ALBERT

Description: The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in some early phase patients while it is more common in advanced disease. Resistance has been generally associated with mutations in the BCR-ABL kinase that effect drug affinity. However patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. Our previous studies suggested a role for Lyn, a Src-related kinase, in imatinib resistance. K562 cells selected for imatinib resistance (K562R) overexpress Lyn kinase and its targeted silencing overcomes imatinib resistance and engages apoptosis. Overexpression of Lyn in K562 cells reduces imatinib sensitivity (3-fold) and patients that fail imatinib therapy in the absence of BCR-ABL mutations express a highly activated Lyn kinase that is not suppressed by imatinib. Silencing Lyn expression in patient specimens induces changes in cell survival that are proportional to the level of Lyn protein reduction. To understand the role of Lyn kinase in imatinib resistance and apoptosis we examined proteins associated with this kinase in imatinib resistant cell lines, leukemic cells overexpressing Lyn and specimens derived from imatinib resistant patients. Lyn overexpression blocked complete suppression of BCR-ABL tyrosine phosphorylation by imatinib and affected BCR-ABL signaling adaptors. Although BCR-ABL forms a stable complex with the leukemogenic-critical adaptor protein Gab2 in imatinib sensitive cells, Lyn overexpression resulted in the formation of Lyn:Gab2 complexed in resistant cells. BCR-ABL kinase inhibition failed to reduce tyrosine phosphorylation of Gab2 in these cells while Lyn silencing or kinase inhibition (with dasatinib) completely suppressed Gab2 tyrosine phosphorylation and correlated with the induction of apoptosis. Lyn silencing in K562R cells also lead to a reciprocal increase in the tyrosine phosphorylation and association with a protein of ~120kDa, identified as the E3 ligase, c-Cbl. Lyn overexpression in K562 cells reduced their imatinib sensitivity and reduced c-Cbl protein levels. Kinase inhibitor and co-transfection studies demonstrated that tyrosine phosphorylation of c-Cbl at a critical signaling site (Y774) is primarily controlled by BCR-ABL and deletion or mutation of the c-Cbl RING domain altered its BCR-ABL phosphorylation. These results suggest that c-Cbl complexes are regulated at both the protein and phosphorylation level by Lyn and BCR-ABL kinase activities, respectively. Overexpression and/or activation of Lyn may disrupt the balance and regulation of critical regulators of leukemogenic signaling (Gab2) or protein trafficking and stability (c-Cbl), resulting in increased cell survival and reduced responsiveness to BCR-ABL kinase inhibition. We conclude that Lyn alters the level and function of critical signaling adaptor proteins in CML cells.

Description: Imatinib mesylate (Gleevec) is effective therapy against Philadelphia chromosome–positive leukemia, but resistance develops in all phases of the disease. Bcr/Abl point mutations and other alterations reduce the kinase inhibitory activity of imatinib mesylate; thus, agents that target Bcr/Abl through unique mechanisms may be needed. Here we describe the activity of WP1130, a small molecule that specifically and rapidly down-regulates both wild-type and mutant Bcr/Abl protein without affecting bcr/abl gene expression in chronic myelogenous leukemia (CML) cells. Loss of Bcr/Abl protein correlated with the onset of apoptosis and reduced phosphorylation of Bcr/Abl substrates. WP1130 did not affect Hsp90/Hsp70 ratios within the cells and did not require the participation of the proteasomal pathway for loss of Bcr/Abl protein. WP1130 was more effective in reducing leukemic versus normal hematopoietic colony formation and strongly inhibited colony formation of cells derived from patients with T315I mutant Bcr/Abl–expressing CML in blast crisis. WP1130 suppressed the growth of K562 heterotransplanted tumors as well as both wild-type Bcr/Abl and T315I mutant Bcr/Abl–expressing BaF/3 cells transplanted into nude mice. Collectively, our results demonstrate that WP1130 reduces wild-type and T315I mutant Bcr/Abl protein levels in CML cells through a unique mechanism and may be useful in treating CML.

Description: Lyn kinase functions as a regulator of imatinib sensitivity in chronic myelogenous leukemia (CML) cells through an unknown mechanism. In patients who fail imatinib therapy but have no detectable BCR-ABL kinase mutation, we detected persistently activated Lyn kinase. In imatinib-resistant CML cells and patients, Lyn activation is BCR-ABL independent, it is complexed with the Gab2 and c-Cbl adapter/scaffold proteins, and it mediates persistent Gab2 and BCR-ABL tyrosine phosphorylation in the presence or absence of imatinib. Lyn silencing or inhibition is necessary to suppress Gab2 and BCR-ABL phosphorylation and to recover imatinib activity. Lyn also negatively regulates c-Cbl stability, whereas c-Cbl tyrosine phosphorylation is mediated by BCR-ABL. These results suggest that Lyn exists as a component of the BCR-ABL signaling complex and, in cells with high Lyn expression or activation, BCR-ABL kinase inhibition alone (imatinib) is not sufficient to fully disengage BCR-ABL–mediated signaling and suggests that BCR-ABL and Lyn kinase inhibition are needed to prevent or treat this form of imatinib resistance.

Description: Albeit most pts with chronic myeloid leukemia (CML) treated with imatinib (IM) have a favorable outcome, some will acquire resistance, mainly due to the development of Abl kinase domain mutations, which confer varying levels of TKI resistance. We describe a novel V304D mutation in pts with Ph+ leukemia who failed TKI therapy. Expression of V304D mutation in BCR-ABL failed to induce cytokine-independence in Ba/F3 cells. Studies in Cos-7 cells demonstrated that this mutant did not induce autophosphorylation and was deficient in kinase activity. We detected V304D mutation in 13 (18%) of 70 IM-resistant pts screened (12 CML, 1 Ph+ acute lymphoblastic leukemia [ALL]), and it was present in a median of 37% (range, 20% to 80%) resistant clones. Median age was 60 years (range, 30 to 81) and median time from diagnosis to IM therapy was 39 months (range, 1 to 91). Eleven (92%) of 12 pts with CML were in chronic phase (CP) at IM start and 1 was in blast phase (BP). Pts received IM for a median of 35 months (range, 2 to 66). Nine pts with CML had failed interferon and 2 (1 CML, 1 Ph+ALL) allogeneic stem cell transplantation prior to IM. Ten (83%) of 12 pts started IM at 400 mg/d but all eventually received ≥600 mg/d. Six pts with CML achieved a complete hematologic response (CHR), 1 BP returned to chronic phase (RCP), and 6 (5 CML, 1 Ph+ALL) had primary hematologic resistance (HR). No cytogenetic (CG) responses were observed and 7 pts with CML CP progressed (4 to AP and 3 to BP) after IM discontinuation. Four pts with CML (1 CP, 2 AP, 1 BP) received nilotinib after IM failure for a median of 2 months (range, 1 to 3.5). Two pts (1 CP, 1 AP) showed primary HR, 1 AP progressed to BP, and 1 BP (on 600 mg twice daily) had a transient (6 weeks) CHR before showing secondary HR. Twelve pts (11 CML, 1 Ph+ALL) received dasatinib: 7 at 70 mg twice daily, 1 at 90 mg daily, 1 at 140 mg daily, 1 at 180 mg daily, 1 at 90 mg twice daily, and 1 at 120 mg twice daily. Dasatinib was administered for a median of 8 months (range, 1 to 23). Two pts achieved CHR and a minor CG response in 1 analysis (75% and 65% Ph+ cells, respectively), 1 RCP, 1 no evidence of leukemia, and 8 (67%) primary HR. One of 4 pts who started dasatinib in CP progressed to AP. Responders to dasatinib had V304D mutation in 20%, 20%, and 25% of clones, respectively. Four pts exhibited concomitant Abl kinase mutations developed prior to dasatinib therapy: 3 with F317L and 1 with G250E. One pt had a 6 base pair in-frame insertion in the TK domain. T315I mutation evolved in 1 pt after dasatinib discontinuation. Eight pts discontinued dasatinib due to disease progression (7 died), 2 were lost to follow-up, and 2 remain on CHR after 17+ and 23+ months on dasatinib. In vitro studies of cells from one pt in CP with V304D mutation (50% of clones) failed to detect CrkL phosphorylation despite detectable expression of the Bcr-Abl protein. In summary, the V304D mutation in the Abl kinase domain results in kinase inactivation and is associated with high-level resistance to TKI therapy, transformation to AP/BP in CML and a particularly poor prognosis. Loss of kinase activity by mutation represents a very unique mechanism of kinase inhibitor resistance and predicts acquisition of other transforming events that support CML cell survival.

Description: The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in a small subset of early stage patients and is very common in advanced stage patients. Resistance is associated with overexpression and/or mutations in the BCR-ABL gene but patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. To circumvent or overcome resistance novel kinase inhibitors have been synthesized and tested clinically. However, while investigators have designed models to measure and predict activity of novel compounds in mutation-mediated imatinib resistance, other mechanisms of resistance have not been modeled or shown clinical relevance. In this report, the activity of four novel kinase inhibitors (norlotinib, dasatinib, SKI-606, ON012380) was compared in two distinct models of imatinib resistance. These models include cell lines established from natural resistant variant clones selected from imatinib sensitive cell lines and associated with a T315I BCR-ABL mutation (BV-173R) or overexpressed Lyn kinase (K562R). Kinase inhibitory activity in these models was compared to transfectant-based resistance models including BaF3 cells expressing the T315I mutant form of BCR-ABL and overexpression of Lyn kinase in K562 cells. K562R cells were completely resistant to imatinib and norlotinib but highly sensitive to Src/Abl inhibitors (dasatinib, SKI-606) but only partially sensitive to ON012380. Overexpression of Lyn in K562 cells reduced imatinib and norlotinib sensitivity (3-fold) but did not affect sensitivity to the other kinase inhibitors. BV-173R cells expressing the T315I mutant form of BCR-ABL were completely resistant to Abl-selective kinase inhibitors (imatinib, norlotinib) and ~100-fold less sensitive (IC50 ~ 2 microM) to Src/Abl-directed inhibitors (dasatinib, SKI-606) while the non-ATP competitive kinase inhibitor, ON012380, was equally effective against both BV-173 and BV-173R cells. Expression of the T315I mutant form of BCR-ABL in BaF3 cells completely blocked kinase inhibitory activity of all inhibitors except ON012380. IL-3 dependent BaF3 cells were not inhibited by imatinib, norlotinib or SKI-606 but were equally sensitive to ON012380 when compared to IL-3 independent BCR-ABL transfectants. Cellular sensitivity was associated with reduced phosphorylation of BCR-ABL, CrkL and Lyn kinase with all inhibitors except ON012380, which mediated apoptosis in the absence of alterations in tyrosine phosphorylation. Together, our results suggest that imatinib resistant cell models are useful in evaluating the activity of novel kinase inhibitors but need to be carefully interpreted and mechanistically tested. Additional mediators of imatinib resistance need to be defined and modeled so that an appropriate individualized therapy can be applied to most effectively overcome resistant disease.

Description: BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of a reciprocal chromosomal translocation that is common in chronic myelogenous and acute lymphocytic leukemia. BCR-ABL induces transformation of hematopoetic stem cells through tyrosine phosphorylation of multiple substrates. The src-family kinases (SFKs), Lyn and Hck, are highly activated by BCR-ABL in leukemic cells and recent studies suggest that they are substrates and essential mediators of BCR-ABL signal transduction and transformation. In cells selected for resistance to the BCR-ABL inhibitor, imatinib mesylate, Lyn kinase is overexpressed and its activation is not dependent on or regulated by BCR-ABL, suggesting that autonomous regulation of SFKs may play a role in imatinib resistant. In this report, activation of Lyn and Hck was compared in CML specimens derived from imatinib responsive and resistant patients that did not express a mutant BCR-ABL protein as their primary mediator of resistance. In imatinib sensitive cell lines and specimens derived from imatinib responsive CML patients imatinib effectively reduced activation of both BCR-ABL and SFKs. However, in multiple specimens from resistant patients, imatinib reduced BCR-ABL kinase activation but failed to reduce SFK activation. The dual ABL/SRC inhibitor, BMS-354825, blocked activation of both BCR-ABL and SFKs expressed in leukemic cells and correlated with clinical responsiveness to this agent. Animal models demonstrated that loss of imatinib-mediated inhibition of Lyn kinase activation significantly impaired its anti-tumor activity which was recovered by treatment with BMS-354825. Direct silencing of Lyn or Hck reduced CML cell survival in imatinib resistant patient specimens and cell models, suggesting a direct role for these kinases in cell survival. Our results show that SFK activation is mediated by BCR-ABL in imatinib responsive cells but these kinases escape control by BCR-ABL in CML patients that develop imatinib resistance in the absence of BCR-ABL point mutations. This form of resistance can effectively be overcome by BMS-354825 through its dual SRC and ABL kinase inhibitory activities. Dual specificity kinase inhibitors may be indicated for the treatment and prevention of imatinib resistance in CML when it is associated with constitutively activated src-family kinases.