Lyn regulates BCR-ABL and Gab2 tyrosine phosphorylation and c-Cbl protein stability in imatinib-resistant chronic myelogenous leukemia cells by Ji Wu, Feng Meng, Henry Lu, Ling Kong, William Bornmann, Zhenghong Peng, Moshe Talpaz, and Nicholas J. Donato Blood Volume 111(7):3821-3829 April 1, 2008 ©2008 by American Society of Hematology

Cellular and biochemical response to tyrosine kinase inhibitors in imatinib-sensitive and -resistant cells. Cellular and biochemical response to tyrosine kinase inhibitors in imatinib-sensitive and -resistant cells. (A) K562 and K562R cells were treated with imatinib (left) or dasatinib (right) at the indicated concentration for 48 hours before assessment of cell viability as previously described.19 Each point represents the average plus or minus SEM (error bar) of 4 determinations. (Inset) Immunoblot analysis of BCR-ABL, Lyn, Src, Yes, Fyn, and actin in equal protein lysates from K562 and K562R cells. BCR-ABL sequencing did not detect mutations in the Abl kinase domain in either cell line (data not shown). (B) K562 (left) or K562R (right) cells were incubated with imatinib (5 μM) or dasatinib (0.5 μM) for 0, 2, or 24 hours before lysates were analyzed for tyrosine-phosphorylated (activated) BCR-ABL, Lyn, and CrkL. Lyn and PARP protein (uncleaved [uPARP] = 116 kDa or cleaved [cPARP] = 85 kDa) were also detected by immunoblotting. (C) K562 and K562R cells were left untreated (control) or electroporated (AMAXA) with control (Con) or Lyn siRNA and analyzed for activated Lyn (pY396-Lyn) or Lyn protein levels by immunoblotting after 48 hours. Lysates were also probed for PARP as a marker of apoptosis. Table 1 summarizes the effect of siRNA on viability in K562 and K562R cells. The results represent the average and standard deviation from 3 independent experiments. Ji Wu et al. Blood 2008;111:3821-3829 ©2008 by American Society of Hematology

Lyn regulates BCR-ABL tyrosine phosphorylation in CML cells. Lyn regulates BCR-ABL tyrosine phosphorylation in CML cells. (A) Cos-7 cells were transfected with a kinase-inactive mutant of BCR-ABL (K271R) or cotransfected with kinase-active Lyn. After 24 hours, cotransfectants were treated with the indicated concentration of kinase inhibitor for 2 hours. Cell lysates were prepared and immunoblotted for site-specific (Y177) BCR-ABL phosphorylation, BCR-ABL, Lyn, and actin levels. Low-level Y177-BCR-ABL was detected in BCR-ABL (K271R)–transfected Cos-7 cells (lane 2) that were not affected by imatinib or dasatinib. The vertical cut line between lanes 2 and 3 denotes merging of the original image to eliminate experimentally irrelevant sample lanes. (B) A leukopheresis specimen from a CML lymphoid blast crisis patient who progressed on imatinib therapy was left untreated (control) or was treated with imatinib (5 μM) or dasatinib (0.25 μM) for 2 hours. Equal protein cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted for the antigen described. For detection of tyrosine-phosphorylated Lyn, 1 mg protein lysate from control or treated cells was subjected to Lyn immunoprecipitation and phosphotyrosine immunoblotting (pY-Lyn). The blot was stripped and reblotted for Lyn (bottom). (C) Specimens from 4 imatinib-resistant myeloid blast crisis patients (Res1-Res 4) that retained wild-type BCR-ABL expression were treated with imatinib or dasatinib (at the concentration indicated) for 2 hours. Equal protein cell lysates were subjected to immunoblotting for phospho-specific and total protein levels (as indicated). K562R cell lysate was used as a positive control for Lyn and other phosphoproteins. Ji Wu et al. Blood 2008;111:3821-3829 ©2008 by American Society of Hematology

Lyn regulates Gab2 tyrosine phosphorylation, BCR-ABL signaling complexes, and imatinib activity in CML cells. Lyn regulates Gab2 tyrosine phosphorylation, BCR-ABL signaling complexes, and imatinib activity in CML cells. (A) Gab2 was immunoprecipitated from K562 and K562R equal protein (1 mg) cell lysates and immunoblotted for phosphotyrosine (pY-Gab2, pY-BCR-ABL), BCR-ABL, Gab2, and Lyn. (B) K562R cells were treated with imatinib or dasatinib at the indicated concentration for 2 hours prior to Gab2 immunoprecipitation from equal protein cell lysates (0.5 mg) and blotting for phosphotyrosine (top) or Gab2 (bottom). Cell lysates subjected to immunoprecipitation media in the absence of anti-Gab2 were used to detect any nonspecific proteins (last lane). (C) K562R cells were electroporated with the indicated siRNA for 48 hours before immunoprecipitation of Gab2 and blotting for phosphotyrosine content and Gab2 levels. Lyn levels were measured by immunoblotting cell lysate. SHP1 was immunoblotted and used as a protein loading control. (D) K562R cells were electroporated with control or Gab2 siRNA (100 nM) and cell death was assessed by trypan blue exclusion (below) after 48 hours. Cell lysates were also examined for Gab2 protein levels by immunoblotting. Actin was probed as a protein loading control. (E) K562 or K562R cells were subjected to electroporation with 100 nM of either control or Lyn siRNA as described in “Methods.” After 24 hours, cells were treated with 2.5 μM imatinib (+) or vehicle alone (−) for an additional 2 hours before analysis of BCR-ABL, Lyn, and Y177-BCR-ABL levels by immunoblotting. (F) K562R cells were electroporated with 100 nM control, Hck, or Lyn siRNA (as indicated). After 24 hours, electroporated cells were treated with vehicle alone (none) or 2.5 μM imatinib for an additional 24 hours. Cells were also treated with dasatinib alone (0.2 μM) (without electroporation) for 24 hours before all cells were collected and cell death was estimated by trypan blue staining. The results represent the average plus or minus SEM of triplicate assays. Ji Wu et al. Blood 2008;111:3821-3829 ©2008 by American Society of Hematology

Lyn is associated with c-Cbl and regulates its stability in CML cells. Lyn is associated with c-Cbl and regulates its stability in CML cells. (A) K562 and K562R cells were subjected to electroporation with control or Lyn-specific siRNA, and after 48 hours changes in phosphotyrosine levels were accessed in equal protein lysates by immunoblotting. Migration of molecular mass standards is shown on the left, and individual targets are shown on the right. In addition to a reduction in pY-Lyn levels in Lyn siRNA-treated cells, Lyn down-regulation in K562R cells was associated with differential regulation of 2 phosphoproteins of 75 kDa and 130 kDa. (B) Lyn and Hck were found to be highly expressed in a lymphoid blast crisis CML patient who failed imatinib therapy (Figure 2B). A specimen was obtained and treated with imatinib (5 μM) or dasatinib (0.5 μM) for 2 hours before lysates (1 mg) were prepared and subjected to Lyn (left) or Hck (right) immunoprecipitation. Immune complexes were resolved by SDS-PAGE and immunoblotted for phosphotyrosine. In addition to recovery of Lyn or Hck by immunoprecipitation, 2 phosphotyrosine protein bands (130 kDa, 75 kDa) were detected in control untreated cells. Imatinib-reduced phosphorylation or recovery of the 130-kDa protein in either Lyn or Hck immunoprecipitates, while dasatinib reduced phosphorylation or recovery of both the 130-kDa and 75-kDa phosphoproteins. In a parallel experiment, the Lyn coprecipitating 130-kDa band was excised from a silver-stained gel, subjected to trypsinization, and liquid chromatography/mass spectrometry (LC/MS) analysis. The 130-kDa protein was identified as c-Cbl. The 75-kDa protein has not yet been identified. (C) To confirm c-Cbl as a Lyn-associated and -regulated protein, K562R cells were subjected to Lyn silencing and (first panel) total lysates were immunoblotted for phosphotyrosine, (second panel) total lysates were immunoblotted for c-Cbl, (third panel) c-Cbl immunoprecipitates or total lysate (TL) was immunoblotted for phosphotyrosine, and (fourth panel) total lysates were subjected to c-Cbl immunoprecipitation and Lyn immunoblotting. The migration of specific target proteins is shown on the right. (D) Lyn was immunoprecipitated from protein lysates (250 mg) derived from 2 CML myeloid blast crisis patient specimens and immunoblotted for c-Cbl. Ji Wu et al. Blood 2008;111:3821-3829 ©2008 by American Society of Hematology

Kinase-mediated regulation of c-Cbl in CML cells. Kinase-mediated regulation of c-Cbl in CML cells. (A) K562 (left) and K562R (right) cells were electroporated with the indicated siRNA or left untreated (−) for 48 hours before cell lysates were analyzed for total tyrosine phosphorylation (top) or c-Cbl protein levels (bottom). Molecular mass marker migration is shown on the left, and specific target phosphoproteins are depicted on the right. (B) Cos-7 cells were transfected with cbl, bcr-abl, or lyn expression vectors alone or in combination (as indicated), and 24 hours later cells were lysed and changes in pY744-c-Cbl levels were monitored by phospho–site-specific immunoblotting. The membrane was stripped and reprobed for c-Cbl, BCR-ABL, and Lyn protein levels. Actin blotting was used as a protein loading control. (C) K562 cells were electroporated with a GFP-IRES Lyn expression vector (pMX-Lyn) or empty vector (pMX). After 7 days, cells were flow sorted for GFP positivity and equal numbers of positive cells were compared with untransfected K562 and K562R cells for Lyn, c-Cbl, and actin protein levels. (D) K562R cells were electroporated with control or c-Cbl–specific siRNA, and after 48 hours cell lysates were examined for c-Cbl, Lyn, and actin (as a protein loading control). Both a short and long exposure for Lyn detection is shown. Ji Wu et al. Blood 2008;111:3821-3829 ©2008 by American Society of Hematology

Lyn regulation in “normal” and Lyn-overexpressing CML cells. Lyn regulation in “normal” and Lyn-overexpressing CML cells. Lyn expression and activation are heterogeneous in CML cells and may be altered by imatinib therapy. In early stage or untreated (“normal”) CML, BCR-ABL is upstream of Lyn and other Src-family kinases. BCR-ABL–mediated Lyn (or related kinase) activation provides essential, possibly lineage-specific, support for BCR-ABL–mediated transformation.54,55 In this setting, BCR-ABL inhibition (with imatinib) reduces Lyn kinase activation and loss of signaling through BCR-ABL– and Lyn-mediated phosphorylation. Lyn complexes with c-Cbl, inducing partial control of Lyn stability, but Lyn does not regulate c-Cbl phosphorylation. Imatinib exposure alters Lyn expression or induces changes in upstream control of its activation. Through overexpression, autoactivation, or association with other adaptor proteins, Lyn activation acquires full or partial BCR-ABL independence, resulting in loss of control of Lyn activation with imatinib alone. Unregulated Lyn alters BCR-ABL signaling complexes, associating with Gab2, inducing its tyrosine phosphorylation, and maintaining or directing Y177 phosphorylation of BCR-ABL. These changes suppress or delay kinase inhibition by imatinib. Lyn overexpression also destabilizes c-Cbl, resulting in a reduction in its protein level. Tyrosine phosphorylation of c-Cbl is mediated by BCR-ABL in both normal and stressed CML cells, allowing it to maintain scaffold or recruitment activity. Lyn may function in a feedback loop with c-Cbl to maintain ubiquitination of substrates, altering protein stability. Stabilization of key ubiquitin-targeted proteins in leukemic cells may independently contribute to BCR-ABL–mediated transformation.56,57 Ji Wu et al. Blood 2008;111:3821-3829 ©2008 by American Society of Hematology