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

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 2017

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 CSC Symmetric Extended divisions self-renewal Viale et al, Nature 2010 CSC Cicalese et al, Cell 2011 Tomilov et al, Aging Cell 2011 Pece et al, Cell 2012 CSC Pasi et al, Cell Death & Diff, 2012 Gambino et al, Aging Cell 2012 Insinga et al, PNAS 2013 Migliaccio et al, Aging Cell 2013 CSC A Santoro, T Vlachou; under revision Progenitor Pr. reprogramming into CSCs

Cancer Stem Cells (AMLs, Breast Cancer): • Altered self-renewal of CSCs is due to aZenuated p53 signalling and acTvaTon of Myc CSC Symmetric Extended p53 divisions self-renewal Viale et al, Nature 2010 CSC Myc Cicalese et al, Cell 2011 Tomilov et al, Aging Cell 2011 Pece et al, Cell 2012 CSC Pasi et al, Cell Death & Diff, 2012 Gambino et al, Aging Cell 2012 Insinga et al, PNAS 2013 Migliaccio et al, Aging Cell 2013 CSC A Santoro, T Vlachou; under revision Progenitor Pr. reprogramming into CSCs

The p53:Myc expression signature is predicTve of clinical outcome, independently of other known risk factors (4 independent cohorts; 892 pa4ents) significant decrease in Same power in ER+ the disease-free survival paTents of the UP cohort

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 • CSC Symmetric Extended divisions self-renewal Viale et al, Nature 2010 CSC Cicalese et al, Cell 2011 Tomilov et al, Aging Cell 2011 Pece et al, Cell 2012 CSC Pasi et al, Cell Death & Diff, 2012 Gambino et al, Aging Cell 2012 Insinga et al, PNAS 2013 Migliaccio et al, Aging Cell 2013 CSC A Santoro, T Vlachou; under revision Progenitor Pr. reprogramming into CSCs

High Clonal Heterogeneity within the pool of LSCs X1 No 5-FU + 5-FU X2 X2 No 5-FU No 5FU X3 X3 100% 90% Human AMLs grown 80% 70% in immunodeficient mice (PDX) 60% 50% 40% 30% 20% 10% 0% � 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

High Clonal Heterogeneity within the pool of LSCs Serial transpl. X1 No 5-FU + 5-FU X2 X2 No 5-FU No 5FU X3 X3 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% � 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

High Clonal Heterogeneity within the pool of LSCs Serial transpl. Serial transpl. + 5FU X1 No 5-FU + 5-FU X2 X2 No 5-FU No 5FU X3 X3 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% � 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

Limited geneTc heterogeneity within the pool of LSCs X1 M3 AMLIEO20_X2_no5FU_ID2_freq.vaf AMLIEO20_X2_5FU_ID2_freq.vaf AMLIEO20_CT_X1_M3_freq.vaf X2 No 5FU X2 + 5FU X2 + 5FU X2 No 5FU X1 M3 ID2 ID2 ID2 ID2 Biological heterogeneity exceeds geneTc heterogeneity by one order of magnitude Thalia Vlachou, unpublished

� 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

Effects of obesity on the self-renewal of Leukemia Stem Cells Anna Giulia Sannarico, PhD Luca Mazzarella MD-PhD

Body-mass index (BMI) correlates with worse prognosis in the Acute PromyelocyTc Leukemia (APL) subtype of AMLs Incidence of relapse Gray test p-value: 0.029 Overweight/Obese Underweight/Normal Years from induc4on Breccia et al., Blood 2012

Higher incidence of FLT3-ITD mutaTons in the obese APL-paTents Incidence of relapse FLT3-ITD Gray test p-value: 0.029 Incidence: Overweight/Obese 28% 9% Underweight/Normal χ2 test p-value: 0.0005 • the most frequent Years from induc4on mutation in AMLs (~33%) • linked to bad prognosis

Driver mutaTons in AMLs are frequently found in rare subclones, including FLT3 VAF: 0,001%-5% Duplex Sequencing 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 / (Schmi' MW et al., PNAS 2012; PER1 / / 2 TOTAL 26 28 8 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

Obesity confers a compeTTve advantage to the FLT3-ITD BM And increases disease aggressiveness of FLT3-ITD transgenic mice ITD-SD ITD-SD ITD-HFD EE Myeloid compartment 100% Spleen weight 80% (2yrs old mice) Ki67 60% grams PBL 40% ITD-HFD 20% 0% sd12 sd22 sd11 sd15 sd02 sd03 sd19 sd13 sd10 sd01 sd09 hfd08 hfd23 hfd17 hfd19 hfd18 hfd07 hfd01 hfd15 hfd22 hfd20 hfd02 EE HFD SD HFD SD 3/21 11/23 FLT3-ITD WT * p < 0.05 Competitive BM transplantation Ki67 Spleen FLT3-ITD mice from G. Gilliland

Obesity might select FLT3-ITD mutaTons through adipose-Tssue - secreted adipokines. A B Cytokine X Lean Obese IL6 IGF1 IL6 IGF1 IL1 TNFa IL1 TNFa Leptin Insulin Leptin Insulin Legend PML/RARa PML/RARa FLT3ITD

The oncogenic potenTal of the FLT3-ITD mutaTon depends on Insulin/IGF1 signalling IL3R IL3 BaF3 Depend. IL3: - +

The oncogenic potenTal of the FLT3-ITD mutaTon depends on Insulin/IGF1 signalling IL3R ITD51 and ITD78 IL3 BaF3 Depend. IL3 BaF3 Independ. FLT3-ITD IL3: - + - +

The oncogenic potenTal of the FLT3-ITD mutaTon depends on Insulin/IGF1 signalling IL3R IL3 BaF3 Depend. IL3 BaF3 Independ. FLT3-ITD IL3: - + - + BaF3 + IGF1/Insulin IL3 FLT3-ITD OSI-906 Depend. inhibitor

The oncogenic signaling of the FLT3-ITD mutaTon depends on Insulin/IGF1 signalling OSI uM 0 1.2 2.5 5 10 0 1.2 2.5 5 10 0 1.2 2.5 5 10 0 1.2 2.5 5 10 Vinculin p-Akt p-S6 p-STAT5 FLT3-ITD 78 Ba/F3 FLT3-WT FLT3-ITD 51

IGF1 potenTates the signaling potenTal of FLT3-ITD IGF1 ng/ml: 0 10 100 0 10 100 0 10 100 0 10 100 Vinculin p-Akt p-FoxO p-S6 Ba/F3 FLT3-WT ITD 51 ITD 78

The ITD mutaTon increases the immature, non-glycosylated form of FLT3, and targets FLT3-ITD to the ER (Ergic compartment) FLT3wt Mature Surface expressed FLT3 Immature “ER”-retained Unglycosylated Ac4n Ba/F3 FLT3-ITD induces ER stress FLT3-ITD p-eIF2a p-PERK PERK eIF2a Vinculin Vinculin DMSO Thapsigargin DMSO Thapsigargin stress-sensing kinase PERK

FLT3-ITD induces a sustained UPR response Ba/F3 ITD ATF4 () - + - + BrefeldinA BiP SREBP1 imm p-eIF2a Vinc SREBP1 mat eIF2a ATF Transcr. Fact. SREBP2 imm BiP Ba/F3 ITD78 Brefeldin - + - + SREBP2 mat BiP Vinculin Tubulin PDIA6 Tunicamycin (Hrs) 0 1 24 TranslaTonal iniTaTon factor 2a (eIF2a) PDIA3 SREBP2 transcripTon factor (cap-dependent protein translaTon) (sterol regulatory element- Tubulin binding protein) PDIA3/6 disulphide isomerases the BiP ER-chaperone TranscripTonal TranslaTonal Colesterol Reprogramming Reprogramming Reprogramming

Obesity increases the UPR response to FLT3-ITD induced ER stress RNAseq on FLT3-HO mice Gene SD (TPM) HFD (TPM) 9 Sqle 38,56 46,76 8 Hmgcr 48,20 65,97 SD 7 Hmgcs1 27,43 35,89 HFD 6 Srebf2 120,62 147,23 5 4 Gene WT (TPM) FLT3 (TPM) 3 Me2 52,16 83,99 2 RNAseq on SD mice Sqle 23,71 38,56 1 0 Hmgcs1 21,84 27,43 Hspa1a Hspa1b Scd2 93,40 188,13 Chaperone synthesis Fasn 53,06 80,04 Slc16a1 24,29 50,39 Srebf2 124,44 120,62 Cholesterol metabolism

Obesity favors resoluTon of ER-stress induced by the ITD mutaTon (UPR response) and acTvates the full oncogenic potenTal of FLT3-ITD FLT3-unfolding ITD-mutaTon (defecTve signaling) ER-stress HSF1 ResoluTon of Obesity UPR GSH ER stress SREBPs AcTvaTon of the FLT3-ITD Full signalling potenTal