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Pheno Pheno'pic 'pic He Heter erog ogen eneit eity of y of - - PowerPoint PPT Presentation

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Pheno Pheno'pic 'pic He Heter erog ogen eneit eity of y of Leukemi mias Pier Giuseppe Pelicci Milan, Italy Universita degli Studi di Milano 7 th INTERNATIONAL SYMPOSIUM ON ACUTE PROMYELOCYTIC LEUKEMIA Rome, September 24 th -27 th

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SLIDE 1

Pheno Pheno'pic 'pic He Heter erog

  • gen

eneit eity of y of Leukemi mias

Pier Giuseppe Pelicci

Milan, Italy

7th INTERNATIONAL SYMPOSIUM ON ACUTE PROMYELOCYTIC LEUKEMIA Rome, September 24th-27th 2017

Universita’ degli Studi di Milano

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SLIDE 2

Cancer Stem Cells (AMLs, Breast Cancer):

  • Have unlimited self renewal potenTal
  • Divide both asymmetrically and symmetrically
  • Symmetric divisions prevail
  • Progenitors are conTnuously reprogrammed into CSCs

A Santoro, T Vlachou; under revision Viale et al, Nature 2010 Cicalese et al, Cell 2011 Tomilov et al, Aging Cell 2011 Pece et al, Cell 2012 Pasi et al, Cell Death & Diff, 2012 Gambino et al, Aging Cell 2012 Insinga et al, PNAS 2013 Migliaccio et al, Aging Cell 2013 Pr. CSC CSC CSC CSC

Extended self-renewal Symmetric divisions Progenitor reprogramming into CSCs

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SLIDE 3

Cancer Stem Cells (AMLs, Breast Cancer):

  • Altered self-renewal of CSCs is due to aZenuated p53 signalling and

acTvaTon of Myc

Pr. CSC CSC CSC CSC

Extended self-renewal Symmetric divisions Progenitor reprogramming into CSCs

p53 Myc

A Santoro, T Vlachou; under revision Viale et al, Nature 2010 Cicalese et al, Cell 2011 Tomilov et al, Aging Cell 2011 Pece et al, Cell 2012 Pasi et al, Cell Death & Diff, 2012 Gambino et al, Aging Cell 2012 Insinga et al, PNAS 2013 Migliaccio et al, Aging Cell 2013

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SLIDE 4

The p53:Myc expression signature is predicTve of clinical outcome, independently of other known risk factors

(4 independent cohorts; 892 pa4ents)

significant decrease in the disease-free survival

  • f the UP cohort

Same power in ER+ paTents

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SLIDE 5

A Santoro, T Vlachou; under revision Viale et al, Nature 2010 Cicalese et al, Cell 2011 Tomilov et al, Aging Cell 2011 Pece et al, Cell 2012 Pasi et al, Cell Death & Diff, 2012 Gambino et al, Aging Cell 2012 Insinga et al, PNAS 2013 Migliaccio et al, Aging Cell 2013 Pr. CSC CSC CSC CSC

Extended self-renewal Symmetric divisions Progenitor reprogramming into CSCs

Symmetric divisions, progenitor reprogramming, extendend self-renewal:

  • Maintenance and conTnuous expansion of the pool of Cancer Stem Cells:

Asymmetric Divisions:

  • Maintenance of biological heterogeneity

Loss of p53 and Myc acTvaTon:

  • General mechanism of self-renewal de-regulaTon in CSCs
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SLIDE 6

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

+ 5-FU

X1 X2 X2 X3

No 5FU No 5-FU

X3

No 5-FU

High Clonal Heterogeneity within the pool of LSCs

Hundreds of LSCs with heterogenous growth potenTal in vivo Strong clonal selecTon during leukemia growth (serial passaging) The process of clonal selecTon can be perturbed by environmental signals (5-FU)

Andrea Cammarata, unpublished

Human AMLs grown in immunodeficient mice (PDX)

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SLIDE 7

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

+ 5-FU

X1 X2 X2 X3

No 5FU No 5-FU

X3

No 5-FU

Serial transpl. Andrea Cammarata, unpublished

Hundreds of LSCs with heterogenous growth potenTal in vivo Strong clonal selecTon during leukemia growth (serial passaging) The process of clonal selecTon can be perturbed by environmental signals (5-FU)

High Clonal Heterogeneity within the pool of LSCs

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SLIDE 8

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

+ 5-FU

X1 X2 X2 X3

No 5FU No 5-FU

X3

No 5-FU

Andrea Cammarata, unpublished Serial transpl. Serial transpl. + 5FU

Hundreds of LSCs with heterogenous growth potenTal in vivo Strong clonal selecTon during leukemia growth (serial passaging) The process of clonal selecTon can be perturbed by environmental signals (5-FU)

High Clonal Heterogeneity within the pool of LSCs

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SLIDE 9

X1 M3 X2 No 5FU ID2 X2 + 5FU ID2

AMLIEO20_CT_X1_M3_freq.vaf AMLIEO20_X2_no5FU_ID2_freq.vaf AMLIEO20_X2_5FU_ID2_freq.vaf

X1 M3 X2 No 5FU ID2 X2 + 5FU ID2

Limited geneTc heterogeneity within the pool of LSCs

Thalia Vlachou, unpublished

Biological heterogeneity exceeds geneTc heterogeneity by one order of magnitude

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SLIDE 10

How biological heterogeneity is generated? Is it generated by phenotypic adaptaTon of Leukemia Stem Cells to (micro)environmental signals? How geneTc and non-geneTc (epigeneTc) mechanisms interact in the selecTon of best-fiZed cancer phenotypes by environmental cues? Model systems:

  • Macrophage-acTvated CD4+ cells
  • Obesity
  • Nutrient deprivaTon
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SLIDE 11

Effects of obesity

  • n the self-renewal of Leukemia Stem Cells

Anna Giulia Sannarico, PhD

Luca Mazzarella MD-PhD

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SLIDE 12

Body-mass index (BMI) correlates with worse prognosis in the Acute PromyelocyTc Leukemia (APL) subtype of AMLs

Incidence of relapse Years from induc4on

Underweight/Normal Overweight/Obese

Gray test p-value: 0.029

Breccia et al., Blood 2012

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SLIDE 13

Higher incidence of FLT3-ITD mutaTons in the obese APL-paTents

Incidence of relapse Years from induc4on

Underweight/Normal Overweight/Obese

Gray test p-value: 0.029

28% 9%

FLT3-ITD Incidence:

χ2 test p-value: 0.0005

  • the most frequent

mutation in AMLs (~33%)

  • linked to bad prognosis
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SLIDE 14

Driver mutaTons in AMLs are frequently found in rare subclones, including FLT3

number of muta4ons per gene Mutated genes UD6 UD5 TO1 primary NRAS 1 / / FLT3 1 1 / IDH2* 3 1 / TP53§ 7 2 3 BRD4* 2 1 / U2AF2* 1 1 / DNMT3A 1 / / ASXL1 1 3 2 TET2 3 7 / KIT 2 / / EZH2 1 3 / GNAQ 1 / / JAK2 2 5 / AXIN1 / 1 / CEBPA / 1 / RUNX1 / 1 1 EGFR / 1 / PER1 / / 2 TOTAL 26 28 8

Duplex Sequencing

(Schmi' MW et al., PNAS 2012; Kennedy SR et al., Nature Protocols 2014)

* exact same muta4on iden4fied in both pa4ents in UD6 and UD5 § exact same muta4on iden4fied in both pa4ents in UD6, UD5 and TO1

VAF: 0,001%-5%

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SLIDE 15

Obesity confers a compeTTve advantage to the FLT3-ITD BM And increases disease aggressiveness of FLT3-ITD transgenic mice

0% 20% 40% 60% 80% 100% sd12 sd22 sd11 sd15 sd02 sd03 sd19 sd13 sd10 sd01 sd09 hfd08 hfd23 hfd17 hfd19 hfd18 hfd07 hfd01 hfd15 hfd22 hfd20 hfd02

PBL

Myeloid compartment

3/21 11/23

* p < 0.05

ITD-SD ITD-HFD Competitive BM transplantation

EE Ki67

ITD-SD ITD-HFD

EE Ki67

grams

Spleen weight

(2yrs old mice) FLT3-ITD WT

HFD SD HFD SD

FLT3-ITD mice from G. Gilliland

Spleen

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SLIDE 16

Legend

PML/RARa PML/RARa FLT3ITD

Obese Lean

IL6 TNFa Insulin Leptin IL1 IGF1

IL6 TNFa Insulin Leptin IL1 IGF1

Cytokine X A B

Obesity might select FLT3-ITD mutaTons through adipose-Tssue - secreted adipokines.

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SLIDE 17

IL3:

The oncogenic potenTal of the FLT3-ITD mutaTon depends on Insulin/IGF1 signalling

  • +

IL3R IL3 Depend.

BaF3

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SLIDE 18

IL3:

  • + - +

ITD51 and ITD78

IL3R IL3 Depend. IL3 Independ.

BaF3

BaF3 FLT3-ITD

The oncogenic potenTal of the FLT3-ITD mutaTon depends on Insulin/IGF1 signalling

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SLIDE 19

IL3R IL3 Depend. IL3 Depend. IL3 Independ.

BaF3

BaF3 FLT3-ITD BaF3 FLT3-ITD + IGF1/Insulin OSI-906 inhibitor

IL3:

  • + - +

The oncogenic potenTal of the FLT3-ITD mutaTon depends on Insulin/IGF1 signalling

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SLIDE 20

Vinculin

p-Akt p-S6

Ba/F3 FLT3-WT FLT3-ITD 78 FLT3-ITD 51

OSI uM

0 1.2 2.5 5 10

p-STAT5

0 1.2 2.5 5 10 0 1.2 2.5 5 10 0 1.2 2.5 5 10

The oncogenic signaling of the FLT3-ITD mutaTon depends on Insulin/IGF1 signalling

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SLIDE 21

IGF1 potenTates the signaling potenTal of FLT3-ITD

0 10 100 0 10 100 0 10 100 0 10 100

Ba/F3 FLT3-WT ITD 78 ITD 51

Vinculin

p-Akt p-FoxO p-S6

IGF1 ng/ml:

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SLIDE 22

The ITD mutaTon increases the immature, non-glycosylated form of FLT3, and targets FLT3-ITD to the ER (Ergic compartment)

FLT3 Ac4n Ba/F3

Mature Immature Unglycosylated

Surface expressed “ER”-retained

FLT3-ITD induces ER stress

p-PERK p-eIF2a eIF2a PERK Vinculin Vinculin

DMSO Thapsigargin DMSO Thapsigargin

stress-sensing kinase PERK

FLT3wt FLT3-ITD

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SLIDE 23

FLT3-ITD induces a sustained UPR response

p-eIF2a eIF2a BiP Vinculin

Tunicamycin (Hrs) 1 24

TranslaTonal iniTaTon factor 2a (eIF2a) (cap-dependent protein translaTon) Vinc BiP ATF4 ()

BiP PDIA6 PDIA3 Tubulin Ba/F3 ITD78 Brefeldin

  • +
  • +

PDIA3/6 disulphide isomerases the BiP ER-chaperone

SREBP1 imm SREBP1 mat SREBP2 mat SREBP2 imm Tubulin Ba/F3 ITD

BrefeldinA

  • +
  • +

SREBP2 transcripTon factor (sterol regulatory element- binding protein)

TranslaTonal Reprogramming TranscripTonal Reprogramming Colesterol Reprogramming

ATF Transcr. Fact.

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SLIDE 24

Obesity increases the UPR response to FLT3-ITD induced ER stress

1 2 3 4 5 6 7 8 9

Hspa1a Hspa1b SD HFD

Chaperone synthesis

Gene SD (TPM) HFD (TPM)

Sqle 38,56 46,76 Hmgcr 48,20 65,97 Hmgcs1 27,43 35,89 Srebf2 120,62 147,23

RNAseq on FLT3-HO mice

Gene WT (TPM) FLT3 (TPM)

Me2 52,16 83,99 Sqle 23,71 38,56 Hmgcs1 21,84 27,43 Scd2 93,40 188,13 Fasn 53,06 80,04 Slc16a1 24,29 50,39 Srebf2 124,44 120,62

RNAseq on SD mice

Cholesterol metabolism

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SLIDE 25

UPR ResoluTon of ER stress

HSF1 SREBPs GSH

Obesity favors resoluTon of ER-stress induced by the ITD mutaTon (UPR response) and acTvates the full oncogenic potenTal of FLT3-ITD

Obesity ITD-mutaTon

FLT3-unfolding (defecTve signaling) ER-stress

AcTvaTon of the FLT3-ITD Full signalling potenTal

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SLIDE 26

UPR ResoluTon of ER stress

HSF1 SREBPs GSH

Pharmacological targeTng of the UPR response

Obesity ITD-mutaTon

FLT3-unfolding (defecTve signaling) ER-stress

AcTvaTon of the FLT3-ITD Full signalling potenTal

KRIBB1 KRIBB11: prevents HSF1 F1 interac'on with p- with p-TEFb Fb t to t

  • the

e hsp70 hsp70 promo moter sit site e Bu Buthion

  • nine sulfoximi

mine (BS (BSO): O): inhibits gamma-glutamylcysteine synthetase and reduces intracellular GSH

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SLIDE 27

Modest effects of BSO or KRIBB1 single-agents; >95% cell death upon combinaTon

  • 2
  • 1

1 2 50 100 150

Log (KRIBB11) uM Percent survival

72 hours after plating

Ba/F3 BSO - Ba/F3 BSO 0.4 mM ITD51 BSO 0.4 mM ITD51 BSO -

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SLIDE 28

FLT3-ITD sensiTzes AMLs to BSO-KRIBB11 combined treatment

THP-1 NB4 KG-1 U937 MOLM13 MOLM14 MV4-11 0.0001 0.0010 0.0100 0.1000 1 10

Log IC50

AMLs

FLT3- neg / WT FLT3- ITD

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SLIDE 29

Conclusions

  • Obesity acTvates the oncogenic potenTal of FLT3-ITD by releasing FLT3-ITD –

induced ER stress (through insulin/IGF1 signaling)

  • This adapTve response to FLT3-ITD – induced ER stress creates selecTve

vulnerabiliTes of FLT3-ITD AMLs, unraveled by inhibiTon of chaperone acTvity or GSH depleTon

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SLIDE 30

Luca Mazzarella, MD-PhD

Rani Pallavi, PhD

Effects of nutrient deprivaTon

  • n Leukemia Stem Cells

Saverio-Minucci’s Group

  • The tumor micro-environment is characterized by a chronic state of

nutrient and oxygen deprivaTon

  • Nutrient scarcity is among the criTcal environmental condiTons

driving phenotypic plasTcity

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SLIDE 31

Standard Diet Caloric Crestric4on weeks 1 2 3 4 5 6 … Leukemia InjecTon CR before Leukemia injecTon CR aqer Leukemia injecTon Caloric Crestric4on Control (SD)

500000 1000000 SD CR IGF-1 (pg/ml)

CirculaTng IGF-1

20 40 0 1 2 3 4 5 6 7 8 9 10 Body weight Week SD CRD

Change in Body Mass

~15% body- weight reducTon in the CR group

Caloric restricTon (CR) as model system to study nutrient deprivaTon

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SLIDE 32

5 10 15 20 25 30 III IV V VI

% of APL blast

Week aqer InjecTon

SD CR

Bone Marrow

Early Tme points: CR markedly reduces leukemic burden

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SLIDE 33

Later Tme points: CR-treated Leukemias re-expand leading to leukemia associated mouse death

20 40 60 80 100 120 20 40 60 80 100

Days % APL blast in PB SD CR SD 4wk CR 4wk SD 6wk CR 6wk 20 40 60 80 100

% CD45.2 in BM

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SLIDE 34

Depleted in CR KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION KEGG_MAPK_SIGNALING_PATHWAY KEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_PHOSPHATIDYLINOSITOL_SIGNALING_SYSTEM KEGG_NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY Enriched in CR KEGG_PPAR_SIGNALING_PATHWAY KEGG_PARKINSONS_DISEASE KEGG_SPLICEOSOME KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION KEGG_RNA_DEGRADATION KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS KEGG_AMINOACYL_TRNA_BIOSYNTHESIS KEGG_PYRIMIDINE_METABOLISM KEGG_HUNTINGTONS_DISEASE KEGG_OXIDATIVE_PHOSPHORYLATION KEGG_RNA_POLYMERASE KEGG_PROTEASOME KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION KEGG_CELL_ADHESION_MOLECULES_CAMS KEGG_RIBOSOME KEGG_ALZHEIMERS_DISEASE KEGG_CITRATE_CYCLE_TCA_CYCLE

1 2 3 4

SD CR

Fold change

OXPHOS genes tRNA/rRNA Mitochondrially- encoded genes

TranscripTonal rewiring in CR

Mitochondrial potenTal

Increased transcripTon of oxydaTve phosphorylaTon genes and oxydaTve metabolism

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SLIDE 35

CR-treated leukemias iniTally slow down their cell cycle (3 weeks)

SD CR

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% SD13 SD14 SD6 LSD1 SD9 LSD1 CR4 CR5 CR6 CR18 LSD1 CR19 LSD1 CR20 LSD1 S-G2-M G1 G0

CR-6 wks

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SLIDE 36

And then resume normal (or accelerated) proliferaTon

SD CR

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% SD13 SD14 SD6 LSD1 SD9 LSD1 CR4 CR5 CR6 CR18 LSD1 CR19 LSD1 CR20 LSD1 S-G2-M G1 G0

CR-6 wks

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SLIDE 37

5000 10000 15000 20000 −3.0 −2.0 −1.0 0.0 dose (number of cells)

log fraction nonleukemic

SD 1/26.279 CR Ratio p-val 1/6.877 3.8 0.0005 Estimated LIC frequency

10 20 30 40 50 60 70 80 90 25 50 75 100

5X104 CR APL 2X104 CR APL 5X104 SD APL 2X104 SD APL Days

CR increases frequency and “aggressiveness” of LICs

Frequency Leukemogenic potenTal aqer transplantaTon (equal numbers)

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SLIDE 38

AdaptaTon of leukemia stem cells to Caloric RestricTon

1 10 100 1000 10000 10 20 30 40 50 SD CR

Total leukemic cells Animal death days

AdaptaTon

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SLIDE 39

The LSD1 Lysine demethylase is involved in the adapTve response to CR

Many OXPHOS genes are targets of LSD1 CR or FasTng increases levels

  • f LSD1

in leukemia cells in vivo

α-ac4n α-LSD1 SD CR 1DF 2DF ADF DF NF

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SLIDE 40

SD SD+LSD1 CR CR+LSD1 100 101 102 103 104 105 106 107 108

total blasts in the BM

CR+LSD1 inhibiTon leads to disease eradicaTon

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SLIDE 41

GeneTc evidence for co-operaTon between LSD1 inhibiTon and Caloric RestricTon

NB4 cells (human APL cell line) NB4 LSD1KO cells ( Saverio Minucci’s Lab)

10 X 106 NB4 cells 10 X 106 LSD KO cells

Standard diet Caloric Restric4on 5 20

SD CR

SD NB4 SD LSDKO CR NB4 CR LSDKO 200 400 600 800

Volume of tumor mass (mm3)

* CR * CR

SD NB4 SD LSDKO CR NB4 CR LSDKO 0.0 0.1 0.2 0.3 0.4

Tumor mass (g)

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SLIDE 42

The insulin/IGF1R inhibitor OSI-906, but not Rapamycin, mimics nutrient deprivaTon and synergizes with the LSD1 inhibitor

50 100 150 50 100

days Percent survival ctrl OSI LSD1 RAPA+LSD1 OSI+LSD1

P=0.014

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SLIDE 43

InhibiTon of phenotypic adaptaTon (to nutrient deprivaTon) eradicates leukemias

Caloric restricTon AZenuaTon of IGF1-signall. AdaptaTon

LIC OXPHOS

No AdaptaTon

LIC exhaus4on Glycolisis

Disease eradicaTon Lsd1 inhibiTon

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SLIDE 44

Phenotypic (non-geneTc) adaptaTon to the changing tumor micro-environment:

  • is criTcal for tumor development
  • is influenced by the specific geneTc make-up
  • f each tumor
  • can be exploited to develop innovaTve anT-

cancer strategies.

Working hypotheses