Bcr-Abl Kinase Inhibitor Research Articles

Potential of ponatinib to treat chronic myeloid leukemia and acute lymphoblastic leukemia

Development of BCR-ABL tyrosine kinase inhibitors (TKIs) have improved outcomes for patients diagnosed with chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia. However, resistance or intolerance to these TKIs still leaves some patients without many treatment options. One point mutation in particular, the T315I mutation, has been shown to be resistant to first and second generation TKIs. The third generation TKI, ponatinib, may provide an option for these patients. Ponatinib (Iclusig®), an orally available, pan-tyrosine kinase inhibitor has a unique binding mechanism allowing inhibition of BCR-ABL kinases, including those with the T315I point mutation. A Phase II study evaluated ponatinib in patients who were resistant or intolerant to nilotinib or dasatinib or patients who had the T315I mutation. In the Phase II study, ponatinib produced a major cytogenetic response in 54% of chronic phase chronic myeloid leukemia patients. It further achieved major hematologic response in 52% of patients in the accelerated phase, 31% of patients in the blast phase, and 41% of Philadelphia chromosome positive acute lymphoblastic leukemia patients. Ponatinib also showed efficacy in patients with the T315I mutation. Serious adverse events included arterial thrombosis, hepatotoxicity, cardiovascular risks, pancreatitis, hemorrhage, fluid retention, myelosuppression, rash, abdominal pain, and embryo–fetal toxicity. Due to the risk of these adverse events and potential drug interactions, the use of ponatinib must be carefully weighed against the benefits in treating patients who have limited treatment options.

Association between severe toxicity of nilotinib and UGT1A1 polymorphisms in Japanese patients with chronic myelogenous leukemia

Nilotinib is a BCR-ABL kinase inhibitor approved for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (CML). The UDP-glucuronosyltransferase 1A1 (UGT1A1) polymorphism UGT1A1*28 (*28)/*28 has been linked to an increased risk of hyperbilirubinemia in patients with CML who receive nilotinib. Beside *28, UGT1A1*6 (*6) is another important variant allele in Japanese patients because it is associated with adverse events of irinotecan, metabolized by UGT1A1. We retrospectively investigated the association between severe toxicity of nilotinib and UGT1A1 polymorphisms (*6 and*28) in Japanese patients with CML. Eight patients with cytogenetically confirmed CML who were receiving nilotinib were studied to explore the association of UGT1A1 polymorphisms with severe nilotinib-related toxicity. Genotyping analyses were determined for *6 and *28. All 3 patients with the *6/*6 or *6/*28 genotype had severe toxicity, including QT interval prolongation (grade 3), elevated lipase levels (grade 3) plus hyperbilirubinemia (grade 2), and anemia (grade 3) plus hepatic cyst hemorrhage (grade 2) in 1 patient each. Among the 5 patients with the *6/*1 or *1/*1 genotype, 1 had elevated lipase levels (grade 3) and another had severe pain in the lower extremities (grade 3). These findings suggest that UGT1A1 polymorphisms are important determinants of severe toxicity of nilotinib in Japanese patients.

Abstract 4308: A model of oncogene addiction: BCR-ABL hijacks signaling through a MEK-dependent process and inhibits growth factor-mediated signal transduction.

Abstract Background: Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome and the fusion gene BCR-ABL, which has been shown to activate key signaling pathways, including PI-3 kinase, RAS/ERK and STAT5. ABL tyrosine kinase inhibitors (TKIs) are highly effective clinical agents that inhibit downstream signaling, trigger CML cell apoptosis, and provide clinical evidence for the phenomenon of “oncogene addiction," whereby cell survival becomes critically dependent upon the activity of a particular oncogene product. TKI therapy has largely failed to achieve the same degree of clinical success in other activated kinase-driven malignancies, suggesting that BCR-ABL is relatively unique in its ability to establish a state of oncogene addiction. Results: To dissect the mechanism by which BCR-ABL coordinates STAT5 and RAS/ERK signaling to establish a state of oncogene addiction, JAK2 kinase activity and RAS-GTP levels were assessed in the CML patient-derived cell line K562. Surprisingly, basal levels of RAS-GTP were low and JAK2 kinase activity was not detectable. Treatment of K562 cells with erythropoietin (EPO) only very modestly activated JAK2. Similarly, EPO treatment after one hour of BCR-ABL inhibition also failed to rescue STAT5 or RAS/ERK activation. However, with more prolonged BCR-ABL kinase inhibition, we observed a time-dependent increase in the ability of EPO to rescue STAT5 and ERK phosphorylation. After 24hrs of BCR-ABL inhibition, EPO treatment led to a significant increase in both JAK2 activation and the GTP loading of RAS relative to vehicle treated cells. These data suggest that BCR-ABL kinase activity hampers the ability of JAK2 to activate STAT5 and RAS/MAPK . Further, 24hr treatment with the MEK inhibitor PD0325901 allowed for a comparable degree of EPO-mediated JAK2 activation, suggesting that the negative regulation of JAK2 is mediated at least in part by a MEK/ERK-dependent mechanism. These observations were subsequently validated in an isogenic system utilizing TF-1 cells. Similar to the EPO/JAK2 axis in K562 cells, TF1-BCR-ABL cells exhibit attenuated GM-CSF signaling relative to control TF1-puro cells, which was restored only after prolonged BCR-ABL inhibition. Significantly, we have observed that BCR/ABL-expressing cells irreversibly commit to apoptosis before growth factor signaling is fully restored. Conclusions: Our data suggest that BCR-ABL-mediated oncogene addiction is a consequence of a high level of physiologic negative feedback mechanisms that dampen growth factor receptor signaling. Our findings also help reconcile recent evidence that JAK2 inhibition restores the sensitivity of CML stem/progenitor cells to ABL TKI treatment in vitro with the observation that JAK2 is completely dispensable for BCR-ABL-induced myeloid disease in mice. Citation Format: Jennifer Gajan, Elisabeth Lasater, Cheryl Tajon, Charles Craik, Neil Shah. A model of oncogene addiction: BCR-ABL hijacks signaling through a MEK-dependent process and inhibits growth factor-mediated signal transduction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4308. doi:10.1158/1538-7445.AM2013-4308

Apoptosis in chronic myeloid leukemia cells transiently treated with imatinib or dasatinib is caused by residual BCR–ABL kinase inhibition

Transient, potent BCR-ABL inhibition with tyrosine kinase inhibitors (TKIs) was recently demonstrated to be sufficient to commit chronic myeloid leukemia (CML) cells to apoptosis irreversibly. This mechanism explains the clinical efficacy of once-daily dasatinib treatment, despite the rapid clearance of the drug from the plasma. However, our in vitro data suggest that apoptosis induction after transient TKI treatment, observed in the BCR-ABL-positive cell lines K562, KYO-1, and LAMA-84 and progenitor cells from chronic phase CML patients, is instead caused by a residual kinase inhibition that persists in the cells as a consequence of intracellular drug retention. High intracellular concentrations of imatinib and dasatinib residues were measured in transiently treated cells. Furthermore, the apoptosis induced by residual imatinib or dasatinib from transient treatment could be rescued by washing out the intracellularly retained drugs. The residual kinase inhibition was also undetectable by the phospho-CRKL assay. These findings confirm that continuous target inhibition is required for the optimal efficacy of kinase inhibitors.

Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt–β-catenin signaling

AbstractTyrosine kinase inhibitors (TKIs) are highly effective in treatment of chronic myeloid leukemia (CML) but do not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSCs, suggesting that additional kinase-independent mechanisms contribute to LSC preservation. We investigated whether signals from the bone marrow (BM) microenvironment protect CML LSCs from TKI treatment. Coculture with human BM mesenchymal stromal cells (MSCs) significantly inhibited apoptosis and preserved CML stem/progenitor cells following TKI exposure, maintaining colony-forming ability and engraftment potential in immunodeficient mice. We found that the N-cadherin receptor plays an important role in MSC-mediated protection of CML progenitors from TKI. N-cadherin–mediated adhesion to MSCs was associated with increased cytoplasmic N-cadherin–β-catenin complex formation as well as enhanced β-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated β-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. Our results reveal a close interplay between N-cadherin and the Wnt–β-catenin pathway in protecting CML LSCs during TKI treatment. Importantly, these results reveal novel mechanisms of resistance of CML LSCs to TKI treatment and suggest new targets for treatment designed to eradicate residual LSCs in CML patients.

Imatinib failure and response to dasatinib in a patient with chronic myeloid leukemia in blast crisis and a novel, nine-nucleotide BCR-ABL insertion mutation

Imatinib failure and response to dasatinib in a patient with chronic myeloid leukemia in blast crisis and a novel, nine-nucleotide BCR-ABL insertion mutation

Molecular mechanisms for survival regulation of chronic myeloid leukemia stem cells

Studies on chronic myeloid leukemia (CML) have served as a paradigm for cancer research and therapy. These studies involve the identification of the first cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors (TKIs) that inhibit BCR-ABL kinase activity in CML. It becomes clear that leukemia stem cells (LSCs) in CML which are resistant to TKIs, and eradication of LSCs appears to be extremely difficult. Therefore, one of the major issues in current CML biology is to understand the biology of LSCs and to investigate why LSCs are insensitive to TKI monotherapy for developing curative therapeutic strategies. Studies from our group and others have revealed that CML LSCs form a hierarchy similar to that seen in normal hematopoiesis, in which a rare stem cell population with limitless self-renewal potential gives rise to progenies that lack such potential. LSCs also possess biological features that are different from those of normal hematopoietic stem cells (HSCs) and are critical for their malignant characteristics. In this review, we summarize the latest progress in CML field, and attempt to understand the molecular mechanisms of survival regulation of LSCs.

Strategies to circumvent the T315I gatekeeper mutation in the Bcr-Abl tyrosine kinase

Despite the remarkable success of imatinib against Bcr-Abl, development of secondary resistance, most often due to point mutations in the Bcr-Abl tyrosine kinase (TK) domain, is quite common. Of these, the T315I “gatekeeper” mutation is resistant to all currently registered Bcr-Abl TK inhibitors (TKIs) with the notable exception of ponatinib (Iclusig™), which was very recently approved by the United States Food and Drug Administration (FDA). Besides ponatinib, numerous strategies have been developed to circumvent this problem. These include the protein synthesis inhibitor omacetaxine (Synribo®), and “switch-control” inhibitors. Dual Bcr-Abl and aurora kinase inhibitors represent another promising strategy. Finally, several promising synergistic combinations, such as TKIs with histone deacetylase inhibitors (HDACIs), warrant attention.

Integrative Analysis of Ikaros-Dependent Changes of Transcriptional Regulation and Tyrosine Phosphorylation Events in Ph+ ALL

AbstractAbstract 528 Background:Tyrosine kinase-driven B-cell lineage acute lymphoblastic leukemia such as Ph+ ALL, is associated with particularly poor clinical outcome and defined by deletions of the IKZF1 gene encoding the Ikaros transcription factor in >80% of the cases. While the role of Ikaros in lymphoid differentiation has been extensively studied, it remains elusive how Ikaros functions as tumor suppressor in Ph+ALL. Here we demonstrate that Ikaros orchestrates a set of gene expression changes that affects leukemia cell proliferation and survival at multiple levels. Results:A comprehensive gene expression analysis revealed that reconstitution of Ikaros function in patient-derived Ph+ ALL cells mimics gene expression changes induced by Pax5 (a B cell specific transcription factor), μ-chain and BLNK (pre-B cell receptor components), and treatment with Imatinib (a BCR-ABL1 kinase inhibitor), while it reverses Myc- and Stat5a/b-dependent survival signals. In combining seven experimental perturbations (reconstitution of Ikzf1, Pax5, μ-chain, Blnk, treatment with Imatinib or deletion of Stat5a/bfl/fl and Mycfl/fl), we identified a commonly regulated gene set, including known tumor suppressors SPIB, BTG1, and BTG2 and highly enriched for Myc-related gene signatures. These findings suggest that reconstitution of PAX5 and IKZF1 transcription factors converges with pre-B cell receptor-mediated transcriptional changes. We next connected our analysis at the transcriptional level with Ikaros-dependent changes at the level of tyrosine phosphorylation events: The BCR-ABL1 kinase in Ph+ ALL drives proliferation and survival through phosphorylation of Stat5, AKT, ERK, SRC and multiple other signaling intermediates. A global mass spectrometry analysis of phosphotyrosine proteins in patient-derived Ph+ ALL cells showed that reconstitution of Ikaros leads to global loss of phospho-tyrosine, in a comparable manner as treatment with TKI. The vast majority of Ikaros-mediated changes resulted in loss of tyrosine phosphorylation such as the SRC tyrosine kinase LCK (Y504 and Y192), the proximal BCR-ABL1 adapter molecule NEDD9 (Y166, Y240, Y344 and Y578) and ABL1 itself (Y257). For a small number of molecules, Ikaros-reconstitution resulted in increased tyrosine phosphorylation including the phosphatases Shp2 (Ptpn11Y584) and Ship2 (Inppl1Y1161). A focus phospho-tyrosine analysis of individual signaling molecules analysis revealed that Stat5, SRC, AKT and ERK are all affected by Ikaros-induced de-phosphorylation. These results suggest that that Ikaros induces dephosphorylation of multiple signaling intermediates in BCR-ABL1 signaling. To identify Ikaros-mediated gene expression changes that were directly induced by Ikaros promoter binding, we matched Ikaros-dependent gene expression changes with Ikaros ChIP-seq data. This analysis confirmed enriched binding of Ikaros on Btg1, Myc and Smyd2 promoters. Among Ikaros target genes, we identified a set of genes that predicts clinical outcome for adult and pediatric patients with ALL. According to this predictor, IkarosUP (FBXO33, HBP1, HIVEP1, DTX1, KIF13B, RHOQ) and IkarosDOWN (PFKP, ASNS, ETV6, PSTPIP2, SMYD2, CDKL5), patients with B-ALL were segregated into two groups with significantly different outcomes, with a 5-year RFS of 76% vs 29% (COG P9906 trial, n=110; p=0.006) and a 5-year OS of 39% vs 0% (ECOG E2993 trial, n=25; p=0.007). Further biochemistry and mouse experiments showed that both the ability of Ikaros to dephosphorylate Stat5 and to function as tumor suppressor requires cooperation with the pre-B cell receptor linker molecule BLNK but not the proximal effector kinase Syk. Conclusions:Importantly, Ikaros binds to and transcriptionally activates CDKN2A (Arf) and TP53 loci and thereby opposes the function of BCL6, which acts as transcriptional repressor at these loci. Comparing the gene expression microarray and ChIP-seq or ChIP-on-Chip data of BCL6 and Ikaros, we identified a set of target genes that is regulated by both BCL6 and Ikaros and found that for the vast majority of Ikaros/BCL6 target genes, Ikaros-reconstitution recapitulated a BCL6−/− situation, i.e. genes that are induced by Ikaros are repressed by BCL6. These findings are of potential clinical relevance since pharmacological inhibition of BCL6 restores sensitivity of IKZF1-mutant Ph+ ALL cells to conventional tyrosine kinase inhibitors. Disclosures:Druker:ARIAD: OHSU receives clinical trial funding. Dr. Druker is currently principal investigator or co-investigator on Novartis, Bristol-Myers Squibb, and ARIAD clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and da Other; Bristol-Myers Squibb: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; Novartis: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man Other.

Dasatinib (Sprycel®) and Low Intensity Chemotherapy for First-Line Treatment in Patients with De Novo Philadelphia Positive ALL Aged 55 and Over: Final Results of the EWALL-Ph-01 Study

AbstractAbstract 666 Background.Dasatinib (Sprycel®, Bristol-Myers Squibb) is a potent multi-targeted kinase inhibitor (TKI) of BCR-ABL and SRC family kinases. The EWALL group for adult ALL decided to run a study at the European level evaluating the combination of dasatinib and chemotherapy for Philadelphia positive (Ph+) ALL patients (pts) aged 55 and over. Aim.To analyse efficacy of Dasatinib combined to low intensity chemotherapy and to test factors associated with outcome. (EudraCT 2006–005694-21). Methods.After prephase, dasatinib was administered at 140 mg QD (100 mg over 70y) during the induction period in combination with weekly vincristine (VCR) 1 mg IV and dexamethasone (DEX) 40 mg for 2 days (20 mg over 70y) for 4 weeks. Consolidation [Display omitted] Disclosures:Rousselot:BMS, Novartis: Research Funding. Gambacorti-Passerini:BMS, Novartis: Research Funding.

Apoptosis in Chronic Myeloid Leukemia Cells Transiently Treated with Imatinib or Dasatinib Is Caused by Residual BCR-ABL Kinase Inhibition

AbstractAbstract 1668Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hemopoietic stem cells. The constitutively active tyrosine kinase BCR-ABL causes defects in the proliferation and differentiation of blood cells. CML is currently treated with tyrosine kinase specific inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib. Transient, potent BCR-ABL inhibition with TKIs was demonstrated to commit CML cells to apoptosis irreversibly (Shah et al., Cancer Cell, 2008; Snead et al., Blood., 2009; Hiwase et al., Leukemia, 2009). This mechanism would explain the clinical efficacy of once-daily dasatinib treatment, despite the rapid clearance of the drug from the plasma. The restoration of BCR-ABL activity after TKI washout was demonstrated using phosphorylated CRKL (p-CRKL) protein as a surrogate marker.Our in vitro data challenges this model. We suggest that apoptosis observed in the BCR-ABL-positive cell line K562 and progenitor cells from chronic phase CML patients with transient dasatinib treatment is instead caused by residual kinase inhibition that persists as a consequence of intracellular drug retention. Relatively high intracellular concentrations of imatinib and dasatinib residues were measured in transiently treated K562 cells using a nanoscale liquid chromatography in-line-coupled with ion trap mass spectrometry (nanoLC-MS/MS system) (Table 1). Western blot analysis confirmed that TKI washout restored p-CRKL. However, STAT5 and ERK, two other important BCR-ABL downstream pathways, remained inhibited in K562 cells. These data suggest that p-CRKL, may not have the capacity to detect this residual TKI. Subsequent flow cytometric analysis confirmed that the residual kinase inhibition is not detectable by p-CRKL assay. To prove apoptosis is caused by residual TKI, we rescued apoptosis by performing additional washout steps in K562 cells treated with various concentrations of TKIs and measured Annexin-V expression (Figure 1). The apoptosis was completely reversible in all samples.We conclude that the reason for the equivalent efficacy of the once-daily and twice-daily TKI treatments is the most likely the intracellular retention of a low TKI concentration rather than the induction of irreversible apoptosis under a high-dose pulse treatment. Our data suggest that BCR-ABL is inhibited by residual TKI that cannot be detected by p-CRKL assay. The residual TKI was directly measured intracellularly, and it is possible to remove it by subsequent washout steps, which gradually restore apoptosis. These findings confirm that continuous target inhibition is required for the optimal efficacy of kinase inhibitors.Table 1Intracellular concentrations of imatinib and dasatinib in K562 cells. Cells were incubated with the indicated TKI either continuously or for 0.3 hours followed by drug washout and further culture in TKI-free medium. Aliquots were lysed at defined time points and analyzed by nanoLC-MS/MS (n=3)Hours after initiation of exposure0.32612242.5 ìM imatinib continuous (ìM)76 ± 2225 ± 332.5 ìM imatinib continuous (ìM)4000 ± 2001140 ± 6032.5 ìM imatinib pulse (ìM)3000 ± 10066 ± 370.0 ± 0.260 ± 140 ± 21 nM dasatinib continuous (nM)120 ± 20200 ± 4100 nM dasatinib continuous (nM)4400 ± 1003190 ± 80100 nM dasatinib pulse (nM)4600 ± 100350 ± 20250 ± 10290 ± 10257 ± 8 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Chaetocin Exhibits Anti-Leukemia Activity Against Chronic Myeloid Leukemia Stem Cells

Abstract 1667Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL kinase results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease, and implicating TKI-insensitive CML stem cells in relapse. Thus, there is great interest in identifying new therapeutic agents that can be combined with TKIs to eliminate TKI-insensitive CML stem cells that are not fully addicted to BCR-ABL. Chaetocin, a mycotoxin that inhibits histone methyltransferase SUV39H1, exhibits anticancer properties against myeloma and AML cells. To investigate a potential role for chaetocin in CML treatment, we first compared its cytotoxic effects with those of imatinib on TonB210, a murine hematopoietic cell line with doxycycline-inducible BCR-ABL expression that is dependent on BCR-ABL for survival and proliferation in the absence of exogenous IL-3. As expected, imatinib treatment produced cytotoxic effects in TonB210 cells induced to express BCR-ABL, but not in uninduced TonB210 cells, as assessed by trypan blue viability assays and Annexin V expression by flow cytometry. In contrast, chaetocin, displayed significant cytotoxicity against both TonB210 cells induced to express BCR-ABL and uninduced TonB210 cells. We then used a standard murine retroviral transduction system that models CML blast crisis (BC-CML) to study chaetocin effects on BCR-ABL expressing cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 48 hrs in chaetocin across a range of concentrations (0 – 400 nM). Chaetocin exhibited cytotoxic effects against GFP+/YFP+ LSKs as assessed by trypan blue, Annexin V, and colony forming assays. Chaetocin effects on CML stem cell self-renewal in vivo were assessed by performing secondary transplantation assays and limiting dilution analysis (LDA). GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to IC50 concentrations of chaetocin and/or imatinib. Survival after transplantation and LSC frequency were compared in four treatment cohorts: Group 1 (untreated control), Group 2 (chaetocin), Group 3 (imatinib) and Group 4 (chaetocin + imatinib). Compared to untreated mice, survival was significantly prolonged in mice treated with imatinib alone and the survival benefit of imatinib was enhanced by co-treatment with chaetocin. LDA showed both chaetocin and imatinib treatment alone decreased LSC frequency (1 in 10,143 in chaetocin treated, and 1 in 95,782 in imatinib treated vs. 1 in 4,036 in untreated control) but that the combination of chaetocin and imatinib treatment resulted in a dramatic decrease in LSC frequency (1 in 293,628 in chaetocin + imatinib treatment vs. 1 in 4036 in untreated control). Chaetocin, imatinib, and chaetocin + imatinib treatment of normal murine hematopoietic stem cells resulted in only minimal cytotoxicity as assessed by trypan blue, Annexin V, and colony forming assays. Our findings suggest that chaetocin, or chaetocin-related compounds, might serve to complement TKI therapy in the treatment of CML. Disclosures:No relevant conflicts of interest to declare.

Microenvironmental Protection of CML Stem and Progenitor Cells From Tyrosine Kinase Inhibitors Through N-Cadherin and Wnt Signaling

AbstractAbstract 912BCR-ABL tyrosine kinase inhibitors (TKI) do not eliminate leukemia stem cells (LSC) in chronic myeloid leukemia (CML), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSC, indicating that additional kinase-independent mechanisms contribute to LSC preservation. We investigated the role of signals from the bone marrow (BM) microenvironment in protecting chronic phase (CP) CML stem/progenitor cells from TKI treatment. Culture with human BM mesenchymal stromal cells (MSC), immortalized by ectopic expression of telomerase, significantly inhibited apoptosis in CP CML CD34+CD38- primitive and CD34+CD38+ commited progenitors treated with imatinib (5μM, p<0.01), nilotinib (5μM, p<0.01), or dasatinib(150nM, p<0.05). MSC coculture reduced TKI-mediated apoptosis in both dividing and non-dividing CD34+CD38- cells, defined using CFSE labeling, and increased the percentage of undivided CD34+CD38- cells (p<0.05). MSC coculture also enhanced colony forming ability of CML CD34+CD38- (p<0.05) and CD34+CD38+ (p<0.05) cells treated with TKI. Importantly MSC coculture, with or without imatinib treatment, significantly enhanced engraftment of CML CD34+ cells in immunodeficient NSG mice, both at 4 weeks (without IM: 27.2±5% human CD45+ cells without stroma, 52.8±8% with stroma, p<0.001; with IM: 4.9±2% without stroma, 10.1±2% with stroma, p<0.05) and at 10 weeks post-transplant (without IM : 1.48±0.2% human CD45+ cells without stroma, 2.37±0.4% with stroma, p=0.07; with IM : 0.74±0.2% without stroma, 1.2±0.3% with stroma, p<0.05). CML progenitors cultured in a transwell insert over MSC showed increased apoptosis following TKI exposure compared to cells cultured in direct contact with MSC (p<0.05). An increased proportion of CML progenitors adhered to MSC after 4 days of TKI treatment (22±4% without IM, 42±5% with IM, p<0.05). Antibody or peptide (NCDH) mediated blockade of the N-Cadherin receptor reduced adhesion of CML progenitors to MSC (p<0.05), and increased apoptosis of TKI-treated CML progenitors cocultured with MSC (p<0.05), indicating an important role for N-Cadherin in MSC-mediated protection of CML progenitors from TKI. Although N-Cadherin expression measured by flow cytometry, Western blot and Q-RT-PCR was lower in CML compared to normal CD34+CD38- and CD34+CD38+ cells, we observed significantly increased N-Cadherin expression in CML cells remaining after 4 days culture with IM and MSC (p<0.05). We observed enhanced β-catenin activity in CML progenitors cocultured with MSC, with and without TKI treatment, as manifested by increased β-catenin protein levels and nuclear localization, enhanced expression of β-catenin target genes, and increased TCF/LEF reporter activity. β-catenin levels and target gene expression were increased primarily in MSC-adherent CML progenitors compared to non-adherent cells, and were significantly reduced by antibody or peptide-mediated inhibition of N-Cadherin-mediated adhesion. Using the Duolink labeling technology, we demonstrated increased protein-protein interactions between N-cadherin and β-catenin in CML progenitors cocultured with MSC, with or without TKI treatment. Finally, we showed that addition of exogenous Wnt1 protected CML progenitors from TKI treatment, whereas inhibition of Wnt receptors by DKK1, or inhibition of β-catenin signaling by the small molecule inhibitor ICG001, enhanced apoptosis of CML progenitors cocultured with MSC, suggesting an important role for exogenous Wnt signaling in MSC-mediated protection of CML progenitors from TKI-induced apoptosis. Microarray analysis of gene expression confirmed enrichment of Wnt/β-catenin and Cadherin related gene sets in CML CD34+ cells cultured on MSC with or without IM, as well as enrichment of genes related to hematopoietic stem cell (HSC) self-renewal, HSC quiescence, and cytokine signaling. In conclusion, our studies reveal an important role for exogenous Wnt-β-catenin signaling, and a close interplay between N-Cadherin and β-catenin, in microenvonment-mediated resistance of CML stem and progenitor cells to TKI treatment. These findings support further development of novel treatment strategies to eradicate residual LSC in TKI-treated CML patients through inhibition of Wnt secretion or blockade of Wnt or N-Cadherin receptors. Disclosures:No relevant conflicts of interest to declare.

Dasatinib targets chronic myeloid leukemia-CD34+ progenitors as effectively as it targets mature cells

Dasatinib is effective in most chronic phase chronic myeloid leukemia patients both in first-line therapy and following imatinib failure. While imatinib uptake into CD34(+) cells is low compared to mononuclear cells, few data evaluate how well dasatinib targets primitive CML cells. This study compares intracellular concentration of dasatinib and Bcr-Abl kinase inhibition in CML-CD34(+) progenitors and mononuclear cells induced by dasatinib. The intracellular concentrations of dasatinib were similar between CML-CD34(+) and mononuclear cells (P=0.8). Similarly, there was no significant difference in the degree of dasatinib-mediated Bcr-Abl kinase inhibition. ABCB1 (MDR1) and ABCG2 inhibitors neither increased dasatinib intracellular concentration nor enhanced dasatinib-mediated Bcr-Abl kinase inhibition. In contrast to nilotinib, we show that dasatinib is not an ABCB1 inhibitor. Thus, dasatinib targets CML-CD34(+) progenitors as effectively as it targets mononuclear cells. ABCB1 and ABCG2 efflux pumps do not appear to influence the intracellular dasatinib concentration in CML-CD34(+) progenitors.

Rationally Designed Aberrant Kinase-Targeted Endogenous Protein Nanomedicine against Oncogene Mutated/Amplified Refractory Chronic Myeloid Leukemia

Deregulated protein kinases play a very critical role in tumorigenesis, metastasis, and drug resistance of cancer. Although molecularly targeted small molecule kinase inhibitors (SMI) are effective against many types of cancer, point mutations in the kinase domain impart drug resistance, a major challenge in the clinic. A classic example is chronic myeloid leukemia (CML) caused by BCR-ABL fusion protein, wherein a BCR-ABL kinase inhibitor, imatinib (IM), was highly successful in the early chronic phase of the disease, but failed in the advanced stages due to amplification of oncogene or point mutations in the drug-binding site of kinase domain. Here, by identifying critical molecular pathways responsible for the drug-resistance in refractory CML patient samples and a model cell line, we have rationally designed an endogenous protein nanomedicine targeted to both cell surface receptors and aberrantly activated secondary kinase in the oncogenic network. Molecular diagnosis revealed that, in addition to point mutations and amplification of oncogenic BCR-ABL kinase, relapsed/refractory patients exhibited significant activation of STAT5 signaling with correlative overexpression of transferrin receptors (TfR) on the cell membrane. Accordingly, we have developed a human serum albumin (HSA) based nanomedicine, loaded with STAT5 inhibitor (sorafenib), and surface conjugated the same with holo-transferrin (Tf) ligands for TfR specific delivery. This dual-targeted "transferrin conjugated albumin bound sorafenib" nanomedicine (Tf-nAlb-Soraf), prepared using aqueous nanoprecipitation method, displayed uniform spherical morphology with average size of ∼150 nm and drug encapsulation efficiency of ∼74%. TfR specific uptake and enhanced antileukemic activity of the nanomedicine was found maximum in the most drug resistant patient sample having the highest level of STAT5 and TfR expression, thereby confirming the accuracy of our rational design and potential of dual-targeting approach. The nanomedicine induced downregulation of key survival pathways such as pSTAT5 and antiapoptotic protein MCL-1 was demonstrated using immunoblotting. This study reveals that, by implementing molecular diagnosis, personalized nanomedicines can be rationally designed and nanoengineered by imparting therapeutic functionality to endogenous proteins to overcome clinically important challenges like molecular drug resistance.

Effects of plerixafor in combination with BCR-ABL kinase inhibition in a murine model of CML

AbstractSequestration in the bone marrow niche may allow leukemic stem cells to evade exposure to drugs. Because the CXCR4/SDF-1 axis is an important mechanism of leukemic stem cell interaction with marrow stroma, we tested whether plerixafor, an antagonist of CXCR4, may dislodge chronic myeloid leukemia (CML) cells from the niche, sensitizing them to tyrosine kinase inhibitors. We initially treated mice with retrovirally induced CML-like disease with imatinib plus plerixafor. Plerixafor mobilized CXCR4+ cells, but no difference was observed in leukemia burden, possibly reflecting insufficient disease control by imatinib. In a second series of experiments, we tested the combination of plerixafor with dasatinib in the same as well as an attenuated CML model. Despite much improved leukemia control, plerixafor failed to reduce leukemia burden over dasatinib alone. In addition, mice receiving plerixafor had an increased incidence of neurologic symptoms in association with CNS infiltration by BCR-ABL–expressing cells. We conclude that plerixafor is ineffective in reducing leukemia burden in this model but promotes CNS infiltration. Beneficial effects of combining tyrosine kinase inhibitors with plerixafor may be observed in a situation of minimal residual disease, but caution is warranted when disease control is incomplete.

1482PD - Dasatinib First-Line Treatment in Gastrointestinal Stromal Tumors. a Multicenter Phase II Trial of the SAKK (SAKK 56/07)

ABSTRACT Introduction First line imatinib has improved the outcome of patients (pts) with gastrointestinal stromal tumor (GIST), but primary and secondary resistance remain a problem. Dasatinib is a second generation tyrosine kinase inhibitor (TKI) of bcr-abl, src-family kinases and a number of other oncogenic kinases including kit. Dasatinib has shown activity against imatinib-resistant cell lines in vitro. 18F-fluorodeoxyglucose positron-emission-tomography (FDG-PET) measures tumor metabolic activity for early response assessment, which might be of particular use in the clinical trial setting. Methods This two-stage phase II trial investigated dasatinib in patients with histologically proven, FDG-PET positive, TKI-naive GIST. Dasatinib starting dose was 2x70mg/day. Response evaluation was done by serial CT and FDG-PET using EORTC PET response criteria (Young et al. 1999). PET was reviewed centrally. Elective surgery was allowed after 6 months of trial treatment. Primary endpoint was response (CR + PR) by FDG-PET after one month of dasatinib. Results 47 of planned 52 pts have been enrolled from December 2007 to November 2011, when the trial was terminated due to slow accrual. 43 pts were eligible. Median age was 61 years, 24 pts were male, 19 female. 30 pts had a performance status of 0 and thirteen of 1. Mutational data are available for 28 pts so far. 20 had a KIT exon 11, one a KIT exon 9 mutation, and 7 were wild-type. At a median follow-up of 12.4 months, 15 pts were still on treatment. Pts went off trial for elective surgery (n = 6), progression (n = 13), toxicity (n = 4), other reasons (n = 2) and three patients died. 5% of pts experienced G4, and 38% of pts G3 toxicity, which were most often gastrointestinal or pulmonary. 28 pts had their dose reduced or interrupted. The primary endpoint, FDG-PET response rate (CR + PR) at 4 weeks was 72% (14 CR, 17 PR, 7 SD, 3 PD, 2 not evaluable). The response rate in patients with a KIT Exon 11 mutation was 80%, in wild-type patients 57%. Median progression-free survival is 11.1 months and median overall survival not yet reached. Conclusions Dasatinib shows promising efficacy in TKI-naive pts with FDG-PET positive GIST. Disclosure M. Montemurro: Speakers fees and travel grants - Bayer, Pfizer, Novartis. P. Rutkowski: Consultant - BMS, Novartis, MSD Speakers fees and travel grants - Novartis, Pfizer, MSD, BMS, Roche. R. von Moos: Consultant/Advisor - AMGEN, Novartis, Roche, BMS, Pfizer, Merck Speakers fees - Roche, Amgen institutional Research Funds - AMGEN Expert Testimony - AMGEN. All other authors have declared no conflicts of interest.

Degree of kinase inhibition achieved in vitro by imatinib and nilotinib is decreased by high levels of ABCB1 but not ABCG2

Imatinib and nilotinib interact with ABCB1 and ABCG2. However, whether they are substrates or inhibitors is a source of conjecture. Here, in vitro, Bcr–Abl kinase inhibition was used to elucidate the impact of ABCB1/ABCG2 overexpression on imatinib and nilotinib transport. High levels of ABCB1 protein in K562-Dox cells resulted in a significantly increased 50% inhibitory concentration (IC50) compared with parental K562 cells for imatinib (IC50IM; 9 µM to 19 µM, p = 0.002) and nilotinib (IC50NIL; 345 nM to 620 nM, p = 0.013). This difference was abrogated by ABCB1 inhibitors. However, overexpression of ABCG2 did not significantly increase IC50IM or IC50NIL or significantly decrease IC50 upon ABCG2 inhibition. Inhibition of ABCB1 but not ABCG2 resulted in a substantial increase in intracellular nilotinib when used at 150 nM but no increase when used at 2 µM. Imatinib and nilotinib appear to be transported by ABCB1 but do not interact strongly with ABCG2. Furthermore, ABCB1 efflux of nilotinib may be concentration-dependent with transport occurring at clinically relevant concentrations.

Label‐free MSE proteomic analysis of chronic myeloid leukemia bone marrow plasma: disclosing new insights from therapy resistance

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic disorder that is currently considered incurable. The tyrosine kinase product of the Philadelphia chromosome, P210 BCR-ABL, provided a pathogenetic explanation for the initiation of the CML chronic phase and is the molecular therapeutic target for the disease. Imatinib mesylate, an orally available BCR-ABL kinase inhibitor, can induce haematologic and cytogenetic remission of CML. However, imatinib resistance occurs frequently, resulting in relapse. New treatment strategies are focusing on resistant CML stem cells and the bone marrow stroma. The identification of novel pathways and mechanisms in the bone marrow microenvironment could significantly contribute to the development of such strategies. In this work, we used a high-resolution label-free MS(E) proteomic approach to identify differential protein expression in the CML bone marrow plasma of responsive and resistant patients. Oxidative lipid metabolism and regulation of the switch from canonical to noncanonical WNT signaling may contribute to CML resistance in the bone marrow compartment.

UGT1A1*28 and *6 polymorphisms and nilotinib-induced unconjugated hyperbilirubinemia in a Japanese patient with chronic myelogenous leukemia

Nilotinib, a second-generation tyrosine kinase inhibitor of BCR-ABL, has shown superior efficacy compared with imatinib for the treatment of chronic myelogenous leukemia (CML). Unconjugated hyperbilirubinemia has been the most frequent adverse event with laboratory abnormality observed in clinical trials of nilotinib. The homozygosity for uridine diphosphate glucuronosyltransferase (UGT) 1A1*28 polymorphism has been reported to increase the risk of nilotinib-induced unconjugated hyperbilirubinemia in Caucasians. However, the frequency of UGT1A1*28 is low in Asians, including Japanese. On the other hand, the UGT1A1*6 allele mutation, which is extremely rare in Caucasians, is more frequent than the UGT1A1*28 allele in the Japanese population. Herein, we present a patient with CML who developed grade 3 unconjugated hyperbilirubinemia after being treated with nilotinib. We found that the patient was heterozygous for both UGT1A1*28 and *6. Our findings suggest that the compound heterozygosity for UGT1A1*28 and *6 could be a cause of unconjugated hyperbilirubinemia during nilotinib treatment.