell growth of IRS1-expressing Ba/F3 cells from (D) after interleukin-3 withdrawal. (F) Quantitative reverse transcription polymerase chain reaction of Nf1 and Nf2 expression after anti-Nf1 or -Nf2 short hairpin (shRNA) knockdown. The numbers below each shRNA indicate the shRNA oligonucleotide sequence (as shown in supplemental Methods). (G) IC50, 50% inhibitory concentration (IC₅₀) of BRAFV600E-expressing Ba/F3 cells to vemurafenib with or without knockdown of mNf1 or mNf2.