OTKs→RAC→PI3K/PAK→AKT→RAF→MEK→ERK/c-CBL pathway enhances the expression of POLθ. (A) FLT3(ITD)-positive MOLM14 cells, JAK2(V617F)-positive HEL cells, and BCR-ABL1–positive 32Dcl3 cells^4-15 were treated with PI3K inhibitor buparlisib, AKT inhibitor perifosine, RAF1 inhibitor LY3009124, RAC inhibitor NSC23766, MEK inhibitor midrametinib, ERK inhibitor SCH772984, PAK1/2 inhibitor IPA-3 after western analysis of the total cell lysates. (B) Western blot of total cell lysates from 32D-FLT3(ITD), BAF3-JAK2(V617F), and 32D-BCR-ABL1 cells untreated (control) and treated with MG132 and/or SCH772984. (C-G) Total cell lysates were obtained from 32D-FLT3(ITD) cells treated with SCH772984 or diluent (control). (C) Western blot detecting ubiquitinated proteins in anti-POLθ immunoprecipitates. (D) Western blot detecting POLθ in antiubiquitin (Ub) immunoprecipitates. (E) Western blot detecting CBL proteins in anti-POLθ immunoprecipitates (IP:POLθ) and in total cell lysates (input) using pan-CBL and c-CBL and CBL-b specific antibodies. (F) Western blot of total cell lysates from 32D-FLT3(ITD) cells treated or not with SCH772984 and c-CBL siRNA or scrambled RNA. (G) Western blot of total cell lysates from 32D-FLT3(ITD cells treated or not with SCH772984 and pYTPEP CBL inhibitory peptide. (H) A diagram illustrating OTK-induced signaling pathways responsible for overexpression of POLθ.