The Mechanisms of TKI Resistance in CML. BCR-ABL dependent and Independent mechanisms. Newer therapies for TKI resistant CML. ELN 2020 recommendations

1. Mechanisms of TKI resistance in
Chronic Myeloid Leukemia
Dr. Nihar Desai
Moderator : Dr. Anshul Gupta

2. oIntroduction
oDefinition of Resistance
oTypes of Resistance in CML
oPrimary vs Secondary Resistance
oBCR-ABL dependent vs BCR-ABL independent mechanisms
oLeukemia Stem Cells and MRD
oTreatment options for resistant disease
oDrug Combinations to target Leukemia Stem Cells
oConclusion
OVERVIEW OF SEMINAR

3. Introduction
• CML is a clonal myeloproliferative neoplasm of
pluripotent stem cells.
• Hallmark is a single acquired genetic abnormality
which produces a chimeric fusion protein.
• This protein is necessary and sufficient for CML
pathogenesis.
• This fusion protein leads to a constitutive
activation of tyrosine kinase.
• Tyrosine Kinase Inhibitors (TKI) are the frontline
of CML management.
Hematol Oncol Clin N Am 31; doi.org/10.1016/j.hoc.2017.04.007

4. • Imatinib mesylate(Gleevac) was the 1st Tyrosine Kinase
Inhibitor approved for the treatment of CML.
• It has revolutionised the treatment of a once lethal
disease.
• At 8 year follow up of the IRIS trial , patients on the
imatinib arm had :
EFS : 81 %
OS : 93 %
• Despite these excellent numbers , a subset of patients
will develop resistance to this TKI.
Imatinib – Just in TIME

5. Mr. A, a 50 year old man, is diagnosed with chronic phase CML.
He is started on TKI therapy (Imatinib 400mg OD) after his BCR-ABL is + (p210)
He is monitored for response at :
o3 months : BCR-ABL qPCR : 8%
o 6 months : BCR-ABL qPCR : 4%
o 12 months : BCR-ABL qPCR : 3%
What are the possible reasons for imatinib failure ?
How do we manage this patient ?
Clinical Case Scenario 1

6. Timepoints Optimal Warning Failure
Baseline NA High risk ACA, High ELTS NA
3 months ≤ 10 % >10% >10% / No CHR
6 months ≤ 1% >1–10% >10% / Ph + >35%
12 months ≤ 0.1% >0.1–1% >1% / No CCyR
Anytime ≤ 0.1% >0.1–1%, loss of MMR >1% , resistance mutations,
high-risk ACA
Milestones for treating CML – ELN 2020
Leukemia 2020 ; doi.org/10.1038/s41375-020-0776-
2

7. • Resistance is defined as the failure to achieve time dependent endpoints upon
initiation of TKI therapy or loss of these attained responses.
• Despite good initial response , TKI therapy eventually fails in ~ 30% of patients.
Trial Name CcyR rate at 18 months (400mg Imatinib dose)
IRIS 75%
TOPS 76%
German CML IV 69%
Leukemia (2011) ; doi:10.1038/leu.2010.238
Resistance to Tyrosine Kinase Inhibitors
~30 % patients
do not achieve
CCyR

8. Primary Resistance vs Secondary Resistance
Primary / Intrinsic Resistance Secondary / Acquired Resistance
denovo lack of treatment response
Primary Hematological
resistance (2-4%)
Primary Cytogenetic
resistance (15-25%)
Loss of an achieved clinical /molecular response
Most often due to BCR-ABL independent pathways Most often due to BCR-ABL dependent pathways

9. Classification of TKI Resistance Mechanisms
BCR-ABL Dependent
mechanisms
BCR-ABL Independent
mechanisms
Mutations in the ABL
Kinase Domain
(most common mechanism of resistance)
Increased expression of BCR-ABL1
Pharmacological:
Poor intestinal absorption
Drug interactions
Plasma protein binding
Poor Compliance
Drug Influx and Efflux pumps
Alternate Pro-survival pathways
Clonal Evolution &
Epigenetic dysregulation
Hematol Oncol Clin N Am 31; doi.org/10.1016/j.hoc.2017.04.007
Persistence of MRD
is also attributed to
this mechanism.

10. BCR-ABL Dependent Resistance

11. Mechanism Of Action Of TKIs
Handbook of Experimental Pharmacology ; doi.org/10.1007/164_2017_81

12. Types of Tyrosine Kinase Inhibitors
Types Of Tyrosine Kinase Inhibitors
Type I TKI Type II TKI
Bind to the kinase in “active”
conformation (DFG in)
Less stringent binding requirement
Less selective
Reduced chance of mutational escape
Eg. Dasatinib / Bosutinib
Bind to the kinase in “inactive”
conformation (DFG out)
More stringent binding requirement
More selective
Higher chance of mutational escape
Eg. Imatinib/Nilotinib/Ponatinib
Hematology Oncol Clin North Am. 2017 ; doi:10.1016/j.hoc.2017.04.007
Aspartate-
Phenylalanine-
Glycine
(DFG)

13. Hematology Oncol Clin North Am. 2017 ; doi:10.1016/j.hoc.2017.04.007
Types of Tyrosine Kinase Inhibitors
In
Closed Position
In
Open Position
Known as
Induced Fit
State
H Bond formation
with T 315

14. • The most common mechanism of resistance to TKI therapy (Point Mutations)
• >100 mutations are now identified at various structural subunits of the Kinase
• Imatinib has the broadest spectrum of mutational vulnerabilities (>50 mutations)
• 1st and 2nd generation TKI need to form a Hydrogen Bond with Threonine 315 to
bind at the TKI binding site.
Kinase Domain Mutations
TKI binding site ATP binding site (P Loop) Activation Loop (A Loop) Catalytic Domain (C Loop)

15. doi.org/10.1016/j.drudis.2019.05.007
Kinase Domain Mutations
Stabilizes the
“active” conformation
of the kinase

16. 2% 4% 12% 14%6% 8% 10%
T 315 I (Gatekeeper Mutation)
Y 253 F / H
E 255 D/K/R/V
M 351 T
G 250 A/E
F 359 C/L/V
H 396 P/R
Percentage of total mutations
o Imatinib Binding Site
o P loop or ATP Binding Site
o Catalytic Domain
o Activation Loop
7 Most Common Mutations in the Kinase Domain
Jane F Apperley; Lancet Oncol 2017

17. • Clinically , the type of Kinase Domain mutation guides the selection of subsequent TKI
Kinase Domain Mutations
TKI Contraindicated Mutations
Dasatinib T315I/A ; F317L/V/I/C ; V299L
Nilotinib T315I , Y253H ; E255K/V ; G250E
Bosutinib T315I ; V299L ; G250E ; F317L
Ponatinib Compound Mutations
Compound Mutations : 2 mutations within the same BCR-ABL protein
Sequential TKI
therapy leads to
acquisition of
Compound
Mutations

18. Methods to Detect Kinase Domain Mutations
Method Sensitivity Advantage Disadvantage
Direct Sequencing Lowest o Mutation characterization o Lowest sensitivity
Pyrosequencing High o High sensitivity and specificity
o Quantitate the KD mutation
burden
o Prior knowledge of
mutations required
o Labour intense
Denaturing-HPLC High o High throughput
o Cost effective
o Suited for large scale screening
o Prior knowledge of
mutations required
ASO-PCR Very High o Highly sensitive
o Quantitate the KD mutation
burden
o Prior knowledge of
mutations required
o False positives
Mary Alikian ;American Jo Haematology ; DOI: 10.1002/ajh.22272

19. Timepoint NCCN recommendation Comment
At diagnosis
o Only for patients presenting
with Accelerated Phase or
Blast Crisis.
o Extremely unusual to find a mutation by
conventional methods in CP-CML patients
Failure to reach
response “milestones”
o Recommended o Primary resistance is usually due to BCR-ABL
independent pathways.
o Low likelihood of finding TKD mutations
Loss of Response
(Hematological or
Cytogenetic relapse)
o Recommended o Secondary resistance is usually BCR-ABL
dependent.
o High likelihood of finding TKD mutations
Loss of MMR o Recommended o Helps guide choice of subsequent TKI
Disease progression
(on TKI)
o Recommended o Secondary resistance is usually BCR-ABL
dependent.
o High likelihood of finding TKD mutations
When to test for Kinase Domain mutations ?
NCCN Guidelines for CML ; 2020

20. o The patient was screened for a Tyrosine Kinase Domain mutation (Sanger Sequencing)
o He was found to have a G 250 E mutation.
oHe was started on T. Dasatinib 70mg BD dose and was followed up after 1 month.
o His BCR-ABL qPCR was 1.03% on the IS .
o He was continued on Dasatinib and attained an MMR 6 months after starting
Dasatinib.
Clinical Case Scenario 1

21. Other Reasons for TKI Failure
Drug Influx and Efflux Pumps
Drug interactions / CYP3A4 Polymorphisms
Increased Expression of BCR-ABL
BCR-ABL independent mechanisms
Poor Compliance

22. Drug Influx and Efflux Pumps
OCT 1
Ph +
cell
OCT 1 levels impart imatinib resistance.
OCT is not involved in cellular uptake of 2nd and 3rd generation TKI’s
Low OCT1 levels and imatinib trough plasma levels <1200ng/ml benefit from imatinib dose escalation.
Deborah L. White; Blood 2007
Imatinib
OCT : Organic Cationic Transporter

23. Difference in achievement of MMR by 24 months in
Low vs High OCT 1 activity
Drug Influx and Efflux Pumps
Deborah L. White; Blood 2007
o OCT-1 activity : Important determinant of response
to Imatinib.
o Accessing OCT1 levels before therapy identifies
those who are likely to have suboptimal response
to imatinib.
o This subset of patients with Low OCT1 levels may
benefit from dose escalation strategies.
o OCT1 levels at baseline are not part of current
routine clinical practice.

24. oABCB-1/ Pgp was the 1st efflux pump identified (1990)
o ABCB-1 as a mechanism of TKI resistance was first recognised by Mahon et.al
o CML patients in AP/BP phases have higher expression of ABCB1.
o The efflux of imatinib via ABCB1 is less efficient than efflux of other anticancer
drugs.
o The role of ABCB-1 in clinical resistance is unclear.
Drug Influx and Efflux Pumps
Jane F Apperley; Lancet Oncol 2007

25. Drugs Imatinib Dasatinib Nilotinib
Pantoprazole Inhibits Pgp/ intracellular
levels
solubility & absorption __
Ranitidine Inhibits Pgp/ intracellular
levels
solubility & absorption __
Metformin OCT1 level intracellular
levels
__ __
Rifampicin
Carbamazepine
Phenytoin
Voriconazole
Fluconazole
Drug Interactions
Amina Haouala ; Blood 2011

26. Increased BCR-ABL1 expression
Gene
amplification
Duplication
of
Ph
Differential
regulation of
oncogene
transcription
David J. Barnes ; Cancer Res 2005
Increased BCR-ABL 1 expression

27. Why is Blast Crisis More Resistant to TKI therapy ?
• BCR-ABL expression is higher in progenitor cells of Blast crisis > CP
• Phosphorylation of STAT5 is higher in progenitor cells of Blast Crisis > CP
• The rate at which a cell develops TKI resistance is determined by basal BCR-ABL levels
• Over expression of BCR-ABL precedes development of KD mutations
• Expression of KD mutations coincides with reduction in BCR-ABL
expression
David J. Barnes ; Cancer Res 2005

28. Mr. B, a 50 year old man, is diagnosed with chronic phase CML.
He is started on TKI therapy (Imatinib 400mg OD) after his BCR-ABL is + (p210)
He is monitored for response at :
o 3 months : BCR-ABL qPCR : 18%
o6 months : BCR-ABL qPCR : 21%
• No Kinase Domain mutation is detected and patient is compliant with therapy.
• Review of his drug chart does not reveal any drug with potential interaction
with his TKI.
• He is started on T.Nilotinib 400mg BD
Clinical Case Scenario 2
PRIMARY RESISTANCE

29. Mr C , 60 year old male , diabetic (well controlled on OHA) is on T. Imatinib 400mg OD
for his Chronic Phase CML.
He has tolerated therapy well and has achieved MMR at 12 months.
On follow up at 15 months his BCR-ABL qPCR is 0.28%
He is continued on the same TKI and at 18 months his BCR-ABL qPCR is 0.86%
What are the reasons for low level persistence of BCR-ABL ?
Are Leukemic Stem Cells at play ??
Clinical Case Scenario 3

30. BCR-ABL Independent Resistance

31. BCR-ABL
dependent
Pathways
(60%)
BCR-ABL
independent
Pathways
(40%)
BCR-ABL Independent Mechanisms of Resistance
Alternate Pro-Survival pathways are an important
mechanism of BCR-ABL independent resistance.
The common alternate pathways are
Leukemia
Cell
Survival
PI3K/AKT
JAK/STAT RAS/MAPK
RAF/MEK/EFK
Overt
Resistance
&
Persistence of
MRD

32. BM Microenvironment & STAT 3 Mediated Resistance
Produce Cytokines like
IL6, G-CSF, VEGF
Increased
phosphorylation of
STAT 3
Increased expression
of BCL-xl, MCL1 &
Survivin
HS 5 conditioned
media
K 562
CML cell line
Nadine N. Bewry; doi:10.1158/1535-7163.MCT-08-0314
Apoptotic Arrest
CM protects CML
Cell Line from TKI
mediated apoptosis
Cell Proliferation,
Angiogenesis &
Cell Survival

33. PI3K / AKT/ mTOR Pathway
M Wagle ; Leukemia (2016); doi:10.1038/leu.2016.51
A Burchert ; Leukemia (2005) ;doi:10.1038/sj.leu.2403898
Imatinib
naïve cell
Activation of PI3K/AKT/mTOR
Pathway
Early Imatinib resistance
Overt Imatinib resistance
and
Development of KD mutations
Imatinib
FOXO are downstream
transcription factors
Phosphorylation of FOXO1 by
AKT l/t cytoplasmic retention
and proteosomal degradation
Elevated FOXO1 = BCR-ABL
independent Resistance
P
P
P
P
P
FOXO : Forkhead Box Transcription Factors
Tumour
Suppression &
Cell Death

34. RAF/ MEK/ ERK Pathway
Leyuan Ma; Cancer Journal 2014
o PRKCH levels were elevated in
BCR-ABL independent resistance
cell lines & LSC.
o PRKCH encodes PKCη which
activated C-RAF.
o Imatinib + Trametinib
synergistically kills these IM
resistant cells and prolonged
survival in mouse models.
o CML LSC also express high levels
of PRKCH which explains their
intrinsic IM resistance
Elevated PKCn levels lead to IM resistance in CML and CML stem cells
Trametinib

35. Nucleo-Cytoplasic Transport Complex – XPO1 and RAN
XPO and RAN
promote shuttling through Nuclear Pore Complex
XPO = Increased export of nuclear tumour suppressor proteins
XPO is also associated with drug resistance in Multiple Myeloma
CLL and T-ALL
SALINEXOR
has shown
pre-clinical
activity in CML
mouse models
Khorashad et.al ; Blood 2015 ; DOI 10.1182/blood-2014- 08-588855

36. Characteristics of Leukemia Stem Cells
Quiescence
BCR-ABL independent survival
Resistance to TKI therapy
Characteristic Immunophenotype

37. Leukemia Stem Cells
Leukemic Stem Cell
Leukemic Cell
TKI
discontinuation
(TFR)
Relapse
LSC Targeted
Therapy + TKI
Disease
Eradication
CML

38. CD 26 LSC +
BCR-ABL +
29%
CD 26 LSC +
BCR-ABL -
38%
CD 26 LSC -
BCR-ABL +
7%
CD 26 LSC -
BCR-ABL -
26%
Bocchia M et.al ; Frontiers in Oncology 2018
Loscocco F et.al ; Frontiers in Oncology 2019
o CML-LSC have aberrant expression of CD 25 /CD26(DPP4)/
CD33 and CD123
o These transcriptionally quiescent LSC are not detected by
BCR-ABL qPCR.
o q-RT-PCR accurately measures transcript level but cannot
estimate the actual CML cell number
o The persistence of CD26+ LSC in the peripheral blood of CML
patients with MMR elegantly confirms that LSC are resistant
to TKIs (66% patients had persistence)
o ~ 30% are CD26 LSC – : stem cell exhaustion / poor
sensitivity of test
Immunophenotype of Leukemic Stem Cells
PB CD26+ LSC & BCR-ABLIS
in the cohort of TFR patients
Median number of CD26+ LSC : 0.015/μL

39. PP2A and Leukemia Stem Cell Survival
Tumour
Suppressor
Protein
PP2A
Leukemia
Stem Cells
JAK2 / β Catenin
Tumour
Suppressor
Protein
PP2A
Leukemia
Stem Cell
Survival
JAK2 / β Catenin
BCR-ABL
FTY 720 (Fingolimod)
PP2A activating Drug
JAK2 / β Catenin Loscocco F et.al ; Frontiers in Oncology 2019
Miki Kiyota; Blood 2012
PP2A : Protein Phosphatase 2A

40. Role of Epigenetics in Resistance
SIRT1
deacetylase
Acy
P53
FOXO
KU 70
Cell Survival
Cell Cycle Arrest
DNA Repair
Acquisition of
mutations
LSC Persistence
TKI Resistance
Christopher L. Brooks; Nature Reviews ; 2009
Other Epigenetic Pathways involved in TKI
Resistance
o Polycomb Repressive Complex 1 and 2 (PRC)
o Aberrant EZH2 activity
L
S
C
S
U
R
V
I
V
A
L
SIRT1 (Sirtuin 1) Type 1
Histone Deacetylase

41. Other BCR – ABL independent pathways
Activation of SRC kinase
P 53 / MYC
Hedgehog Pathway
IL2 / CD25 signalling circuit
RAD 21 Heat Shock Proteins
Hypoxia Inducing Factor 1 a
King Pan Ng; Blood 2014 ;DOI 10.1182/blood-2013-07-511907
Loscocco F et.al ; Frontiers in Oncology 2019
Hypoxia mediates TKI Resistance
Hypoxia enhances LSC survival

42. The number of theoretical synergistic lethality approaches is destined to grow as
more resistance mechanisms are uncovered.
The clinical relevance of most remain unknown as of today
What to target ?

43. Cost
Poor
Physician-
Patient
Relation
Poor Compliance
Adverse
Events
Treatment
Duration
Reasons For Poor Compliance
Compliant
Compliant
Non-Compliant
Non-Compliant
Implications of Poor Compliance
Lucien Noens; Haematologica 2014

44. Newer Treatment Options for
TKI Resistant CML

45. Asciminib (ABL 001)
Allosteric Inhibitor
Binds to Myristoyl Site of BCR-ABL
Locks the BCR-ABL in an
Inactive Conformation
Phase I , dose escalation study in patients
with CML Resistant to ≥ 2 TKI
T.P.Hughes; N Engl J Med 2019
Patients with
T315I were
included
Grade 3
toxicity
Lipase &
Cytopenias
MMR at 12
months: 48%
(Including those with
T315I)

46. Mechanism of Action of Asciminib
T.P.Hughes; N Engl J Med 2019

47. Axitinib – Repurposed for T315I Resistant CML
Axitinib – VEGF-R inhibitor
Approved for Renal Cell Carcinoma
Selectively inhibits T315I BCR-ABL
(Drug Sensitivity & Resistance Profiling)
Has mutation
selective binding
ability
Dose Response curve of various TKI on
T315I mutant BCR-ABL

48. Axitinib – Repurposed for T315I Resistant CML
Phase I/II trial is ongoing at MDACC
To study the MTD and Adverse Effect
Profile of Axitinib + Bosutinib
combination in TKI resistant CML
Bosutinib doesn’t inactivate its own
metabolizing enzymes(unlike other TKI)
Thus is a safer option for combination with
Axitinib
MDACC ; P.Bose et.al ;clinicaltrials.gov
Results of this trial are awaited

49. Other Kinase Inhibitors for Resistant CML
Bafetinib (BCR-ABL1 / Lyn)
Rebastinib (BCR-ABL1 / TIE2)
Danusertib (Aurora Kinase Inhibitor)
Radotinib (BCR-ABL1)
Except for Radotinib which is approved in South Korea as 2nd line treatment for CML , all the others have
been sidelined due to poor efficacy in early phase trials.

50. Drug combinations to eradicate LSC – make TFR a reality
CML in DMR
(Deep Molecular Response)
60% maintain TFR 40% don’t maintain TFR
Combine TKI
with other drugs
to eliminate LSC
Role of Immune
Surveillance in
maintaining TFR
Hematology Oncol Clin North Am. 2017 ; doi:10.1016/j.hoc.2017.04.007

51. Drug Combination Target Rationale Phase Patient
Population
Primary Endpoint Comments
IFN + Nilotinib
(PETALs)
Immune
Activation
Restoration of
immune surveillance
III Newly dx
CML-CP
MR 4.5 at 12
months
Recruiting
Ruxolitinib
+
Nilotinib
JAK/STAT Elimination of CD34+ LSC
& inhibiting
downstream signalling
I/II CML-CP
w/o CMR
CMR at 24 months Recruiting
Pioglitazone
+
Imatinib
(ACTIM)
PPAR γ Pioglitazone
downregulates STAT5A/B
LSC survival
II CML-CP in
MMR
MMR to MR4.5
over 12 months
Cumulative
incidence of
MR 4.5 was
56%
Pioglitazone
+
TKI
(PIO2STOP)
PPAR γ Pioglitazone
downregulates STAT5A/B
LSC survival
II CML-CP
after failure
of 1st TFR
attempt
Treatment free
survival after
TKI + Pio
discontinuation at
24months
Recruiting
Drug combinations to eradicate LSC – make TFR a reality
Curr Opin Hematol 2017; doi:10.1097/MOH.0000000000000403

52. Homoharringtonine (HHT) and Omacetaxine
HHT/ Omacetaxine
Plant alkaloid/ derived from Cephalotaxus species
Inhibit Ribosomal Protein Translation
Induce Apoptosis via of oncoproteins ,
activation of caspase
US FDA approved for
CML-CP/AP after
failing ≥2 TKI
(2012)
Jorge Cortes et.al ; Blood 2012
o Active against CML
with T315I mutation
o Can be administered
subcutaneously

53. PD-L1 inhibitors in CML
CML is sensitive to immunological control
PD-L1 upregulation is an
immunological escape for CML LSC
Phase Ib trial of
Dasatinib + Nivolumab
in CML-CP/AP failing ≥ 2TKI is awaiting results
P.E.Westerweel et.al ; Frontiers in Oncology 2019

54. Failure Of TKI Therapy
Take Home Message
Compliance
Drug Interactions
Drug Resistance

55. Take Home Message
BCR-ABL dependent BCR-ABL independent

56. Take Home Message
Leukemia Stem Cells & Hypoxic BM niche

57. Take Home Message
Newer Treatment Approach
Asciminib
Axitinib
Danusertib
PD-L1 inhibitors
+
TKI’s
Interferon
+
TKI’s
Ruxolitinib
+
TKI’s
Pioglitazone
+
TKI’s
Radotinib