ALBERT

Description: Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.

Description: BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCFSKP2 (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G1 arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2V617F, and TEL-PDGFRβ, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL–infected SKP2−/− marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2+/+ marrow. SKP2−/− leukemic cells demonstrated higher levels of nuclear p27 than SKP2+/+ counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL–induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCFSKP2 may be therapeutically useful.

Description: Chronic myeloid leukemia (CML) is caused by the tyrosine kinase BCR-ABL and inhibition of BCR-ABL by imatinib correlates with clinical response. In addition to BCR-ABL, imatinib inhibits c-ABL, ARG, PDGFR, c-FMS, LYN, KIT and possibly other kinases. It is presently unclear whether there are therapeutic benefits derived from simultaneous inhibition of BCR-ABL and non-BCR-ABL targets within CML cells. KIT is a receptor tyrosine kinase expressed by both normal and leukemic hematopoietic progenitors. Its ligand, stem cell factor (SCF), supports proliferation of CML progenitors in the absence of other cytokines and thus KIT signaling in conjunction with BCRABL signaling may be central to leukemogenesis. We hypothesized that the capacity of imatinib to inhibit KIT may contribute to its efficacy in CML. In this study, we use a panel of sole BCR-ABL, sole KIT and dual BCR-ABL/KIT inhibitors to explore: the requirement for BCR-ABL and KIT signaling in the survival and growth of primary CML stem and progenitor cells and the degree to which dual BCR-ABL/KIT inhibition by imatinib contributes to its efficacy in CML. We evaluated CFU-GM colony formation from newly diagnosed CML cells cultured in SCF, IL-3, and GM-CSF in the presence of the following BCR-ABL and KIT inhibitors: imatinib (dual BCR-ABL/KIT), PPY-A (sole BCR-ABL), SCF-blocking antibody K44.2 (SCF-block, sole KIT) and PPY-A+SCF-block (BCR-ABL+KIT). Enumeration of CFU-GM revealed minimal suppression by PPY-A (30%) and somewhat more significant suppression by SCF-block (50%). Maximal colony suppression achieved during treatment with imatinib (80%) could only be reproduced under conditions of simultaneous BCR-ABL and KIT inhibition using PPY-A+SCF-block (p=0.4 relative to imatinib) or PPY-A upon removal of SCF (p=0.5 relative to imatinib). Removal of IL-3 or GM-CSF did not significantly impact the growth of CFU-GM under conditions of sole BCR-ABL inhibition. We additionally cultured CML progenitors on human or murine stroma for 1–3 weeks under conditions of BCRABL and/or KIT inhibition and evaluated colony forming cell (CFC) growth. Neither sole BCR-ABL nor sole KIT inhibition suppressed growth of week 1 or week 3 CFC to the degree of dual inhibition by either imatinib or PPY-A+SCF-block. Our findings indicate that targeted therapy aimed at BCR-ABL without simultaneously targeting KIT may not be as effective at eliminating proliferative progenitors that are largely responsible for the expansion of leukemic cells. To evaluate whether KIT supports CML stem cell survival in a similar manner, we performed 6-week LTC-IC on murine stroma with BCR-ABL and KIT inhibitors. Sole BCR-ABL inhibition was more effective at blocking CML stem cell growth than progenitor cell growth (98% versus 71% suppression respectively) and KIT inhibition did not affect this population. The requirement of dual BCR-ABL and KIT inhibition to suppress primary CML cell growth was therefore cell context dependent and restricted to multipotent proliferative progenitors and CFU-GM. We conclude that despite the presence of BCR-ABL in all cell types associated with the malignant clone, survival depends on both oncogenic and physiological signaling pathways. Survival of CML stem cells, as seen in patients harboring residual disease, is independent of KIT. This may offer a partial explanation of why imatinib is less effective against this population than KIT-dependent progenitors. The degree to which specific oncogene-independent pathways contribute to survival impacts the efficacy of targeted therapy.

Description: Abstract 2778 In chronic myeloid leukemia (CML), imatinib and other tyrosine kinase inhibitors (TKIs) inhibit BCR-ABL1 tyrosine kinase activity but also target additional kinases including KIT. The role of KIT inhibition in the therapeutic efficacy of TKIs is controversial. We used TKIs with selective activity against ABL (PPY-A) or KIT (BAW667) and genetic tools to assess the role of KIT signaling for growth of CML cell lines and primary CML progenitor and stem cells. In Mo7eBCR-ABL1 or newly diagnosed CML CD34+ progenitor cells, immunoblotting confirmed that PPY-A (1 μM) suppresses BCR-ABL1 phosphorylation but not KIT tyrosine phosphorylation. In contrast, treatment of cells with a KIT-blocking antibody (K44.2, 200ng/mL), shRNA targeting KIT (shKIT), or the KIT selective inhibitor BAW667 (1 μM), suppressed KIT activity without affecting BCR-ABL1 kinase activity. Therefore, these systems are suitable to isolate the role of BCR-ABL1 vs. KIT inhibition. Treatment of Mo7eBCR-ABL1 cells with PPY-A resulted in suppression of growth by 91.7% (p

Description: Chronic myeloid leukemia is effectively treated with imatinib, but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain mutations. The additional approved ABL tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib, along with investigational TKIs such as ponatinib (AP24534) and DCC-2036, support the possibility that mutation-mediated resistance in chronic myeloid leukemia can be fully controlled; however, the molecular events underlying resistance in patients lacking BCR-ABL point mutations are largely unknown. We previously reported on an insertion/truncation mutant, BCR-ABL35INS, in which structural integrity of the kinase domain is compromised and all ABL sequence beyond the kinase domain is eliminated. Although we speculated that BCR-ABL35INS is kinase-inactive, recent reports propose this mutant contributes to ABL TKI resistance. We present cell-based and biochemical evidence establishing that BCR-ABL35INS is kinase-inactive and does not contribute to TKI resistance, and we find that detection of BCR-ABL35INS does not consistently track with or explain resistance in clinical samples from chronic myeloid leukemia patients.

Description: Background : Systemic mastocytosis (SM) is characterized by infiltration of extracutanous tissues by neoplastic mast cells. Primary target organs are liver, spleen and bone marrow. In some instances, SM progresses to aggressive systemic mastocytosis (ASM) or mast cell leukemia (MCL), which are associated with extensive mast cell infiltration into various organs and their failure. Almost all cases of SM exhibit point mutations at codon 816 of Kit, a receptor tyrosine kinase. These mutations (most commonly D816V) lead to constitutive activation of the kinase and are the causative lesion of SM. Here, we describe a novel murine model of SM/ASM that shares many characteristics with the human disease and may be useful for in vivo drug testing, including targeted therapy of D816 mutant Kit with small molecule inhibitors. Materials and methods : P815 cells, a cell line expressing D814Y Kit (homologous to human D816V kit) that was established in DBA2 mice (Dunn T, Bap M. J Natl Cancer Inst1957;18:587–95) were injected retro-orbitally into two groups (n = 4) of 8–10 week-old syngeneic mice, at a dose of 1x102 and 5x104 cells. Using an automated animal cell counter, the mice were monitored at 48h intervals with full blood counts, including white cell differential and platelets. The presence of mast cells was assessed by FACS for mast cell markers (CD117-PE and CD45-APC) as well as Giemsa staining. The animals were evaluated daily for signs of morbidity. Moribund mice were sacrificed and subjected to autopsy. Liver, spleen and bone marrow were analyzed by histopathology, and the expression and phophorylation status of Kit was assessed by FACS and immunoblotting. Results : Both cell doses induced an aggressive disease, with all animals reaching a moribund stage on day 9 (5x104 cells) and 16 (1x102 cells). A significant (p

Description: Imatinib mesylate (Gleevec, formerly STI571) is an effective therapy for all stages of chronic myelogenous leukemia (CML). While responses in chronic-phase CML are generally durable, resistance develops in many patients with advanced disease. We evaluated novel antileukemic agents for their potential to overcome resistance in various imatinib-resistant cell lines. Using cell proliferation assays, we investigated whether different mechanisms of resistance to imatinib would alter the efficacy of arsenic trioxide (As2O3) or 5-aza-2-deoxycytidine (decitabine) alone and in combination with imatinib. Our results indicate that resistance to imatinib induced by Bcr-Abl overexpression or by engineered expression of clinically relevant Bcr-Abl mutants does not induce cross-resistance to As2O3 or decitabine. Combined treatment with these agents and imatinib is beneficial in cell lines that have residual sensitivity to imatinib monotherapy, with synergistic growth inhibition achieved only at doses of imatinib that overcome resistance. In some imatinib-resistant cell lines, combination treatments that use low doses of imatinib lead to antagonism. Apoptosis studies suggest that this can be explained in part by the reduced proapoptotic activity of imatinib in resistant cell lines. These data underline the importance of resistance testing and provide a rational approach for dose-adjusted administration of imatinib when combined with other agents.

Description: Oncogenic mutations of the KIT receptor tyrosine kinase have been identified in several malignancies including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), seminomas/dysgerminomas and acute myelogenous leukemia (AML). Mutations in the regulatory juxtamembrane domain are common in GIST, while mutations in the activation loop of the kinase (most commonly D816V) occur predominantly in SM and at low frequency in AML. Several ATP-competitive kinase inhibitors, including imatinib, are effective against juxtamembrane KIT mutants, however, the D816V mutant is largely resistant to inhibition. We analyzed the sensitivities of cell lines expressing wild type KIT, juxtamembrane mutant KIT (V560G) and activation loop mutant KIT (D816V,F,Y and murine D814Y) to a potent Src/Abl kinase inhibitor, AP23464, and analogs. IC50 values for inhibition of cellular KIT phosphorylation by AP23464 were 5–11 nM for activation loop mutants, 70 nM for the juxtamembrane mutant and 85 nM for wild type KIT. Consistent with this, IC50 values in cell proliferation assays were 3–20 nM for activation loop mutants and 100 nM for wild type KIT and the juxtmembrane mutant. In activation loop mutant-expressing cell lines, AP23464, at concentrations ≤50 nM, induced apoptosis, arrested the cell cycle in G0/G1 and down-regulated phosphorylation of Akt and STAT3, signaling pathways critical for the transforming capacity of mutant KIT. In contrast, 500 nM AP23464 was required to induce equivalent effects in wild-type KIT and juxtamembrane mutant-expressing cell lines. These data demonstrate that activation loop KIT mutants are considerably more sensitive to inhibition by AP23464 than wild type or juxtamembrane mutant KIT. Non-specific toxicity in parental cells occurred only at concentrations above 2 μM. Additionally, at concentrations below 100 nM, AP23464 did not inhibit formation of granulocyte/macrophage and erythrocyte colonies from normal bone marrow, suggesting that therapeutic drug levels would not impact normal hematopoiesis. We also examined in vivo target inhibition in a mouse model. Mice were subcutaneously injected with D814Y-expressing (D816V homologous) murine mastocytoma cells. Once tumors were established, compound was administered three-times daily by oral gavage. One hour post treatment we observed 〉90% inhibition of KIT phosphorylation in tumor tissue. Following a three-day treatment regimen, there was a statistically significant difference in tumor size compared to controls. Thus, AP23464 analogs effectively target D816-mutant KIT both in vitro and in vivo and inhibit activation loop KIT mutants more potently than the wild type protein. These data provide evidence that this class of kinase inhibitors may have therapeutic potential for D816V-expressing malignancies such as SM or AML.

Description: Resistance to current Bcr-Abl kinase inhibitors in CML patients is associated with the emergence of Bcr-Abl point mutations, including the T315I variant which represents 15–20% of clinically observed mutants and is resistant to all approved agents (e.g. imatinib and dasatinib). AP24534 is a novel, orally active Bcr-Abl inhibitor that also potently inhibits T315I and other clinically relevant mutants. We previously showed that daily oral administration of AP24534 can induce complete regression of T315I-expressing tumors in mice. In this study, we set out to characterize the antitumor activity of AP24534 through correlative in vitro and in vivo efficacy and pharmacodynamic studies. AP24534 inhibited the kinase activity of unmutated and T315I Abl enzymes with IC50s of 12 and 58 nM respectively, and inhibited the proliferation of their respective Bcr-Abl Ba/F3-derived cell lines with IC50s of 1 and 8 nM in a 3-day assay. Exposure to AP24534 for three hours led to the inhibition of Bcr-Abl phosphorylation with IC50s of 25 and 78 nM, respectively, with substantial apoptosis observed within 24 hours, suggesting that short term exposure to AP24534 is sufficient for efficacy. In mononuclear cells isolated from 3 patients with the T315I mutation (2 CML patients in blast crisis and a Ph+ ALL patient), treatment with 50 nM AP24534 resulted in a 〉50% decrease in phospho-CrkL levels. To further investigate the relationship between dose, exposure and response, we tested the activity of AP24534 on the human CML cell line K-562. In vitro, exposure of K-562 cells to 60 nM AP24534 for only 3 hr was sufficient to reduce cell proliferation by 〉90% when measured 3 days later. Oral administration of AP24534 to mice bearing K-562 xenografts inhibited tumor growth in a dose-dependent manner; a 2.5 mg/kg daily dose induced complete tumor regression. Bcr-Abl phosphorylation was only transiently inhibited at this dose, confirming that sustained target inhibition is not required for antitumor activity. Indeed, complete regression could also be achieved by intermittent (twice-weekly) dosing. Compared to AP24534, dasatinib required significantly higher transient exposure levels to achieve 〉90% K-562 cell killing in vitro (300–600 nM), and showed reduced efficacy in the xenograft model when dosed daily or intermittently. Pharmacokinetic analyses showed that the longer half-life of AP24534 versus dasatinib results in higher levels of exposure in mice. The in vitro kinase specificity profile of AP24534 suggests that Bcr-Abl inhibition is the dominant mechanism for its activity in CML models. Specifically, it is inactive against Aurora kinases (IC50 〉 1000 nM). AP24534 potently inhibits a discrete subset of other kinases, including Flt3 and members of the Src, VEGFR and FGFR families (IC50s = 0.4–58 nM), activities which may contribute to its potency against CML and suggest the potential for efficacy in other tumor types. Together these data indicate that AP24534 has the potential to be an effective treatment for CML, including in patients refractory to current targeted agents. A phase 1 clinical trial is being planned.

Description: Oncogenic mutations of the Kit receptor tyrosine kinase occur in several types of malignancy. Juxtamembrane domain mutations are common in gastrointestinal stromal tumors, whereas mutations in the kinase activation loop, most commonly D816V, are seen in systemic mastocytosis and acute myelogenous leukemia. Kit activation-loop mutants are insensitive to imatinib mesylate and have been largely resistant to targeted inhibition. We determined the sensitivities of both Kit mutant classes to the adenosine triphosphate (ATP)–based inhibitors AP23464 and AP23848. In cell lines expressing activation-loop mutants, low-nM concentrations of AP23464 inhibited phosphorylation of Kit and its downstream targets Akt and signal transducer and activator of transcription 3 (STAT3). This was associated with cell-cycle arrest and apoptosis. Wild-type Kit–and juxtamembrane-mutant–expressing cell lines required considerably higher concentrations for equivalent inhibition, suggesting a therapeutic window in which cells harboring D816V Kit could be eliminated without interfering with normal cellular function. Additionally, AP23464 did not disrupt normal hematopoietic progenitor-cell growth at concentrations that inhibited activation-loop mutants of Kit. In a murine model, AP23848 inhibited activation-loop mutant Kit phosphorylation and tumor growth. Thus, AP23464 and AP23848 potently and selectively target activation-loop mutants of Kit in vitro and in vivo and could have therapeutic potential against D816V-expressing malignancies.