In Contrast to Imatinib, Dasatinib Intracellular Concentration In CML-CD34+ Progenitors Is Not Significantly Different Than That Observed In CD34- Mature Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1205-1205
Author(s):  
Devendra K Hiwase ◽  
Jane Engler ◽  
Verity Saunders ◽  
Deborah L. White ◽  
Timothy Hughes
Keyword(s):  
Mrna Expression ◽  
Research Funding ◽  
Kinase Activity ◽  
Intracellular Uptake ◽  
Leukemic Stem Cells ◽  
Kinase Inhibition ◽  
Abl Kinase ◽  
Psc 833 ◽  
Cd34 Cells ◽  
Expression And Activity

Abstract Abstract 1205 Long term follow up of imatinib (IM) clinical studies and in vitro studies suggest that tyrosine kinase inhibitors (TKI) do not eradicate leukemic stem cells. Refractoriness of leukemic stem cells is postulated to be due to inadequate Bcr-Abl kinase inhibition which in turn could be due to low intracellular uptake and retention (IUR) of TKI. We have previously demonstrated that IM cellular uptake is predominantly mediated by the organic cation transporter protein (OCT-1) and patients with low OCT-1 activity have suboptimal response as compared to patients with high OCT-1 activity. More recently Engler et al (Leukemia 2010) demonstrated that IM IUR is significantly lower in CML-CD34+ cells compared to CD34- cells. This could be due to low expression and activity of the OCT-1 protein and/or high expression of ABCB1 and/or ABCG2 (Jiang et al Leukemia 2007). Although dasatinib is clinically available there are no published data assessing dasatinib IUR in CML-CD34+ progenitors. We and others have previously demonstrated that dasatinib cellular uptake is predominantly OCT-1 independent and dasatinib is a substrate of ABCB1 and ABCG2. We hypothesized that dasatinib IUR would be lower in CML-CD34+ cells compared to CD34- cells. In this study we compare dasatinib and IM IUR; and OCT-1 and ABCB1 mRNA expression in CML-CD34+ and CD34- cells of newly diagnosed CML-CP patients. CD34+ and CD34- cells were incubated with 14C-dasatinib (100 nM and 1 μM) or 14C-IM (2 μM) for 2h and IUR was assessed as described previously (Hiwase et al Clin Cancer Res. 2008). As shown previously, the OCT-1 expression and activity was lower in CML-CD34+ cells, and resulted in lower IM IUR in CML-CD34+ cells compared to CML-CD34- cells (15±5 vs. 27±5; p=0.04; Fig 1A and C). However at a therapeutically achievable concentration (100 nM dasatinib) and at higher concentration (1 μM dasatinib), there was no significant difference in dasatinib IUR in CML-CD34+ and CD34- cells (Fig 1B). Low OCT-1 expression and activity in CML-CD34+ cells did not influence the dasatinib IUR; further confirming that dasatinib cellular influx is predominantly OCT-1 independent. Despite higher ABCB1 mRNA expression in CML-CD34+ cells, the dasatinib IUR was not lower in CML-CD34+ cells compared to CD34- cells. High ABCB1 mRNA expression may not necessarily translate into high ABCB1 activity. To this end, we assessed the effect of PSC-833, an ABCB1 inhibitor, on dasatinib IUR and dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells. The baseline p-Crkl, a surrogate marker of Bcr-Abl kinase activity, was significantly higher in CML-CD34+ compared to CML-CD34- cells (67±5% vs. 55±8%; p=0.002; n=9). PSC-833 neither increased dasatinib IUR, nor enhanced dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells (% p-Crkl at 10 nM dasatinib: 20±6 vs. 27±10). Similarly, Ko143, an ABCG2 inhibitor, did not significantly change dasatinib IUR or Bcr-Abl kinase inhibition (% p-Crkl at 10 nM dasatinib: 21±3 vs. 27±10). In summary, although dasatinib is an ABCB1 and ABCG2 substrate, ABCB1 and ABCG2 inhibitors neither increase dasatinib IUR, nor enhance dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells. This data suggest that dasatinib IUR in CML-CD34+ cells is not influenced by ABCB1 and ABCG2. Hatziieremia et al (Exp. Hematology, 2009) reported that ABCB1 activity is low in CML-CD34+ cells and suggested that it did not influence IM level in CML-CD34+ cells. We further demonstrated that 100 nM dasatinib inhibited ≥95% Bcr-Abl kinase activity in CML-CD34+ and CD34- cells. In summary our data demonstrates that in contrast to IM, the intracellular concentration of dasatinib is equivalent in mature and immature CML cell compartments which may contribute to better targeting of early CML progenitors with dasatinib. Fig. 1: In contrast to IM, dasatinib intracellular uptake and retention (IUR) is not significantly different in CML-CD34+and mature CD34-cells: (A) OCT-1 activity and IM IUR is significantly lower in CML-CD34+ than CD34- cells (p=0.04). (B) However, dasatinib IUR is not significantly different in CD34+ and CD34- cells (p=0.8) (C) OCT-1 mRNA expression is lower in CML-CD34+ cells than CD34- cells. While ABCB1 expression is significantly higher in CML-CD34+ compared to CD34- cells (p=0.007). Fig. 1:. In contrast to IM, dasatinib intracellular uptake and retention (IUR) is not significantly different in CML-CD34+ and mature CD34- cells: (A) OCT-1 activity and IM IUR is significantly lower in CML-CD34+ than CD34- cells (p=0.04). (B) However, dasatinib IUR is not significantly different in CD34+ and CD34- cells (p=0.8) (C) OCT-1 mRNA expression is lower in CML-CD34+ cells than CD34- cells. While ABCB1 expression is significantly higher in CML-CD34+ compared to CD34- cells (p=0.007). Disclosures: White: Novartis: Honoraria, Research Funding; BMS: Research Funding. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

Blocking of Cytokine Survival Signals along with Intense Bcr-Abl Kinase Inhibition May Eradicate CML Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3250-3250
Author(s):  
Devendra K Hiwase ◽  
Deborah L White ◽  
Jason A Powell ◽  
Verity A Saunders ◽  
Stephanie Zrim ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Activity ◽  
Short Term ◽  
Kinase Inhibition ◽  
Gm Csf ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Short Term Culture

Abstract Abstract 3250 Poster Board III-1 Preclinical studies of imatinib set the paradigm of continuous Bcr-Abl kinase inhibition for optimal response in chronic myeloid leukemia (CML). However, the clinical success of once daily dasatinib, despite its short serum half life, implies that intermittent inhibition of Bcr-Abl kinase activity is sufficient for clinical response. In vitro studies also demonstrated that short-term intense (≥90%) Bcr-Abl kinase inhibition triggers cell death in BCR-ABL + cell lines, demonstrating their oncogene addiction. However, the effect of short-term intense kinase inhibition on CD34+ CML progenitors is not studied. Clinical, mathematical modelling and in vitro studies suggest that leukemic stem cells (LSC) are difficult to eradicate and hence the majority of CML patients may not be cured with tyrosine kinase inhibitors (TKI). Inadequate Bcr-Abl kinase inhibition has been postulated to cause refractoriness of LSC to TKI's. This may be due to increased expression of ABCB1 and ABCG2 efflux proteins, or the quiescent state of LSC. However, the phenomenon could be independent of Bcr-Abl kinase activity. In vivo leukemic progenitors live in a cytokine rich environment which may be providing a mechanism for Bcr-Abl independent resistance. We have assessed the impact of short-term intense Bcr-Abl kinase inhibition on CML cell lines and CML CD34+ primary cells in the presence and absence of cytokines. In CML cell lines, short-term (cells were cultured with dasatinib for 30 min and following thorough drug washout, cells were recultured in drug free media for 72 hr) intense Bcr-Abl kinase inhibition with 100 nM dasatinib triggers cell death. In CML-CD34+ cells 30 min of culture with 100 nM dasatinib (n=13) or 30 μM IM (n=7) reduced the level of p-Crkl (surrogate marker of Bcr-Abl kinase activity) by 97±3% and 96±4% respectively. In the presence of either a six growth factors cocktail (6-GF; n=10) or GM-CSF (n=11) or G-CSF (n=4) alone, despite 97% inhibition of p-Crkl, short-term culture with 100 nM dasatinib (D100ST) reduced colony forming cells (CFC) by only 24%, 32% or 5%, respectively. However without cytokines, D100ST reduced CML-CD34+ CFCs by 70%. Consistent with the results observed with dasatinib, short-term culture with 30 μM imatinib (IM) (n=3) also reduced 90% CFC in the absence of cytokines but by only 38% in the presence of 6-GF. These results suggest that in CML-CD34+ cells, GM-CSF, G-CSF or 6-GF mediate Bcr-Abl independent TKI resistance. It is possible that cytokines may be promoting cell survival via signalling pathways that are refractory to dasatinib. To examine this possibility, we assessed the effect of D100ST on p-STAT5 signalling in CML-CD34+ cells, in the presence and absence of GM-CSF, G-CSF or 6-GF. STAT5 was constitutively phosphorylated in CML-CD34+ cells, and in the absence of cytokines, D100ST reduced the p-STAT 5. STAT5 phosphorylation was not inhibited by D100ST when cells were cultured with 6-GFs or GM-CSF however, the combination of D100ST and a Janus kinase (Jak) inhibitor dramatically reduced p-STAT5. Similarly, in the presence of GM-CSF (32.35±5.16% vs. 68.33±14.90%) or G-CSF (58.13±13 vs. 94.68±21.12) combination of D100ST and JAK inhibitor significantly reduced CFC compared to D100ST only. Thus our data suggest that in contrast to CML cell lines, primary CML progenitors may not be completely dependent on the BCR-ABL oncogene and that activation of the cytokine mediated JAK-2/STAT-5 pathway may circumvent the need for BCR-ABL signalling for maintenance of survival. Thus a therapeutic strategy based on short-term intense kinase inhibition may have limited success unless critical redundant cytokine-induced survival pathways are also inhibited. We postulate that blockade of cytokine signalling along with short-term intense Bcr-Abl kinase inhibition with a potent second generation TKI may provide a novel strategy to eradicate primitive CML cells. Fig 1 In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Fig 1. In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Disclosures: White: Novartis and Britol-Myers Squibb: Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Proton Pump Inhibitors Augment Nilotinib and Dasatinib Mediated Bcr-Abl Kinase Inhibition

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3991-3991 ◽  
Author(s):  
Devendra K Hiwase ◽  
Laura Eadie ◽  
Verity Saunders ◽  
Timothy Hughes ◽  
Deborah L. White
Keyword(s):  
Proton Pump Inhibitors ◽  
Proton Pump ◽  
Research Funding ◽  
Kinase Activity ◽  
Surrogate Marker ◽  
K562 Cells ◽  
P Value ◽  
Intracellular Uptake ◽  
Kinase Inhibition ◽  
Abl Kinase

Abstract Abstract 3991 Proton pump inhibitors (PPIs) are widely used for the treatment of gastro-oesophageal reflux and peptic ulcer disease. PPIs may not only alter absorption and metabolism of tyrosine kinase inhibitors (TKIs) but could also alter intracellular uptake and retention (IUR) into leukemic cells by interacting with ABCB1 and ABCG2 pump. There is limited literature assessing the interaction of PPI with TKI's at the cellular level. Here we compare the interaction of PPI with dasatinib, imatinib and nilotinib. Mononuclear cells (MNC) of CML-CP patients, K562 and K562-Dox (ABCB1 overexpressing), K562-ABCG2 cells were cultured with 14C-labelled dasatinib, imatinib and nilotinib with or without PPI for 2h and IUR were assessed. The effect of combining PPI with TKI's on Bcr-Abl kinase activity was assessed by measuring p-Crkl (surrogate marker of Bcr-Abl kinase activity). High concentrations of pantoprazole (1 and 2 mM) significantly increased dasatinib IUR in K562-Dox cells (p=0.004 and p=0.0006 respectively) but not in K562 cells (p=0.7 and p=0.1). Similarly, 1 mM (p=0.01) and 2 mM (p=0.007) esomeprazole significantly increased dasatinib IUR in K562-Dox cells but not in K562 cells. These data suggest that high concentration of PPI inhibit ABCB1 mediated dasatinib efflux. This was further supported by significant reduction in IC50dasatinib in K562-Dox cells by 1 mM pantoprazole (112±37 nM to 28±6; p=0.02; n=4). Similarly, 1 mM esomeprazole reduced IC50dasatinib (112±37 nM to 12±3 nM; n=2). This demonstrates that PPI mediated increase in IUR translates to increased kinase inhibition and lower IC50dasatinib. At lower concentrations (100 to 400 μM), neither pantoprazole nor esomeprazole significantly changed the dasatinib IUR or the IC50dasatinib in K562-Dox cells. In K562-ABCG2 cell line 100, 200, 400, 500, 1000 μM pantoprazole reduced IC50dasatinib from 27 nM to 15, 7, 11, 9.5 and 6.5 nM respectively. Similarly, 100, 200, 400, 500 and 1000 μM esomeprazole reduced IC50dasatinib from 21 nM to 15, 11, 8.5, 4.5 and 3.5 nM respectively. These data suggest that PPI enhances dasatinib mediated Bcr-Abl kinase inhibition in ABCG2-overexpressing cells. Although PPI did not change dasatinib IUR significantly in primary CML-MNC (n=10, Table I), it reduced IC50dasatinib (n=4, Table II). Similarly, pantoprazole and esomeprazole (5 to 400 μM) significantly increased nilotinib IUR (n=10, Table I) and significantly reduced IC50nilotinib in CML-MNC (n=3, Table II). Pantoprazole also increased the nilotinib IUR in K562 and K562-Dox cells, and reduced the IC50nilotinib in K562 (500 vs. 250 nM) and K562-Dox (600 vs. 230 nM) cells. Similarly esomeprazole reduced the IC50nilotinib in K562 (500 vs. 250 nM) and K562-Dox (600 vs. 150 nM) cells. The effect of PPI on IC50nilotinib was dose dependent. Pantoprazole and esomeprazole reduced imatinib IUR in K562, K562-Dox and CML-MNC. Pantoprazole increased IC50imatinib in K562 (3.8 to 4 μM) and K562-Dox (6.5 to 8 μM) cells. Similarly, 200 μM of pantoprazole and esomeprazole significantly reduced IM IUR in CML-MNC. However, the effect of pantoprazole on IC50imatinib in CML-MNC was variable and modest (Table II). Our data provide evidence that PPI might interfere with TKI activity. PPI's can enhance the dasatinib and nilotinib mediated Bcr-Abl kinase inhibition in primary CML cells. Table I: Effect of PPI on dasatinib, imatinib and nilotinib intracellular uptake and retention (IUR) in CML-MNC (n=10) TKI at 1μM TKI at 2μM Pantoprazole 0 μM 100μM 200μM 400μM 0uM 100μM 200μM 400μM Imatinib 13.3 10.9 9.9 8.8 23.7 20.4 18.9 16.3 p-value 0.06 0.01 0.004 0.2 0.09 0.01 Nilotinib 13.0 16.4 16.6 17.4 30.5 56.9 66.2 74.7 p-value 0.04 0.02 0.03 0.004 0.001 <0.001 Dasatinib 10.3 11.1 9.4 7.9 18.9 18.3 17.7 18.9 p-value 0.6 0.1 0.08 0.4 0.4 0.2 Table II: Effect of PPI on IC50 of nilotinib, dasatinib and imatinib in CML-MNC Nilotinib Nilotinib + 200 μM pantoprazole (% change) Nilotinib + 200 μM esomeprazole (% change) CML1 62.5 nM 30 nM (−52) 55 nM (−12) CML2 80 nM 32 nM (−60) 17 nM (−78) CML3 75 nM 68 nM (−9.4) 27 nM (−64) Dasatinib Dasatinib + 200 μM pantoprazole (% change) Dasatinib + 200 μM esomeprazole (% change) CML1 1.8 nM 0.7 nM (−61) 1.2 nM (−33) CML2 3 nM 2.6 nM (−13) – CML3 7 nM 2.5 nM (−64) – CML4 2.25 nM 1.7 nM (−25) 1.5 nM (−33) Imatinib Imatinib + 200 μM pantoprazole (% change) Imatinib + 200 μM esomeprazole (% change) CML1 1.05 μM 0.325 μM (−69) 0.8 μM (−23) CML2 1.8 μM 1.35 μM (−25) CML3 0.83 μM 1.15 μM (+38) 0.65 μM (−21) Disclosures: Hughes: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Research Funding.

Short –Term Intense Bcr-Abl Kinase Inhibition Is Adequate to Trigger Cell Death in CML Cell Lines but Not in CML-CD34+ Cells Unless They Are Growth Factor Deprived.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1086-1086
Author(s):  
Devendra K. Hi Wase ◽  
Deborah L. White ◽  
Verity A. Saunders ◽  
Junia V. Melo ◽  
Sharad Kumar ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Growth Factors ◽  
Growth Factor ◽  
Cell Lines ◽  
Kinase Activity ◽  
Short Term ◽  
Kinase Inhibition ◽  
Abl Kinase ◽  
Cd34 Cells

Abstract After 5 years of imatinib treatment, only a minority of newly diagnosed chronic myeloid leukemia chronic phase (CML-CP) patients achieve complete molecular response. Imatinib has antiproliferative effects, but may not be able to eradicate CML-stem cells. Preclinical studies of imatinib suggested that sustained BCR-ABL kinase inhibition was required to block proliferation and induce apoptosis in CML cells. This formed the rationale for treatment regimens that maintain continuous kinase inhibition. Clinical studies with dasatinib suggested that daily dosing achieves equivalent response to twice daily even though ABL kinase inhibition only persists for 4–6 hours. We have demonstrated that 30 minutes of exposure to 100 nM dasatinib or 30 μ M imatinib (equipotent) inhibit p-Crkl (surrogate marker of Bcr-Abl kinase activity) by 80 to 90% in Bcr-Abl +ve cell lines and CML-CD34+ cells (n=8). We then sought to compare antiproliferative and pro-apoptotic effects of short term (ST; cells were cultured with dasatinib/imatinib for 30 minutes and after thorough wash, were recultured without dasatinib/imatinib for 72 hours) and continuous (CT, cells were cultured with drugs continuously) dasatinib or imatinib in BCRABL +ve cell lines (K562, Meg 01) and CD34+ cells of CML-CP patients. Although Bcr- Abl kinase reactivated within 30 minutes of drug removal, ST 100 nM dasatinib (D100ST) or 30μ M of imatinib (IM30ST) induced apoptosis (~80%) and blocked cell proliferation equivalent to continuous dasatinib (10 nM; D10CT) or imatinib (2μ M, IM2CT) in Bcr-Abl +ve cell lines. The kinetics of cell death and caspase-3 activation over 72 hours of culture were similar in D100ST and D10CT. In the presence of 6-growth factors (GFs; IL-3, IL- 6, G-CSF, SCF, TPO, Flt-3) D100ST and IM30ST reduced cell viability and CFU-GM colonies of CML-CD34+ cells by only 25 to 30% of no drug control. Moreover in the presence of GFs, 30 to 40% CD34+ve cells were viable and retained CFU-GM potential in spite of continuous dasatinib 100 nM (D100CT) or 30 μ M of imatinib (IM30CT). However, in the absence of GFs, D100ST and IM30ST reduced viability by 60 to 70%, and CFU-GM by 95% of control (with GFs, no TKI control; Fig 1). Figure 1: Survival of CFU-GM according to growth factor and dasatinib exposure: Figure 1:. Survival of CFU-GM according to growth factor and dasatinib exposure: Conclusion: Short term intense inhibition of BCR-ABL kinase activity triggers apoptosis in CML cell lines, which demonstrate their Bcr-Abl oncogene dependence. However, in spite of &gt;80% kinase inhibition, D100ST and D100CT did not eliminate the majority of CML-CD34+ cells in the presence of GFs. In the absence of GFs, D100ST and IM30ST were able to inhibit cell proliferation, induce cell death and eliminate 95% of CFU-GM. This data suggests that oncogene dependence of CML CD34+ cells can be overcome by cytokines. Unlike CML cell lines where transient intense kinase inhibition leads to cell death, primary CML cells are only sensitive to this short term kinase inhibition in the absence of cytokines. Strategies that block cytokine pathways in combination with Bcr-Abl kinase inhibition may eliminate leukemic stem cells in-vivo even if only applied intermittently. CFU-GM colonies expressed as % of control. CML-CD34+ cells (n=3) were cultured with dasatinib in the presence (With GFs) or absence (No GFs) of 6-growth factors (GF) and CFU-GM colonies were plated on D3, using Methocult 4230 (Invitrogen) along with growth factors in all cases. Colonies were read after 14 days. In each patient values were normalised to cells cultured with GFs and no dasatinib. Short term (ST) and continuous (CT), Dasatinib 10 nM (D10), 100 nM (D100).

Low Level BCR-ABL Mutations Below the Detection Limit of Current Standard Screening Techniques Occur Predominantly in the CD34+ Progenitor Cell Compartment in Chronic Phase CML Patients At Diagnosis. A Substudy of the ENEST1st Trial

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1671-1671
Author(s):  
Jacqueline Maier ◽  
Karoline Schubert ◽  
Michael Cross ◽  
Sabine Leiblein ◽  
Kathrin Wildenberger ◽  
...  
Keyword(s):  
Detection Limit ◽  
Research Funding ◽  
Progenitor Cell ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Cell Compartment ◽  
Screening Techniques ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Kinase Domain Mutations

Abstract Abstract 1671 The presence of BCR-ABL kinase domain mutations below the detection limit of conventional screening techniques (low level mutations, LLM) predicts outcome of subsequent therapy in patients with imatinib resistance (Parker et. al JCO 2011 and Blood 2012). We have further evaluated LLM in the context of the ENEST1st trial, which addresses the frequency of complete molecular responses after 18 months on nilotinib 300mg BID (NI) in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). Here, we have investigated the incidence of detectable LLM in the CD34+ progenitor cell compartment in comparison to total white cells (TWBC). Sixty nine ENEST1st study patients with CP CML provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34+ selection was carried out by MACS® (Miltenyi Biotec) and the CD34+ purity was subsequently determined by fluorescent activated cell sorting (FACS). The results were compared to those derived from stored TWBC from 23 of the same patients and a further 16 patients at diagnosis. Aliquots of 105 CD34+ or at least 106 TWBC were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to <0.1% mutant allele (3–3.5 log). No patients showed BCR-ABL kinase domain mutations detected by Sanger sequencing spanning ABL exons 4–9. Forty five of 69 patients (65%) with 105 CD34+ cells and a documented CD34+ purity of >50% were available for BCR-ABL amplification. Amplification was successful from 36 (52%) of these CD34+ samples and from 38 of the 39 (97%) TWBC samples. A total of 180 L-PCR assays of CD34+ cells identified 29 (16%) mutations (T315Ix12, Y253Hx7, E255Kx8/Vx1 and F359Vx1) in CD34+ cells from 21/36 patients (58%). In comparison, 190 assays of TWBC identified 10 (5%) mutations (T315Ix3, Y253Hx6, E255Vx1, p=0.0005) in 8/38 patients (21%, p=0.001 Fishers exact test). Significantly more T315I (33%) and E255K (22%) mutations were observed in CD34+ cells than in TWBC (8%, p=0.007 and 0% p= 0.003 respectively). The quantitative levels of all mutant alleles were median 0.135 (range 0.06–0.535) and 0.1 (range 0.04-0, 25) BCR-ABLmutant/ BCR-ABLunmutated for mutations in CD34+ cells and TWBC, respectively and were not significantly different. Where both CD34+ and TWBC were available from the same patient (n=23), 11 patients showed a total of 18 mutations in the CD34+ fraction but only one of these mutations was confirmed in TWBC. One additional mutation was detectable in the TWBC. The remaining 12 patients with no detectable mutation in the CD34+ fraction showed 3 mutations (2x Y253H, T315I) in 2 patients in TWBC only. In conclusion, LLM with either no (T315I) or intermediate (Y253H, E255K/V, F359V) sensitivity to nilotinib are detectable in CP CML patients at a frequency of 21% in the TWBC but with a significantly higher frequency of 58% in the enriched CD34+ progenitor cell compartment. Longterm patient follow up on the ENEST1st and ENESTobserve studies will allow analysis of the relationship between LLM and clinical outcomes on nilotinib. Disclosures: Hochhaus: Novartis, BMS, MSD, Ariad, Pfizer: Consultancy Other, Honoraria, Research Funding. Frank:Novartis: Employment. Lange:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Potent Transient Inhibition of BCR-ABL by Dasatinib Leads to Complete Cytogenetic Remissions in Patients with Chronic Myeloid Leukemia: Implications for Patient Management and Drug Development.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2166-2166 ◽  
Author(s):  
Neil P. Shah ◽  
John M. Nicoll ◽  
Eric Bleickardt ◽  
Claude Nicaise ◽  
Ronald L. Paquette ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Biology ◽  
Kinase Activity ◽  
Kinase Inhibition ◽  
Once Daily ◽  
Minute Exposure ◽  
Abl Kinase ◽  
Short Term Exposure ◽  
Dose Modifications

Abstract Dasatinib (Sprycel; formerly BMS-354825) is an oral multi-targeted SRC/ABL tyrosine kinase inhibitor that is approximately 325-fold more potent than imatinib, and has been recently approved by the US FDA for the treatment of imatinib-resistant and -intolerant Philadelphia chromosome-associated leukemias. In contrast to other tyrosine kinase inhibitors, pharmacokinetic analyses reveal that dasatinib has a short half-life (5–6 hours), with near-complete loss of BCR-ABL kinase activity inhibition eight hours after drug administration in patients with CML. Remarkably, patients treated with dasatinib as infrequently as once daily, five days per week, have achieved complete cytogenetic remission (CCyR). We tested transient BCR-ABL inhibition in vitro by assessing the sensitivity of the CML cell line K562 to short-term exposure of dasatinib. After as short as a 20-minute exposure to a clinically-relevant dasatinib concentration (100 nM), the majority of cells undergo apoptosis when analyzed after 48 hours, whereas a clinically-relevant concentration of imatinib (5 uM) failed to result in substantial cytotoxicity under these conditions. When higher concentrations (>=12.5 uM) of imatinib were used that correct for differences in potency between imatinib and dasatinib, cytotoxicity at 48 hours after a 20-minute exposure was similar to that observed with dasatinib. These results exclude SRC family inhibition as a potential mediator of this effect, and were reproducible in a second CML cell line, KU-812. Importantly, cytotoxicity was not observed in BCR-ABL-negative leukemia cell lines under these conditions. Signiificantly, similar phenomenon was observed when an erlotinib-sensitive non-small cell lung cancer was exposed to high concentrations of erlotinib for 20 minutes. We assessed the degree of BCR-ABL kinase inhibition achieved in 20 patients with chronic phase CML who were treated with dasatinib once daily as part of a phase I clinical trial by quantifying the ratio of phospho-CRKL to CRKL (a BCR-ABL substrate) achieved in PBMCs harvested four hours after the first dose of dasatinib. We found a strong correlation between the magnitude of BCR-ABL kinase inhibition and the depth of response achieved. Notably, CCyR was achieved exclusively in four patients who experienced the deepest inhibition of BCR-ABL kinase activity activity. The current medical management of CML involves assessing the degree of cytogenetic response after 6–12 months of imatinib therapy before considering dose modifications of imatinib. While our findings will need to be validated prospectively in larger cohorts of patients, they suggest that it may be feasible to make early dose modifications based upon the degree of target inhibition following the initial dose of dasatinib, and thus maximize the likelihood of clinical benefit. Together, these findings have potentially significant implications not only for the optimal clinical management of CML patients, but also for the rational development of small molecule inhibitors of other cancer-associated tyrosine kinases, as well as our global understanding of cancer cell biology.

Nilotinib Inhibits Bcr-Abl Kinase Activity in CML Progenitor Cells More Effectively Than Imatinib but Is Equipotent in Inducing Growth Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 744-744 ◽  
Author(s):  
Heiko Konig ◽  
Melissa Holtz ◽  
Hardik Modi ◽  
Paul Manley ◽  
Tessa L. Holyoake ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Inhibition ◽  
Progenitor Cells ◽  
Drug Exposure ◽  
Kinase Activity ◽  
Dose Range ◽  
Therapeutic Success ◽  
Abl Kinase ◽  
Signaling Mechanisms ◽  
Cd34 Cells

Abstract The therapeutic success of imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by persistence of malignant stem cells. Mechanisms contributing to incomplete elimination of CML stem cells include resistance to proapoptotic effects of IM, persistent signaling through growth stimulating pathways and Abl kinase mutations resulting in IM resistance. We investigated whether nilotinib, a more potent Bcr-Abl tyrosine kinase (TK) inhibitor than IM, could more effectively target CML progenitors. CD34+ progenitors from CML patients and healthy donors were cultured with nilotinib (0–10μM) and IM (0–5μM) in growth factor (GF) containing medium. After 96h incubation, cells were harvested and assayed for colony-forming-(CFC) and long-term culture initiating cells (LTCIC). CD34+38- primitive progenitor cells (PPC) and CD34+38+ committed progenitor cells (CPC) labeled with CFSE were incubated under similar conditions and evaluated for proliferation and apoptosis assays by annexin-V staining and FACS analysis. The IC50 for CML LTCIC-suppression was 0.9μM for nilotinib and 0.8μM for IM. Normal LTCIC were not significantly suppressed at doses below 10μM nilotinib and 5μM IM. The IC50 for CML CFC suppression for nilotinib was 6.4μM and 1.0μM for IM. Normal CFC showed less growth inhibition and the IC50 was not reached at the dose range tested. CFSE assays indicated dose-dependent antiproliferative activity of both compounds with IC50-values of 3.0μM nilotinib and 1.8μM IM for CML PPC, and 8.2μM nilotinib and 4.0μM IM for CML CPC. In CML PPC, apoptosis significantly increased following nilotinib treatment (8±2.1% [control] to 22±2.7% [10μM], n=4, p=.015). IM treatment also increased apoptosis in CML PPC (to 35±3.8% [5μM], n=4, p=.0008). In CML CPC, apoptosis increased from 33±2.3% [control] to 50±1.7% (n=4, p=.002) with 10μM nilotinib, and to 53±2.1% (n=4, p=.001) with 5μM IM exposure. The effects on IM and nilotinib on Bcr-Abl-TK activity were investigated by Western blotting with anti-CrkL antibodies after overnight drug exposure of CML CD34+ cells in GF containing media. Importantly, 0.25μM nilotinib significantly reduced P-CrkL levels (from 85.7±4.1% [control] to 13.9±4.5%, n=4, p=.000002), whereas a higher concentration of IM (1–5μM) was needed to achieve similar inhibition. Nilotinib resulted in increased MAPK activity (to 13.3-fold [5μM]) in CML CD34+ cells. MAPK kinase activity was also increased following treatment with IM (to 22.7-fold [5μM]). P-AKT and P-STAT5 levels were not significantly changed in response to either drug. In contrast incubation with either drug in the absence of GF resulted in inhibition of MAPK, Akt and STAT5 activity in CML CD34+ cells. In conclusion, nilotinib is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors but does not induce greater suppression of progenitor growth. As with IM, inhibition of cell division represents the predominant mode of inhibition of progenitor growth. Nilotinib or IM treatment inhibits Bcr-Abl dependent MAPK, STAT5 and Akt activity, but does not inhibit these signaling mechanisms in CML progenitors in the presence of GF. Our results suggest that combined inhibition of Bcr-Abl TK and additional signaling mechanisms may be required to achieve elimination of CML progenitors by targeted therapies.

Reduced Activity of the OCT-1 Protein in Primitive CML Cells: A Likely Determinant of Stem Cell Resistance in Imatinib Treated CML Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 196-196 ◽  
Author(s):  
Jane R Engler ◽  
Amity Frede ◽  
Andrew C W Zannettino ◽  
Deborah L White ◽  
Timothy P Hughes
Keyword(s):  
Stem Cell ◽  
Standard Deviation ◽  
Mrna Expression ◽  
Functional Activity ◽  
P Value ◽  
Cell Resistance ◽  
Kinase Inhibition ◽  
Normal Individuals ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Despite cytogenetic and molecular remissions, residual chronic myeloid leukemia (CML) cells persist in the primitive CD34+ compartment in the majority of imatinib treated patients. It has been demonstrated that CD34+ CML cells have a reduced sensitivity to imatinib induced apoptosis. Factors which may contribute to this reduced sensitivity are reduced dependence on BCR-ABL, an increase in BCR-ABL transcripts, increased expression of efflux proteins or decreased expression of drug influx proteins. Our previous studies show that a patient’s intrinsic sensitivity to imatinib-induced kinase inhibition (IC50) is related to the intracellular uptake and retention (IUR) of imatinib in peripheral blood mononuclear cells. The organic cation transporter 1 (OCT-1) is the major active influx transporter for imatinib in these cells, and the functional activity of this protein (determined using functional inhibition of OCT-1, in the IUR assay) directly correlates with molecular response to imatinib. In the present study we investigated the role that OCT-1 plays in stem cell resistance to imatinib. Primitive CD34+ and mature CD34− cells were isolated from CML patients and normal individuals using magnetic cell sorting. CML CD34+ cells had a significantly lower IURimatinib than that of CML CD34− cells (Table 1). The addition of the OCT-1 inhibitor, prazosin (100μM), eliminated this difference in IUR (Table 1), indicating that variation in IURimatinib is due to variation in the functional activity of the OCT-1 protein. In addition, the OCT-1 Activity (Table 1) and OCT-1 mRNA expression (expressed as % of BCR: mean CD34+=0.25; CD34−=4.9, p=0.040, n=10) was significantly lower in CML CD34+ cells compared with CML CD34− cells. These differences in IURimatinib and OCT-1 activity between CD34+ and CD34− cells were not observed in normal individuals (Table 1), suggesting this phenomenon is specific to leukemic cells. Furthermore, we isolated the more primitive compartment, CD34+38− cells and less primitive CD34+38+ cells in 4 CML patients. The CD34+38− cells demonstrated a 13% reduction in IURimatinib and a 41% reduction in OCT-1 activity compared with CD34+38+ cells. These data suggest a reduced IUR mediated by low OCT-1 function and/or expression may play a role in the resistance of CML stem cells to imatinib. Table 1: The IUR of 2μM imatinib expressed as ng/200,000 cells (standard deviation) n Imatinib IUR P value + Prazosin P value OCT-1 Activity P value CML CD34+ 14 16.05 (4.53) 0.002 13.02 (3.34) 0.505 3.25 (2.32) &lt;0.001 CML CD34− 14 27.28 (12.92) 14.76 (5.25) 14.01 (12.08) Normal CD34+ 11 11.13 (2.66) 0.212 10.43 (3.80) 0.743 2.03 (2.09) 0.693 Normal CD34− 11 13.92 (5.32) 10.93 (3.30) 3.52 (5.24) Increased expression of efflux transporters of imatinib (i.e. ABCB1 and ABCG2) has been suggested as an important mechanism for drug resistance. The effect of an ABCB1 inhibitor (PSC833) and ABCG2 inhibitor (Ko143) was assessed in CD34+ cells from 3 CML patients, using the IUR assay. Neither of these drugs had any effect on the IURimatinib in CML CD34+ cells. Additionally the mRNA expression of ABCB1 did not differ between CML CD34+ and CD34− cells (expressed as a % of BCR: mean CD34+=33.7; CD34−=33.77, p=0.064, n=10). These data suggest that alterations in imatinib influx (via OCT-1) are more critical for development of stem cell resistance rather than differences in efflux. We have previously demonstrated that, unlike imatinib, the OCT-1 protein is not involved in nilotinib transport, as the addition of OCT-1 inhibitors does not alter IURnilotinib in patients. Assessing the IUR of nilotinib in CD34+ and CD34− CML cells reveals no significant difference between the two populations (Table 2). Additionally, the IURnilotinib is significantly higher than IURimatinib in CML CD34+ cells (Table 2). In summary, the reduced OCT-1 mediated uptake of imatinib in more primitive, CD34+ CML cells may result in inadequate kinase inhibition and contribute to stem cell resistance in CML. Since nilotinib uptake into CML CD34+ cells is not impaired in the same manner as imatinib, more substantial depletion of the primitive CML cells may be achieved. Table 2: The IUR of 2μM imatinib and nilotinib in the same 11 CML patients. Expressed as ng/200,000 cells (standard deviation) n Imatinib IUR P value Nilotinib IUR P value P value between imatinib & nilotinib IUR CML CD34+ 11 17.80 (5.73) 0.006 26.35 (7.54) 0.230 0.007 CML CD34− 11 30.28 (12.33) 22.05 (8.68) 0.076

The Protein Tyrosine Phosphatase STS-1 Interacts with Bcr-Abl and Impairs the Response of Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) to Tyrosine Kinase Inhibitors

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3236-3236
Author(s):  
Marcus Liebermann ◽  
Daniela Hoeller ◽  
Susanne Badura ◽  
Tamara Tesanovic ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Signalling Pathways ◽  
Abl Kinase ◽  
Clinical Resistance

Abstract Abstract 3236 Bcr-Abl is a leukemogenic fusion gene that by itself is sufficient for cellular transformation (Daley et al.) and is the hallmark of chronic myeloid leukemia and Philadelphia chromosome positive (Ph+) ALL. The Bcr-Abl fusion protein is a constitutively active tyrosine kinase (TK) which disrupts multiple cellular signalling pathways controlling apoptosis, cell cycle, proliferation and DNA repair. In Ph+ ALL, a subtype of ALL with a particularly poor prognosis, targeted inhibition of Bcr-Abl activity by Abl kinase inhibitors such as imatinib has improved treatment outcome but has not abrogated the frequent development of clinical resistance. In addition to mutations in the Bcr-Abl tyrosine kinase domain (TKD), it has become apparent that other resistance mechanisms contribute to disease progression. The activity of proteins involved in the above-mentioned signalling pathways and possibly resistance to TK inhibitors (TKI) is controlled at least partially by posttranslational modifications such as phosphorylation, which is regulated by the balance between kinases and protein tyrosine phosphatases (PTP). We previously showed that PTP1B is a negative regulator of Bcr-Abl-mediated transformation and modulates sensitivity to the TKI imatinib (Koyama et al). We hypothesized that other phosphatases for which Bcr-Abl is a substrate may also contribute to resistance, one candidate being Suppressor of T-cell receptor Signalling 1 (STS-1), which negatively regulates the endocytosis of receptor TK involved in a variety of hematologic malignancies. It was the aim of this study to determine whether: i) Bcr-Abl is a substrate of STS-1 ii) STS-1 is able to dephosphorylate Bcr-Abl iii) expression of STS-1 reduces the proliferation of Bcr-Abl expressing cells by inhibiting Bcr-Abl kinase activity iv) the level of STS-1 expression modulates the sensitivity of Bcr-Abl positive cells to TKI In order to answer these questions, we used 293T cells, a human primary embryonal kidney cell line, and the IL3-dependent murine pro B cell line Ba/F3. Both cell lines were modified with constructs encoding both forms of Bcr-Abl (p185/p210) and Sts-1. For experiments with endogenous Bcr-Abl (p185) and Sts-1 we used Sup B15 cells, a human B cell precursor leukemia, and its TKI-resistant subline (Sup B15 RT), which was generated in our lab and is highly resistant not only to imatinib but also to 2nd generation TKIs (Nilotinib & Dasatinib), with no evidence of TKD mutations or transcriptional up-regulation of Bcr-Abl. In all above described cell lines the interaction between Bcr-Abl and Sts-1 could be shown in an overexpressed system (293T & Ba/F3) and on an endogenous level (Sup B15 & Sup B15 RT) by using co-IPs followed by SDS-PAGE and Western blotting. The functional relevance was examined by testing the ability of Sts-1 to dephosphorylate Bcr-Abl. Complete dephosphorylation of Bcr-Abl was shown for p185bcr-abl and p210bcr-abl in 293T cells. To verify that the functional activity was also present in hematopoietic cells, we analyzed the ability of Sts-1 to dephosphorylate Bcr-Abl in Ba/F3 and Sup B15 cells. Dephosphorylation was observed in both cell lines but was less pronounced than in 293T cells. We therefore more closely examined the most important tyrosine (Tyr) residues of Bcr-Abl and identified Tyr245 and Tyr412 as the major targets of Sts-1. Phosphorylation of Tyr245 and Tyr412 was decreased by ∼60% in Ba/F3 cells and ∼39% in Sup B15 cells. These two residues are known to be important for regulating cell proliferation, survival and cell motility. In a competitive proliferation assay in the absence of IL3, the proliferation rate of BA/F3 cells infected with Bcr-Abl and Sts – 1 was reduced compared to a Bcr-Abl infected control population. When treated with imatinib the Sts-1 expressing cells showed an approximately 5-fold reduced proliferation rate compared to cells lacking Sts-1, or to imatinib-resistant cells harbouring the Bcr-Abl “gatekeeper mutation” T315I. The expression level of Sts-1 was found to be approximately 3-fold lower in the Sup B15 RT compared to the WT cell line. Regulation appeared to occur at the transcriptional level as shown by quantitive RT-PCR. These results show that Bcr-Abl is a substrate of Sts-1, that this phosphatase modulates Bcr-Abl kinase activity and may abrogate the response to TKI. This suggests that phosphatases may contribute to the development of clinical resistance of Ph+ leukemias to TKIs. Disclosures: Ottmann: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3167-3174 ◽  
Author(s):  
Su Chu ◽  
Melissa Holtz ◽  
Mamta Gupta ◽  
Ravi Bhatia
Keyword(s):  
Growth Factor ◽  
Imatinib Mesylate ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Combined Treatment ◽  
Myelogenous Leukemia ◽  
Imatinib Treatment ◽  
Abl Kinase ◽  
Mapk Activity ◽  
Cd34 Cells

Abstract Chronic myelogenous leukemia (CML) results from malignant transformation of a primitive hematopoietic cell by the BCR/ABL oncogene. The breakpoint cluster region/ABL (BCR/ABL) tyrosine kinase inhibitor imatinib mesylate (imatinib) is highly effective in inducing remissions in CML. However, the effects of imatinib on intracellular signaling in primary progenitor cells are not well described. We show that imatinib exposure resulted in a significant dose-responsive reduction in BCR/ABL kinase activity in CML CD34+ cells. However, imatinib treatment resulted in an increase in activity of p42/44 mitogen-activated protein kinase (MAPK), an important downstream effector of BCR/ABL. Increased MAPK activity was growth factor dependent. Pharmacologic inhibition of MAPK using MAPK/extracellular signal–regulated kinase kinase–1/2 (MEK-1/2) inhibitors significantly reduced CML progenitor proliferation. Combined treatment with a MEK-1/2 inhibitor and imatinib significantly increased suppression of CML progenitors compared with either inhibitor alone. In contrast, imatinib treatment resulted in a small reduction in AKT activity. Combined treatment with a phosphatidylinositol-3 (PI-3) kinase inhibitor and imatinib significantly increased suppression of CML progenitor growth compared with either inhibitor alone. We conclude that inhibition of BCR/ABL kinase activity in CML progenitors by imatinib results in a growth factor-dependent compensatory increase in MAPK activity and in only partial inhibition of PI-3 kinase activity. These mechanisms may contribute to incomplete elimination of CML progenitors by imatinib. (Blood. 2004;103:3167-3174)

The Oncogenic Role of Tumor Suppressor Protein p27 in Ph+ Chronic Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3276-3276 ◽  
Author(s):  
Anupriya Agarwal ◽  
Ryan Mackenzie ◽  
Dorian LaTocha ◽  
Kavin Vasudevan ◽  
Eduardo Firpo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Chronic Phase ◽  
Abl Kinase ◽  
Blastic Phase ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Abstract 3276 Poster Board III-1 Background: Several studies have indicated that BCR-ABL causes cell cycle defects by interfering with the cell cycle regulatory functions of p27, a Cyclin dependent kinase (Cdk) inhibitor and tumor suppressor. Studies in BCR-ABL positive cell lines have shown that BCR-ABL promotes proteasomal degradation of p27 in a pathway that involves the SCFSKP2 ubiquitin ligase, while cytoplasmic mislocalization has been described in primary CML cells. It has been suggested that the principal effect of this cytoplasmic mislocalization is to remove p27 from the nucleus, thereby relieving Cdks from p27 inhibition. However, recent studies have shown that a p27 mutant (p27CK-), that cannot bind to Cdks or Cyclins, actively contributes to oncogenesis. This raises the question as to whether cytoplasmic mislocalization of p27 in CML cells may in fact promote leukemogenesis rather than merely compromise Cdk inhibition. We therefore hypothesized that the net contribution of p27 in CML is to promote leukemogenesis due to the oncogenic activity of cytoplasmic p27. Experimental approach and results: We determined p27 localization in BCR-ABL positive cell lines and CD34+ progenitor cells from newly diagnosed chronic phase CML patients (N=7) and from CML patients in blast crisis (N=2) by immunoblotting of nuclear and cytoplasmic cellular fractions. We found that p27 is predominantly cytoplasmic in most CML cell lines and in CD34+ cells from 8/9 (89%) patient samples, including patients in blastic phase. Cytoplasmic localization of p27 in CD34+ cells from CML patients was also confirmed by immunofluorescence analysis. Further, we observed that inhibition of BCR-ABL kinase by imatinib, an Abl kinase inhibitor increased nuclear p27 in all cell lines tested and in 4/9 patient samples (3/7 chronic phase and 1/2 blastic phase samples). However, we did not observe a substantial change in the cytoplasmic p27 levels. Similar results were obtained in Ba/F3 and 32D murine hematopoietic cell lines expressing BCR-ABL when compared with the respective parental cells. Further, SKP2 was up-regulated in CD34+ cell from CML patients as compared to the normal patients consistent withSKP2 mediated down-regulation of nuclear p27. These data suggest that nuclear but not cytoplasmic p27 levels are predominantly regulated by BCR-ABL kinase activity. To test whether p27 is crucial for BCR-ABL-driven leukemia, we compared leukemogenesis between recipients of BCR-ABL transduced p27+/+ and p27-/- bone marrow. Mice transplanted with BCR-ABL infected p27-/- marrow had significantly longer median survival (70 days, range 48-150 days) compared to recipients of p27+/+ marrow (37 days, range 14-56 days) (p=0.0123). To exclude that this difference was related to the differences in homing and engraftment capabilities of p27+/+ and p27-/- bone marrow cells, we compared short term homing and long term engraftment of p27+/+ and p27-/- bone marrow cells transplanted into wild-type recipients and found no differences. These data suggest that the net contribution of p27 to BCR-ABL-mediated leukemogenesis is positive. Further, to investigate the contribution of nuclear p27 to leukemogenesis, we utilized marrow from p27S10A mice in the murine CML model. In p27S10A mice, p27 is nuclear to to abrogation of the phosphorylation site implicated in nuclear export. We injected BCR-ABL transduced bone marrow cells of p27S10A and p27+/+ mice into wild-type recipients and compared the disease progression. We observed that mice transplanted with BCR-ABL infected p27S10A marrow had significantly longer median survival (28 days, range 23-79 days) compared to the recipients of p27+/+ marrow (23 days, range 21-38 days) (p=0.0139). This data is consistent with nuclear tumor suppressor function of p27. Combined with the data above, this suggests that cytoplasmic p27 promotes BCR-ABL mediated leukemogenesis. Conclusions: Our data suggest that though nuclear p27 functions as a tumor suppressor, the net contribution of p27 in CML might be oncogenic due to an oncogenic role of the increased cytoplasmic p27. Restoring nuclear p27 or reducing cytoplasmic p27 may be therapeutically useful in malignancies with low nuclear and high cytoplasmic p27 expression. Disclosures: Druker: OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. Deininger:Genzyme: Research Funding; BMS: Consultancy; Novartis: Consultancy, Honoraria; Ariad : Research Funding.

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