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Mechanisms of resistance in CML Vernon Louw Clinical Haematology June 2010 1 Molecular basis CML 1882 – Sir Arthur Conan Doyle published a case in the Lancet Thought that the leukemia was caused by malaria Nowell PC et al. Science 1960; 132:1497 www.siteman.wustl.edu 6 Quintas-Cardama et al. Blood 2009 7 • p210BCR-ABL - CML • p190BCR-ABL - ALL • p230BCR-ABL - CNL UpToDate v14.1 Substrate Effector Bcr-Abl ADP P PPP ATP PPP SIGNALING Savage et al. NEJM 2002; Scheijen et al. Oncogene 2002 9 Quintas-Cardama et al. Blood 2009 10 Net effect •Enhanced cellular proliferation •Resistance to apoptosis •Oncogenesis 11 Quintas-Cardama et al. Blood 2009 12 Imatinib 13 Bcr-Abl P ATP PPP Imatinib mesylate SIGNALING Savage et al. NEJM 2002 14 Resistance 15 16 Primary resistance Failure to achieve responses at defined endpoints 15 – 25% Secondary (acquired) resistance Loss of previously achieved milestones 20-25% 17 1/3 patients may need alternative Rx to imatinib due to: resistance intolerance 18 Mechanisms resistance BCR-ABL dependent BCR-ABL independent 19 BCR-ABL dependent mechanisms 20 BCR-ABL dependent BCR-ABL overexpression BCR-ABL mutation 21 BCR-ABL dependent *BCR-ABL overexpression In vitro demonstration Cell lines from CML blast crisis pts developed resistant clones serial passages in imatinib-containing cultures elevated ABL kinase activity due to genetic duplication of Bcr-Abl sequence Bcr-Abl Le Coutre et al. Blood 2000; Gorre et al. Science 2001 22 BCR-ABL dependent *BCR-ABL overexpression More commonly seen in advanced phase disease Uncommon cause of resistance Bcr-Abl Le Coutre et al. Blood 2000; Gorre et al. Science 2001 23 BCR-ABL dependent *BCR-ABL mutations Imatinib binds to the Abl enzyme in the inactive, closed formation Relatively rigid structural requirement for entry and binding to the ATP-binding site 24 BCR-ABL dependent *BCR-ABL mutations Point mutations seen in 40 - 90% of resistant patients > 100 identified Mutated Bcr-Abl 25 BCR-ABL dependent *BCR-ABL mutations P-loop (residues 244 to 255) docking site for phosphate moieties of ATP most frequent (48% of mutations in resistant cases) Destabilize conformation required for imatinib binding Branford S et al. Blood 2003; Jabbour E et al. Leukemia 2006 26 BCR-ABL dependent *BCR-ABL mutations P-loop controversy poor vs good outcome Branford S et al. Blood 2003; Jabbour E et al. Leukemia 2006 27 BCR-ABL dependent *BCR-ABL mutations Catalytic domain mutations (residues 350 to 363) Can affect imatinib binding Branford S et al. Blood 2003; Jabbour E et al. Leukemia 2006 28 BCR-ABL dependent *BCR-ABL mutations A-loop (residues 381 to 402) key regulatory element of the ABL1 kinase domain Can adopt an open/active or closed/inactive conformation mutations prevent the kinase from adopting the inactive conformation needed for imatinib binding 29 BCR-ABL dependent *Bcr-Abl mutations Imatinib binding site T315I seen in 4-19% of pts with imatinib failure impairs imatinib binding to kinase domain complete resistance to imatinib and 2nd generation TKIs Med survival 12/12 30 BCR-ABL dependent *BCR-ABL mutations Mutations do not always explain resistance May activate different downstream signalling pathways Very sensitive techniques may detect mutations that are not clinically relevant • Mutations have different transforming capabilities, e.g. • Y253F > E255K (P-loop) > unmutated BCR-ABL1 > T315I (binding site)> H396P (activation loop) > M351T (catalytic domain) 31 Mutations at seven residues account for 85% of all resistance-associated mutations P-loop M244V, G250E, Y253F/H, E255K/V Imatinib binding site T315I Catalytic domain M351T, F359V 32 Mutations have been documented prior to TKI Rx Suggests that pre-existing mutations do not acquire a survival advantage until subjected to a TKI No difference in pre-existing mutational status in pts who have relapsed 33 BCR-ABL independent mechanisms 34 BCR-ABL independent Leukemia cell related Patient-related (Pseudoresistance) Heterogeneity of CML cells Reduced levels of hOCT1 Poor compliance Pharmacological Poor absorption GIT Drug interactions Binding with plasma components Increased levels of exporter (PgP) Alternative signalling pathway activation Epigenetic modification Clonal evolution Quiescent stem cells 35 BCR-ABL Independent *Drug concentration 36 BCR-ABL Independent *Drug concentration • Possible association between trough plasma levels and CCyR and MMR rates • No prospective data • Requires further validation 37 Compliance 38 THE GOAL ≥ 95% COMPLIANCE FOR PATIENTS WITH LIFETHREATENING DISEASES 39 Compliance mg taken / mg prescribed 40 Suboptimal compliance Doses < 300-400mg Plasma concentration too low Malignant cells not eliminated 41 In developed countries compliance with prescribed medication WHO reports compliance rates: 51% with antihypertensives averages only 50% in pts with chronic illness 40%-70% with antidepressants 43% with asthma medications 37%-83% with HIV/AIDS medications Estimated that 3-10% of pts never fill their prescriptions, and >30% of all prescription refills are never obtained Haynes, 2002; WHO 2003 42 125,000 AMERICANS DIE EACH YEAR BECAUSE OF MEDICATION NON-COMPLIANCE 43 PRESCRIPTION COMPLIANCE 3502 patients (CML and GIST) 14 months of pharmacy data analyzed Average compliance over 14 months of observation was 75% Only 41% of patients more than 90% compliant On average patients consume about 20% less than their prescribed dose CML patients more compliant than GIST Tsang et al. ASH 2006 44 • Men and women aged 55-70 most compliant • Men < 25 and women 35-45 least compliant Tsang et al. ASH 2006 45 • Pts initially treated with 400 or 300mg/d had the highest compliance (89%) Tsang et al. ASH 2006 46 3500 Patients 3000 COMPLIANCE DECAY 2921 2500 2000 1500 1000 685 500 0 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11 11-12 12-13 13+ Months • Adherence* is near 100% through month 4 • Declines from 94% at month 5 to 23% at month 14 • Only 57% of patients were still on imatinib @ 12 months *Time on therapy without significant gaps in refills. Tsang J et al. J Clin Oncol. 2006;24:330s. Abstract 6119. 47 Compliance decay – why? Unpleasant side-effects Pts do not fully understand importance of taking medication prescribed Forget to take pills Cancer patients overestimate compliance to oral Rx by a factor of 2 in discussion with physicians 48 Intake Similar time each day. With food With fulll glass of water. Can put tablets in 1/2 cup of water or apple juice Drink plenty of noncaff einecontaini ng liquid Take a missed dose ASAP. 49 Absorption Substrate for intestinal ATPbinding cassette transporters ABCB1 and ABCG2 Extent absorption may be affected by Primarily absorbed from small intestine • GI abnormalities or disease • Drugs interfering with transporters • NOT affected by food 50 Distribution Circulating imatinib 95% bound to plasma proteins Mainly albumin and alpha-1-acid glycoprotein (AAG) AAG levels may influence imatinib pharmacokinetics Individual differences in AAG and plasma protein binding may explain some of the inter-patient variability in the observed total plasma exposure to imatinib. 51 BCR-ABL Independent *Drug binding • α1-AGP binds basic drugs • Sequesters imatinib in plasma • Reduces ability of imatinib to inhibit the ABL kinase 52 BCR-ABL Independent *Drug binding • • • Role in resistance implicated in mouse models Elevated AGP in tumourbearing mice associated with PD despite imatinib Rx Competitive binder erythromycin reversed the effect • AGP levels shown to be elevated in pts with CML • Further increased with disease progression • Lack of clinical data demonstrating variable outcomes in patients • Clinical role of AGP remains undefined 53 Distribution Rapidly and extensively distributed into tissues Minimal penetration to the central nervous system 54 Metabolism Metabolized by cytochrome P450 (CYP) 3A4 and 3A5 Imatinib exposure influenced by • drug-drug interactions • CYP3A4 inhibitors or inducers Major metabolite, Ndesmethyl metabolite (CGR74588) • Similar activity to parent drug 55 Metabolism CYP3A4 inhibitors: drugs that may INCREASE imatinib plasma concentrations CYP3A4 inducers: drugs that may DECREASE imatinib plasma concentrations Clarithromycin Carbamazepine Erythromycin Phenytoin Ketoconazole Dexamethasone Itraconazole Phenobarbitone Grapefruit Rifampicin 56 Elimination Therefore once daily dose • Faeces (predominant) • Urine Elimination half-life imatinib +/18 hours Can be dosed twice daily to Steady state reached within one week 57 BCR-ABL Independent *Drug import hOCT1 responsible for imatinib uptake in cell hOCT1 expression and inhibition correlated with intracellular imatinib concentration Differential in vitro susceptibility to imatinib based upon expression and function of hOCT1 Thomas et al. Blood 2004; White et al. Blood 2006 58 BCR-ABL Independent *Drug import Reduced levels of hOCT1 found in BM mononuclear cells in pts failing to achieve at least a minor CyR after 10/12 on imatinib Study of 70 pts demonstrated a significant difference in CCyR @ 6/12, PFS and OS based upon pre-Rx hOCT1 level TOPS trial showed reduced MMR rate @ 12/12 in pts with lower hOCT1 levels receiving standard-dose imatinib compared with highdose imatinib SNPs of hOCT1 associated with poor response to imatinib Crossman et al. Blood 2005; Wang et al. Clin Pharmacol Ther 2008; White et al. Blood 2008 [abstract] 59 BCR-ABL Independent *Drug efflux • • Increased expression PGP efflux pump (MDR gene) associated with exposure to increasing doses of imatinib • Associated with 6-fold amplification of the BCR-ABL gene product • Not differentiated in study between effect of amplification and PGP expression on resistance SNPs in MDR gene with different MMR rates in pts on imatinib Mahon et al. Blood 2000; Dulucq et al. Blood 2008 60 BCR-ABL Independent *Drug efflux • PGP inhibitors could modulate the activity of the export protein and restore susceptibility to previous resistant cell lines • siRNA also successfully used in vitro to • Restore imatinib susceptibility • Increase intracellular imatinib concentration Che et al. Cancer letters 2002; Kotaki et al. Cancer letters 2003; Rumpold et al. Exp Hematol 2005; Widmer et al. Leukemia 2007 61 BCR-ABL Independent *Drug efflux • • Other studies failed to find: • Association clinical response and PGP expression levels • Difference PGP levels in pre-Rx and post-Rx BM mononuclear cells in pts achieving CCyR vs pts with less than a minor CyR Role of PGP as a relevant marker of resistance in population studies questioned Lange et al. Blood 2003; Crossman et al. Blood 2005 62 BCR-ABL Independent *Alternative signaling pathway activation Quintas-Cardama et al. Blood 2009 63 BCR-ABL Independent *Alternative signaling pathway activation • SRC family kinases LYN and HCK upregulation associated with: • Imatinib resistance • Lymphoid blast phase • Stabilizes BCR-ABL1 in the active conformation to which imatinib can not bind 64 BCR-ABL Independent *Epigenetic modification • Imatinib resistance shown in a cell line through the concurrent upregulation of Class I and II deacetylases (HDACs) and downregulation of histone acetyltransferases • Rx of these cells with an HDAC inhibitor restored the acetylation pattern of several proteins and altered the apoptotic threshold • TKIs + HDAC inhibitors showed a synergistic effect on the level of apoptosis in cells from CML pts Lee et al. J Pharmacol Exp Ther 2007; Kircher et al. Eur J Haematol 2009; Fiskus et al. Blood 2006 65 BCR-ABL Independent *Clonal evolution • Usually indicates transformation to an advanced phase • Most common: • Additional Ph+ • Trisomy 8 • Isochromosome 17q Secondary genetic alterations Clonal evolution von Bubnoff et al. Leukemia. 2003;17:829. 66 BCR-ABL Independent *Clonal evolution • Decreased response rate to imatinib • Increased haematological relapse and reduced OS • Reflects genetic instability of CML progenitors • Haematological resistance more common with clonal evolution(58%) than with BCR-ABL1 mutations (45%) • Clonal evolution more common in blast phase (73%) than kinase domain mutations (30%). • More kinase domain mutations in pts with clonal evolution (58%) than in pts with Ph+ metaphases only von Bubnoff et al. Leukemia. 2003;17:829. 67 BCR-ABL Independent *Quiescent stem cells • Primitive CD34+CD38cells in G0 phase cell cycle • <1% of CD34+ cells at dx • Inherent insensitivity to imatinib • Postulated to sustain the disease with the constant potential to escalate Mughal et al. Frontiers in Bioscience 2006 68 BCR-ABL Independent *Quiescent stem cells • Resistance QSCs multifactorial: • Altered drug influx and efflux mechanisms (low OCT1 and high PgP) • • Increased BCR-ABL1 transcript levels in the absence of BCR-ABL1 gene amplification Decreased BCR-ABL1 transcript degradation • Cell migration defects due to down-regulation of CXCR4 by BCR-ABL1 overexpression • Promotes the migration of CML cells to BM stroma • Causes G0-G1 cell cycle arrest • Preserve survival of quiescent CML progenitor cells 69 Summary Bixby et al. Hematology 2009 70 Further investigation needed into the roles of: • Genomic instability • Faulty DNA repair • Quiescent stem cells • Tumor suppressors • Cross-talk between BCR-ABL1 and oncogenic signaling pathways 71 Good news • Many novel agents directed at every area of the pathogenetic pathway in development 72 73 Mechanisms resistance • Bcr-Abl-independent • Successful inhibition Bcr-Abl kinase, yet pt resistant to imatinib • • Most common in primary resistance Bcr-Abl-dependent • Reactivation of Bcr-Abl kinase after successful inhibition before • Most common in pts who relapse on Rx with imatinib 74