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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 (type II)
PROLIFERATION (type I)
Initial leukemogenic events
Adcquired, evolutive events
Involve transcription factors Mutually exclusive
Involve cell signalling pathways
Fusion transcripts PML/RARA AML1(RUNX1)/ETO(RUNX1T1) CBFbeta/MYH11 MLL/partners Uncommon types (DEK/CAN, MOZ/CBP,HOXA9/NUP98) Other mutations NPM1 CEBPA RUNX1
Usually associated with diverse type II mutations
Gene mutations FLT3 (ITD, TKD) Kit Ras …
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 disease
28
23
Kantarjian H, et al., JCO 2012
Pre-‐planned OS Sensi:vity Analysis 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
31
TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients
Mutated gene Adjusted OR (95% CI) MutaPons with VAF ≥10% TET2-‐mut vs TET2-‐WT 1.98 (1.02, 3.85) ASXL-‐mut vs ASXL1-‐WT 0.68 (0.38, 1.19)
P value .044 .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 group Favorable
Molecular subsets
Recommended post-CR1 therapy
t(8;21)/RUNX1(AML)-RUNXT1(ETO)
HiDAC
inv(16)/CBFb-MYH11
Autologous HSCT?
NPM1mut w/o FLT3-ITD Intermediate-I
Double CEBPA mutation
Consider alloHCST if MRD(+)
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 Adverse
t(9;11)/AF9-MLL
AlloHSCT in CR1
t(11;19)/MLL-ENL
AutoHSCT if MRD(-)?
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
1 (MLL-AML)
CSL362
DOT1L inhibitor Anti-IL3Rα (CD123)
SGN-CD33A
Conjugated antiCD33 Ab
1
CAR(T)s
AntiCD123, antiWT1
Pre-clinical
…
...
…
1
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
HOX overexpresion
DOT1L1 inh
DNMT3A
Hypomethilators
FLT3-ITD
FLT3 inhibitors
IDH2
IDH2 inh
AML-NPM1mut
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 ERK-1, MEK- 1, PKA, PKB, PKC, cdk1/cyclinB, pim-1
Receptor tyrosine kinase1,2
150 >10,000 IC50 (nM)
VEGFR-13
26
VEGFR-2
90
VEGFR-3*
20
Flt-3
33
RET4
47
PDGFR-β*
57
c-KIT
68
FGFR-1 c-met, IGFR-1, EGFR, HER2, LCK
580 >10,000
Zarrinkar et al., Blood 2009, Wilhelm SM, et al. Cancer Res 2004, Riedl B, et al. AACR 2001, Carlomagno F, et al. J Natl Cancer Inst
Event-‐Free Survival (ITT, no SCT censoring) 100
Median EFS Placebo vs Sora: 9 m vs 25 m
Probability (%)
80
60 Sorafenib 40
Placebo
20
p=0.01 0
0
12
24
36
60 46
38 25
Time (months) Sorafenib 134 Placebo 133
72 61
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 attachment • Mitotic progression
Plk1 deficiency • Spindle elongation
Midzone Kinetochores
Metaphase
Prometaphase
• Mitotic entry (Cdk1 activation) • Centrosomal microtubule nucleation
Anaphase
Plk1 NH2
• Cleavage furrow formation (cytokinesis)
COOH
Midbody Centrosomes
Telophase
Prophase
Plk1 localization Plk functions
• Checkpoint adaptation and recovery
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 arm
ATO
ATRA 2 weeks on / 2 weeks off
Estey et al, Blood 2006
Induction Chemo Arm
ATO
4 weeks on / 4 weeks off
Until CR
R
ATO
IDA
ATRA
Maintenance
Consolidation IDA
ATRA
Until CR Lo-Coco et al., Blood 2010
MTZ
ATRA
3 monthly cycles
IDA
ATRA
MTX + 6MP ATRA 2 years
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 wholegenome 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) Grup de recerca mieloide, IDIBAPS, IR Josep Carreras
University of Barcelona Alfons Navarro Marià Monzó
CETLAM Group Salut Brunet Jordi Sierra Josep Nomdedéu Josep Ma. Ribera Mar Tormo David Gallardo Olga Salamero Carme de Pedro …. CETLAM centers
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 (type II)
PROLIFERATION (type I)
Initial leukemogenic events
Adcquired, evolutive events
Involve transcription factors Mutually exclusive
Involve cell signalling pathways
Fusion transcripts PML/RARA AML1(RUNX1)/ETO(RUNX1T1) CBFbeta/MYH11 MLL/partners Uncommon types (DEK/CAN, MOZ/CBP,HOXA9/NUP98) Other mutations NPM1 CEBPA RUNX1
Usually associated with diverse type II mutations
Gene mutations FLT3 (ITD, TKD) Kit Ras …
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 disease
28
23
Kantarjian H, et al., JCO 2012
Pre-‐planned OS Sensi:vity Analysis 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
31
TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients
Mutated gene Adjusted OR (95% CI) MutaPons with VAF ≥10% TET2-‐mut vs TET2-‐WT 1.98 (1.02, 3.85) ASXL-‐mut vs ASXL1-‐WT 0.68 (0.38, 1.19)
P value .044 .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 group Favorable
Molecular subsets
Recommended post-CR1 therapy
t(8;21)/RUNX1(AML)-RUNXT1(ETO)
HiDAC
inv(16)/CBFb-MYH11
Autologous HSCT?
NPM1mut w/o FLT3-ITD Intermediate-I
Double CEBPA mutation
Consider alloHCST if MRD(+)
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 Adverse
t(9;11)/AF9-MLL
AlloHSCT in CR1
t(11;19)/MLL-ENL
AutoHSCT if MRD(-)?
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
1 (MLL-AML)
CSL362
DOT1L inhibitor Anti-IL3Rα (CD123)
SGN-CD33A
Conjugated antiCD33 Ab
1
CAR(T)s
AntiCD123, antiWT1
Pre-clinical
…
...
…
1
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
HOX overexpresion
DOT1L1 inh
DNMT3A
Hypomethilators
FLT3-ITD
FLT3 inhibitors
IDH2
IDH2 inh
AML-NPM1mut
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 ERK-1, MEK- 1, PKA, PKB, PKC, cdk1/cyclinB, pim-1
Receptor tyrosine kinase1,2
150 >10,000 IC50 (nM)
VEGFR-13
26
VEGFR-2
90
VEGFR-3*
20
Flt-3
33
RET4
47
PDGFR-β*
57
c-KIT
68
FGFR-1 c-met, IGFR-1, EGFR, HER2, LCK
580 >10,000
Zarrinkar et al., Blood 2009, Wilhelm SM, et al. Cancer Res 2004, Riedl B, et al. AACR 2001, Carlomagno F, et al. J Natl Cancer Inst
Event-‐Free Survival (ITT, no SCT censoring) 100
Median EFS Placebo vs Sora: 9 m vs 25 m
Probability (%)
80
60 Sorafenib 40
Placebo
20
p=0.01 0
0
12
24
36
60 46
38 25
Time (months) Sorafenib 134 Placebo 133
72 61
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 attachment • Mitotic progression
Plk1 deficiency • Spindle elongation
Midzone Kinetochores
Metaphase
Prometaphase
• Mitotic entry (Cdk1 activation) • Centrosomal microtubule nucleation
Anaphase
Plk1 NH2
• Cleavage furrow formation (cytokinesis)
COOH
Midbody Centrosomes
Telophase
Prophase
Plk1 localization Plk functions
• Checkpoint adaptation and recovery
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 arm
ATO
ATRA 2 weeks on / 2 weeks off
Estey et al, Blood 2006
Induction Chemo Arm
ATO
4 weeks on / 4 weeks off
Until CR
R
ATO
IDA
ATRA
Maintenance
Consolidation IDA
ATRA
Until CR Lo-Coco et al., Blood 2010
MTZ
ATRA
3 monthly cycles
IDA
ATRA
MTX + 6MP ATRA 2 years
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 wholegenome 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) Grup de recerca mieloide, IDIBAPS, IR Josep Carreras
University of Barcelona Alfons Navarro Marià Monzó
CETLAM Group Salut Brunet Jordi Sierra Josep Nomdedéu Josep Ma. Ribera Mar Tormo David Gallardo Olga Salamero Carme de Pedro …. CETLAM centers
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