Leukemogenesis:

acute myeloid leukemia (AML)

Jordi Esteve
Hospital Clínic, Barcelona
 Acute  Myeloid  Leukemia  (AML)
• Concept  of  AML
• Leukemic  hierarchy:  leukemia  stem-­‐cell  model
• Muta:onal  landscape
• Biography  of  leukemogenic  muta:ons  of  AML:  from
pre-­‐clonal  lesions  to  clonal  evolu:on
• Epigene:c  disturbance
• Role  of  microevironment
• Transla:on  to  clinic:  risk-­‐adapted  therapy,
unravelling  therapeu:c  targets
 Acute leukemia: behind a name

– Biological meaning
• Enhanced cell kinetics- high proliferation
• Origin in immature hematopoietic progenitors

– Clinical meaning
• Rapid onset of symptoms
• Risk of threatening clinical events
 Acute leukemia: main subtypes

– Acute Myeloid Leukemia (AML)

– Acute Lymphoblastic Leukemia (ALL)

– Acute Leukemia of ambiguous lineage

- Acute indifferentiated leukemia
- Mixed phenotype acute leukemia
- Natural killer-cell leukemia

WHO Classification, 2008

 AML: operative definition (WHO 2008)

• Clonal expansion of myeloid blasts in bone marrow (BM),

peripheral blood (PB) or other tissue

• Minimum threshold of blast cells for AML diagnosis (BM):

– >20% blasts (WHO, 2008)
– Any blast count in cases with RUNX1/RUNX1T1,
CBFb/MYH11 or PML/RARA rearrangement
 Hematopoiesis in AML patients

B cell

T cell

NK cell

DC

HSC AML  BLASTS

Erythrocyte

Platelet
 Acute  Myeloid  Leukemia  (AML):
behind  an  opera2ve  defini:on
• Gene:cally  heterogeneous  clonal  disorder
• Origin  in  hematopoie:c  progenitor  cells
• Due  to  accumula:on  of  soma:c  acquired
gene:c  &  epigene:c  altera:ons
• Altered  mechanisms  of  self-­‐renewal  (↑),
prolifera:on  (↑)  &  differen:a:on  (↓)
• Resul:ng  in  an  impaired  leukemic
hematopoie:c  hierarchy:  the  leukemia  stem-­‐
cell  model
 Acute  Myeloid  Leukemia  (AML)
• Concept  of  AML
• Leukemic  hierarchy:  leukemia  stem-­‐cell  model
• Muta:onal  landscape
• Biography  of  leukemogenic  muta:ons  of  AML:  from
pre-­‐clonal  lesions  to  clonal  evolu:on
• Epigene:c  disturbance
• Role  of  microevironment
• Transla:on  to  clinic:  risk-­‐adapted  therapy,
unravelling  therapeu:c  targets
 WHO  classifica:on  of  AML  (II):  an  increasing
repertoire  of  molecularly-­‐defined  en::es
I. AML with recurring genetic abnormalities
– AML with t(8;21)(q22;q22)/RUNX1-RUNXT1
– AML with inv(16) or t(16;16)(p13;q22)/CBFβ-MYH11
– Acute promyelocytic leukemia [t(15;17) & PML-RAR-α]
– AML with t(9;11)(p22;q23)/AF9(MLLT3)-MLL
– AML with t(6;9)(p23;q34)/DEK-CAN(NUP214)
– AML with inv(3) or t(3;3)(q21;q26)/RPN1-EVI1
– Megakaryoblastic AML with t(1;22)(p13;q13)/RBM15-MKL1
– AML with mutated NPM1
– AML with normal karyotype and CEBPA mutation

WHO Classification, 2008

AML  muta:ons:  distribu:on  into  categories  of  related  genes

The Cancer Genome Atlas Research Network, NEJM 2013

AML  muta:ons:  categories  of  related  genes
– Myeloid transcription factors
• Gene fusions: PML-RARA, CBF-AML,…
• Gene mutations: CEBPA, RUNX1,…
– NPM1
– Activating signalling: FLT3, KIT, KRAS/NRAS,…
– Tumor supressor genes: TP53, WT1, PHF6
– DNA methylation machinery: DNMT (3A,3B,1),
IDH1/2, & TET1/2
– Histone modifiers: MLL-X fusions, MLL-PTD,
NUP98-NSD1, ASXL1, EZH2,…
– Splicing machinery
– Cohesin complex: STAG2, SMC1A/3, RAD21,…

The Cancer Genome Atlas Research Network, NEJM 2013

Gene  muta:ons  in  AML:  a  systema:c  (simplis2c?)  view
MATURATION ARREST PROLIFERATION
(type II) (type I)
Initial leukemogenic events Adcquired, evolutive events
Involve transcription factors Involve cell signalling pathways
Mutually exclusive Usually associated with diverse
type II mutations
Fusion transcripts
PML/RARA
AML1(RUNX1)/ETO(RUNX1T1)
CBFbeta/MYH11 Gene mutations
MLL/partners FLT3 (ITD, TKD)
Uncommon types (DEK/CAN, Kit
MOZ/CBP,HOXA9/NUP98) Ras
…
Other mutations
NPM1
CEBPA
RUNX1
AML  genomic  landscape:  number  of  recurrent
muta:ons  in  coding  sequence  per  sample

The Cancer Genome Atlas Research Network, NEJM 2013

Core-­‐binding  factor  AML:  loss  of  histone  acetyl  transferase
(HAT)  ac:vity  leads  to  transcrip:on  repression

inv(16)(p13q22)/t(16;16)(p13;q11)
CBFbeta/MYH11

t(8;21)(q22;q22)
AML1(RUNX1)/ETO(RUNX1T1)
FLT3  (fms-­‐like  TK)  Internal  Tandem  Duplica:on  (ITD)

Downstream FLT3 signaling

 Muta:ons  in  AML  -­‐  summary
• Rela:vely  low  number  of  muta:ons  to  drive  AML
• Unusual  genomic  inas:bility
• Random  background  muta:ons  before  acquisi:on  of
founding  muta:on
• Only  one  or  two  coopera:ng  muta:ons  are  needed
for  genera:on  of  malignant  founding  clone
• Founding  clone  can  acquire  addi:onal  coopera:ng
muta:ons  –  origin  of  subclones
Changer  le  monde...
 Increasing  incidence  of  AML  with  age:
a  work  of  years

Dores G M et al. Blood 2012 / SEER registry, 2001-2006

Normal,  self-­‐renewing  human  hematopoie:c  stem  cells
accumulate  random  muta:ons  over  :me
as  a  func:on  of  age  –  passenger  muta:ons

John S Welch, et al. Cell 2012

Origin of mutations in AML: pre-leukemic mutations
in healthy hematopoietic stem cells (HSCs)

• Detec:on  of  DNMT3A  muta:ons  in  non-­‐leukemic

cells  (HSCs)  –  pre-­‐leukemic  HSCs
• DNMT3Amut  HSCs  maintain  mul:lineage  (My-­‐Ly)
differen:a:on  poten:al  –  non-­‐leukemic
• DNMT3Amut  HSCs  show  clonal  expansion    -­‐
compe::ve  advantage
• DNMT3Amut  HSCs  survive  chemotherapy,  are  found
in  long-­‐survivors  &  expand  during  remission  –
reservoir  for  clonal  evolu:on  –  relapse?
• Should  pre-­‐leukemic  HSCs  be  therapeu:cally
targeted?
Liran I Slush, et al. Nature 2014
Clonal  hematopoiesis  is  found  in  elderly  persons  and  is  a
strong  risk  factor  for  subsequent  hematologic  cancer

 WGS  of  peripheral  blood  cells  in  a  cohort  of  >12300

persons  &  subsequent  follow-­‐up:
• Clonal  hematopoiesis  with  soma:c  muta:ons  found
in  10%  of  persons  >65  year-­‐old
• Common  soma:c  muta:ons  in  3  genes  commonly
involved  in  myeloid  neopl:  DNMT3A,  ASXL1,  &  TET2
• Clonal  hematopoiesis,  a  strong  risk  factor  for
hematologic  cancer  (HR:  12.99,  5.6-­‐28.7)
• 42%  of  hematologic  cancers  in  persons  with  previous
detectable  clonality
G Genovese, et al. N Engl J Med 2014
 Clonal Expansion of pre-leukemic mutations

Genovese G et al. N Engl J Med 2014;371:2477-2487

 Hematopoietic Clones and further Evolution

Genovese G et al. N Engl J Med 2014;371:2477-2487

 Origin of relapse in AML: evolution from
founding clone / subclone / ancestral clone?

L Ding et al. Nature 2011

 Acute  Myeloid  Leukemia  (AML)
• Concept  of  AML
• Leukemic  hierarchy:  leukemia  stem-­‐cell  model
• Muta:onal  landscape
• Biography  of  leukemogenic  muta:ons  of  AML:  from
pre-­‐clonal  lesions  to  clonal  evolu:on
• Epigene:c  disturbance
• Role  of  microevironment
• Transla:on  to  clinic:  risk-­‐adapted  therapy,
unravelling  therapeu:c  targets
AML  is  a  disease  with  deregulated  epigene:c  program:
role  for  epigene2c  therapy

DNA methylation (CpG islands) – demethylating agents

Histone deacetylation – HDAC inhibitors
Histone methylation
miRNA gene methylation
 AML:  also  an  epigene:c  disease

• Dis:nc:ve  CpG  methyla:on  paeerns

• Frequent  muta:ons  in  genes  involved  in:
– DNA  methyla:on:  DNMT3A,  IDH1-­‐2,  TET2
– Histone  modifiers:  MLL,  ASXL1-­‐PRC2
• Epigene:c  deregula:on  of  non-­‐coding  RNAs?
• An:leukemic  ac:vity  of  hypomethyla:ng  agents
– Azacy:dine
– Decytabine
Dis:nc:ve  CpG  methyla:on  signatures  among  AML  subtypes

Figueroa ME, Cancer Cell 2010

AML  muta:ons:  distribu:on  into  categories  of  related  genes

The Cancer Genome Atlas Research Network, NEJM

2013
 Decitabine in elderly AML – benefit in patients unfit,
unsuitable for intensive chemotherapy

Decitabine Control  arm

CR 16 7
CRp 2 0.4
CR  +  CRp 18 8
CRi 10 3
Stable 28 23
disease

Kantarjian  H,    et  al.,  JCO  2012

 Pre-­‐planned  OS  Sensi:vity  Analysis
31

Censored  for  Subsequent  AML  Tx*

Median  [95%  CI]  OS:  AZA  =  12.1  mos  [9.2,  14.2]  vs.  CCR  =  6.9  mos
[5.1,  9.6]
Unstra:fied  HR=0.75  [95%CI  0.59,  0.95];  log-­‐rank  p=0.0147
Stra:fied†  HR=0.76  [95%CI:  0.60,  0.96];  log-­‐rank  p=0.0190

12.1  mos

6.9  mos

○  =  Censored
*67  AZA  pts  and  75  CCR  pts  in  this  sensi:vity  analysis  were  censored  at  the  :me  they  received  subsequent  AML  Tx
†Stra:fied  by  ECOG  PS  and  cytogene:c  risk
TET2 mutations predict response to hypomethylating
agents in myelodysplastic syndrome patients

Mutated  gene Adjusted  OR  (95%  CI) P  value

MutaPons  with  VAF  ≥10%
 TET2-­‐mut  vs  TET2-­‐WT 1.98  (1.02,  3.85) .044
 ASXL-­‐mut  vs  ASXL1-­‐WT 0.68  (0.38,  1.19) .17
 TET2-­‐mut  +  ASXL1-­‐WT  vs  other 3.64  (1.35,  9.79) .011

 TET2-­‐mut  +  ASXL1-­‐WT  vs  both  WT 3.36  (1.20,  9.38) .013

Bejar R, et al. Blood 2014

AML cells disrupt normal hematopoietic
niche

Lane SW et al, Blood 2009

 Leukemic cells & bone marrow
microenvironment: dangerous liaisons

• Impairment  of  normal  hematopoie:c  niche  by:

– Direct  invasion
– Secre:on  of  substances  (SCF,…)
• Alterna:ve  leukemic  niche,  deregulated  homing
• Protec:ve  effect  of  stroma  on  leukemic  blasts
(quiescence,  chemoresitance,…)
• Drug  interference  with  stroma-­‐LSC:  a  new
therapeu:c  opportunity  (the  plerixafor  paradigm)
 Acute  Myeloid  Leukemia  (AML):
lessons  from  biology
• Defini:on  of  AML
• Recogni:on  of  disease  diversity
• Unraveling  the  underlying  complex  biology  of  AML:
– Leukemic  hierarchy
– Accumula:on  of  gene  muta:ons
– Epigene:c  disturbance
– Origin  of  leukemia  &  relapse
– Role  of  microevironment
• Biology  leads  to  refinement  of  prognosis,  risk-­‐
adapted  therapy  &  therapeu:c  targets
Risk-­‐adapted  therapy  …  what’s  in  a  concept?

• Ini:al  risk  assignment

– Biological  profiling  (gene:cs  of  disease)
– Evidence  generated  on  op:mal  management
• Dynamic  risk  assessment  –  minimal  residual  disease
• Host-­‐adapted  therapy
– Age
– Evalua:on  of  comorbid  condi:ons
AML:  high  clinical  heterogeneity  due  to  biological
diversity

Grimwade D, et al. Blood 2010

 Recommended post-remission therapy for ELN-adapted
genetic categories
ELN genetic Molecular subsets Recommended post-CR1 therapy
group
Favorable t(8;21)/RUNX1(AML)-RUNXT1(ETO) HiDAC
inv(16)/CBFb-MYH11 Autologous HSCT?
NPM1mut w/o FLT3-ITD
Double CEBPA mutation Consider alloHCST if MRD(+)
Intermediate-I Triple negative (NPM1,FLT3-ITD,CEBPA) AlloHSCT?
Consider autoHSCT if MRD(-)
Int-I’ NPM1mut or NPM1wt FLT3-ITD AlloHSCT in CR1
AutoHSCT /HiDAC in FLT3-ITDlow ratio?
Int-II t(9;11)/AF9-MLL AlloHSCT in CR1
t(11;19)/MLL-ENL AutoHSCT if MRD(-)?
Adverse inv(3)/t(3;3)/RPN1-EVI1 AlloHSCT in CR1
t(6;9)/DEK-NUP214(CAN) AlloHSCT in advanced phase?
-5/del(5q), -7, abn(17p), complex or
monosomal karyotype Experimental therapy
t(v;11q23)/Other MLL-rearrangement
 Therapy in AML: state-of the-art

• Induc:on  therapy  based  on  combina:on  of  an

anthracycline  &  ara-­‐C
• High-­‐dose  ara-­‐C  benefits  pts  with  good-­‐prognosis
AML  (cytogene:c  &  molecular  defini:on)
• Early  alloHSCT  benefits  younger  high-­‐risk  pa:ents
• The  APL  paradigm:  differen:a:ng  therapy  (ATRA,
ATO)
• Improved:  suppor:ve  therapy
• Diverse  novel  strategies  are  being  explored
 Therapy  of  AML  –  unmet  needs

• “High-­‐risk”  presenta:on  forms  -­‐  a  “gentle  APL-­‐

like  approach”  with  differen:a:ng  agents?
• Remission  is  based  on  highly  myelotoxic
agents
• Limited  target  popula:on  of  hematopoie:c
stem-­‐cell  transplant
 AML & age: rationale for a different
biological background
• AML is a disease of long-lived progenitor
hematopoietic stem-cells
• Multi-step leukemogenic model: increasing risk
with age
• Increasing proportion of multi-hit AML subtypes
• Accumulation of pre-leukemic mutations
• Stromal aging: a role in AML origin?
• Failing inmune surveillance with age
 On-going trials with novel agents in AML
Drug Target Study phase
Dasatinib Kit 3 (CBF-AML)
Crenolanib PanFLT3mut inhibitor 2
ABT-199 Bcl-2 inhibitor 2
AG-221 IDH2 mutant 1
RG7388 MDM2 antagonist 1
OTX015 BET-Bromodomain inhibitor 1b
ABT-199 Bcl-2 inhibitor 1
Alisertib Aurora kinase inhibitor 2
EPZ-5676 DOT1L inhibitor 1 (MLL-AML)
CSL362 Anti-IL3Rα (CD123) 1
SGN-CD33A Conjugated antiCD33 Ab 1
CAR(T)s AntiCD123, antiWT1 Pre-clinical
… ... …
Potential therapeutic targets within main AML entities

AML subtype Molecular target Drug

APL PML-RARA ATRA, ATO
AML-CBF Kit TKIs (dasatinib)
Ras PI3K + MEK inh
AML-NPM1mut HOX overexpresion DOT1L1 inh
DNMT3A Hypomethilators
FLT3-ITD FLT3 inhibitors
IDH2 IDH2 inh
MLL-r AML Methyltransferase complex DOT1L1inh (EPZ5676)
CDK6 Palbociclib
EVI1-r AML Ras PI3K + MEK inh
t(6;9) AML FLT3 FLT3 inhibition
MRC-AML TP53 MDM2 antagonist?
EZH2 mutation Proteasome inh
MoAbs  in  AML:  Humanized  an:CD33  Ab
Gemtuzumab  +  calicheamicin  (Mylotarg)

1-2. Binding to CD33 Ag

3-4. Internalization & calicheamicin activation
8. Antitumoral effect: induction of DNA breaks
7. Mechanisms of resistance: drug efflux
5. Rapid CD33 re-expression
Mylotarg/GO  in  AML  frontline  therapy:  impact  of
cytogene:cs
Favorable  cytogene:cs Intermediate-­‐risk  cytogene:cs
FLT3  inhibitors:  diverse  an:-­‐TKI  poten:al
 Sorafenib:  InhibiPon  of  MulPple  Kinases
Serine/threonine kinases1,2 IC50 (nM)
Raf-1 6
B-Raf 25
Oncogenic b-raf V600E mutant 38
p38 38
Mnk-2 150
ERK-1, MEK- 1, PKA, PKB, PKC,
cdk1/cyclinB, pim-1 >10,000

Receptor tyrosine kinase1,2 IC50 (nM)

VEGFR-13 26
VEGFR-2 90
VEGFR-3* 20
Flt-3 33
RET4 47
PDGFR-β* 57
c-KIT 68
FGFR-1 580 Zarrinkar  et  al.,  Blood  2009,  Wilhelm  SM,  et  al.
c-met, IGFR-1, EGFR, Cancer  Res  2004,  Riedl  B,  et  al.  AACR  2001,
HER2, LCK >10,000 Carlomagno  F,  et  al.  J  Natl  Cancer  Inst
Event-­‐Free  Survival
(ITT,  no  SCT  censoring)

100

Median  EFS  Placebo  vs  Sora:

80
9  m  vs  25  m
Probability (%)

60
Sorafenib

40
Placebo

20

p=0.01
0
0 12 24 36
Time (months)
Sorafenib 134 72 60 38
Placebo 133 61 46 25
Median  follow-­‐up  36
months
C Röllig, et al./SAL (abst. 6, Plenary session)
 FLT3  inhibitors:  current  evidence
Limited activity in monotherapy (sorafenib, midostaurin,…)
Possible synergy in combination with chemotherapy
- Lestaurtinib: no benefit in relapsed AML
- Midostaurin/PKC-412: on-going trial (front-line tx)
Treatment of relapse after alloHSCT: prolonged responses
in some pts. treated with sorafenib – induction of GvL effect
after cytoreduction?
AC220 (quizartinib): remarkable activity in monotherapy
- Composite response rate (CR+CRp+CRi) of ≈45%
- Differentiating potential in AML blasts
Fischer T, JCO 2010
Levis M, Blood 2011
Cortes J, Haematologica 2011
 Plk1: a key regulator of mitosis
• Microtubule-kinetochore Plk1 deficiency
attachment
• Mitotic progression • Spindle elongation

Midzone
Kinetochores Metaphase

Prometaphase
Anaphase
• Mitotic entry
(Cdk1 activation)
Plk1
• Cleavage furrow
NH2 COOH
• Centrosomal formation (cytokinesis)
microtubule
nucleation
Midbody

Centrosomes

Prophase Telophase

• Checkpoint adaptation Plk1 localization

and recovery Plk functions
Microtubules DNA
Interphase

Modified from Takaki T, et al. Curr Opin Cell Biol 2008;20:650–60.

 Epigenetics effects of IDH mutations (II)
• IDH mutations lead to a neomorphic enzyme activity, converting
alpha-ketoglutarate (α-KG) to 2-hydroxyglutarate (2HG)
• 2HG is an oncometabolite which disrupts enzymes using α-KG:
– TET2, resulting in a 5-OH-methylation block
– Jumonji family of histone demethylase

Ari Melnick, et al (ASH 2011 Scientific Program)

IDH  mutants  are  a  poten:al  target  in  AML

52
AG-­‐221  for  AML  pa:ents  with  mutated  IDH-­‐2

Agresta  S,    et  al.,  EHA  2014

Mechanism of action of ATRA in APL: transcriptional
reactivation by overcoming maturation block
(“relieving repression induces remission”)

Hugues de Thé, Zhu Chen; Nature Review 2010

 Treatment schedule
Induction Consolidation

ATO ATO ATO ATO

ATO 4 weeks on / 4 weeks off
arm
ATRA
Until CR 2 weeks on / 2 weeks off
Estey et al, Blood 2006
R
Induction Consolidation Maintenance
IDA IDA MTZ IDA MTX + 6MP
Chemo
Arm ATRA ATRA ATRA ATRA ATRA
Until CR 3 monthly cycles 2 years
Lo-Coco et al., Blood 2010
U Platzbecker, et al. (abst. 12, session 615)
AG-­‐221  for  AML  pa:ents  with  mutated  IDH-­‐2

• AG-221 induces effective 2-HG inhibition in

pts with R140Q IDH-2 mutation
• Significant clinical activity:
 14 of 25 responses
 9 pts achieved CR/CRp/CRi
 Durable responses: 5 pts with responses
> 2.5 mos.
• Responses observed in AML, MDS & CMML
• Safe & well tolerated
Analyzing  causes  of  failure  -­‐  challenges  for
developing  a  cura:ve  therapy  in  AML

• Biological heterogeneity – not a unique target

• Multi-step process – lessons from whole-
genome sequencing
• Quiescence of leukemia-stem cells confers
chemoresistance – need to target LSCs
• AML: a family of different subclones –
preleukemic & evolutive clones
• BM microenvironment – a protective milieu
 Potential mechanisms for targeting
Leukemia Stem Cells
• Targeting fusion proteins
- High diversity in AML
• Signaling pathways (JAK/STAT, Wnt, Hedghog,…)
- Diversity
- Redundancy-overlapping
• Self-renewal mechanisms
- Similarity HSCs - LSCs
• Inducing differentiation
• MoAbs against specific LSC Ag (CD44,CD123,TIM-3,…)
 New  agents  for  acute  leukemia  –  final
considera:ons
• Progress  in  AML/ALL  biology  knowledge  is
essen:al  for  developing  new  therapies
• Heterogeneity  of  disease  –  analysis  of  benefit
in  specific  popula:ons
• Mul:step  disease  –  need  of  combining  agents
against  diverse  targets
• Targe:ng  LSCs:  hope  for  cure
 Marta  Pratcorona  (FCRB)
Marina  Díaz  Beyá  (HCB,  FCRB  )
Ruth  M.  Risueño  (IRJC)
Meritxell  Nomdedeu  (FCRB)
CETLAM  Group
Grup  de  recerca  mieloide,
Salut  Brunet
IDIBAPS,  IR  Josep  Carreras
Jordi  Sierra
Josep  Nomdedéu
Josep  Ma.  Ribera
University  of  Barcelona
Mar  Tormo
Alfons  Navarro
David  Gallardo
Marià  Monzó
Olga  Salamero
Carme  de  Pedro
….
CETLAM  centers