Translational regulation of Myeloid Cell Differentiation: Novel Mechanisms and Players PDCD4, DAP5 and eif2α

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1 Translational regulation of Myeloid Cell Differentiation: Novel Mechanisms and Players PDCD4, DAP5 and eif2α Bulent Ozpolat,M.D., Ph.D. The University of Texas M.D. Anderson Cancer Center Department of Experimental Therapeutics, Houston, TX

2 LEUKEMIA 33,440 new cases of leukemia will be diagnosed in the US this year 21,700 individuals will die of the disease

3 LEUKEMIA Immature malignant progenitor cells in circulation BM infiltration & failure: RBC- Anemia WBC- Infections Platelets- Hemorrhage Acute Leukemia Chronic Leukemia AML % survival with Standard Rx ALL CML CLL Gleevec (STI-571)

4 Acute Promyelocytic Leukemia (APL) Type of Acute Myeloid Leukemia (M3-AML) The incidence is 3-fold higher in Hispanic/latinos Characterized by translocation (15;17) (q22;q21) that leads to expression of PML-RARα fusion receptor Lin et al, TAG 1999

5 Normal Hematopoiesis G-CSF, GM- CSF ATRA IL-3 Granulocytes (Neutrophils, Basophil, Eosinophil) Monocytes / Macrophages RBC Platelet Mature Blood Cells HSC LEUKEMIA Differentiation block Leukemic blasts Mature Blood Cells

6 All-trans Retinoic Acid (ATRA) is used for the Differentiation therapy of APL Differentiation therapy of APL - A natural derivative of Vitamin A (Retinol) - induces differentiation - First line standard therapy in APL ATRA

7 ATRA treatment of Acute Promyelocytic leukemia (APL) ATRA alone induces Complete remission in 90-95% APL patients Immature APL cell Mature granulocyte ATRA

8 Molecular mechanisms of ATRA induced differentiation is not well understood Immature myeloid cell +ATRA? 1300 increased 1700 decreased Mature myeloid cell Expression of ~3000 gene is altered 85 % inhibition in protein expression

9 GOAL : Determine the molecular mechanism of ATRA induced terminal differentiation of myeloid cells Immature APL cell (NB4) Differentiated granulocyte ATRA? Ozpolat et al, Mol Can Res 2007

ATRA induces differentiation in APL (NB4) cells CD11b CD11c 60 % Differentiation (CD11b + ) 50 40 30 20 10 0 0 12 24 48 72 Time (h) 80 %

10 ATRA induces differentiation in APL (NB4) cells CD11b CD11c 60 % Differentiation (CD11b + ) Time (h) 80 % Differentiation (CD11b + ) ATRA (μm) % Differentiated (CD11c+) Control ATRA (72h)

11 Differentiation in maturation resistant APL cells (NB4.R1) cannot undergo maturation by ATRA NB4 NB4.R1 % CD11b positive ATRA (μm)

12 ATRA suppresses translational regulators during terminal differentiation of APL cells by Proteomics analysis eif4g Hsp70 eif4a hnrnpk eif6 Ribosomal Acidic Protein P2 CRABPII Harris &Ozpolat, Blood. 104 (5):

13 ATRA downregulates expression of translational regulators in APL cells Peptides ATRA Treated/Non Mr Accession Functional (kda) Sequen Treated Number Protein Name Classification (theor) pi ced (8 h) (48 h) (72 h) (96h) Q04637 Translational initiation factor 4G protein synthesis AAH19816 Heat Shock 70 kda protein 8 chaperone * 0.39 P04765 Translation initiation factor 4A protein synthesis Q9Y kDa TATA box-binding protein transcription P Tropomyosin alpha 3 chain cellular organization * * P protein epsilon signal transduction P56537 Translation initiation factor 6 protein synthesis P52597 hnrnp K mrna processing Q14974 Importin beta-1 cellular transport * P50213 Isocitrate dehydrogenase metabolism N/A P47755 F-actin capping protein alpha-2 subunit structural Q9UL46 Proteasome activator complex sub 2 cellular defense P F-actin capping protein beta subunit structural * P78417 Glutathione transferase omega 1 not defined P18669 Phosphoglycerate mutase 1 glycolysis * Q14019 Coactosin-like protein cellular organization * P29373 Retinoic acid-binding protein II cellular defense P27482 Calmodulin-related protein NB-1 signal transduction * * 1.7 P S acidic ribosomal protein P2 protein synthesis N/A P19105 Myosin regulatory light chain 2 cellular organization P27482 Rho GDP-dissociation inhibitor 2 signal transduction P29218 Inositol-(or 4)-monophosphatase signal transduction N/A 2.1* All values are statistically significant (non-treated versus ATRA-treated), p < 0.05 unless indicated * p

14 Central hypothesis: Translational suppression plays a role in ATRA-induced differentiation 40S initiation factors bind eif6, IF-2 eif4ai m 7 G ATP ADP met-trna i-gtp eif2 43S m 7 G AUG ATP (A) n eif4g eif4e eif4ai N 4E eif4f 4A mnk1 C AUG (A) n m 7 G 48S AUG ADP met-trna i -GTP eif2 (A) n PML-eIF4E mrna export removal of initiation factors eif5 eif2-gdp transcription pre-rna synthesis maturation m 7 G Met AUG (A) n mrna splicing Nucleus 60S P2-P1 P0 PML m 7 G Met-aa n 80S trna (A) n + GTP-eEF1A-aminoacyl-tRNA GDP eef1a aminoacyl-trna + eef1b alpha eef1bbeta eef1bgamma

15 Translational Control is lost in Cancer cells Deregulation of mrna translation can contribute to cell transformation and the malignant phenotype Translational factors are overexpressed in different cancers types eif4e- CML, breast, melanoma, neuroblastoma, colon, prostate, lung cancer eif4a- Melanoma eif4g - Lung cancer (SCLC) hnrnpk- CML, Lung cancer eif2- CML, colon eef1α- Pancreas and prostate Transfection of cells with eifs leads to cell transformation eif4e (De Bendetti al, 1997) eif4g (Fukuchi et al, 1997) eif2α (Donze et al, 1995) eef1α (Tatsuka et al, 1992) Normal Cell Malignant

16 Signaling pathways activated by Cytokines, Growth Factors or Stress Regulate Translational control Mitogens, Cytokines, Growth Factors MAPKK MAPK MEK ERK p38 Ras PI3K AKT PKR P eef2 Mnk mtor Rapamycin P eif4e p 4E-BP1 p P eif4g P p70s6k P eef2 S6K P eif4b Ribosomal S6 eif4a Translation Growth & Proliferation

17 Hypothesis Leukemia APL Translational activation proliferation Differentiation? Translational Inhibition

18 Hypothesis: ATRA supresses translation by inducing translational inhibitors (PDCD4, DAP5 and p- eif2α) during the differentiation Differentiation PDCD4 DAP5 p-eif2α Translational Inhibition

19 PDCD4 tumor suppresor and DAP5 are Novel inhibitors of translational initiation AAAAA PABP IF4E mrna IF2 eif4g IF4A eif3 IF4A i AUG 40 S Translation on PABP Ozpolat et al, Gene Ther (in press) AAAAA IF4E IF4A eif4g DAP5 PDCD4 mrna i AUG Translation off

20 PDCD4 (Programmed Cell Death 4) A novel tumor suppressor protein (Yang et al, 2003) Inhibits neoplastic transformation Specific inhibitor of translation initiation - inhibits helicase activity of eif4a Its expression is downregulated in many tumors

21 ATRA induces PDCD4 expression during granulocytic differentiation of APL cells ATRA (1 μm) ATRA (0.1 μm) Time (h): PDCD β-actin ATRA (1 μm) Time (h): PDCD4 β-actin Relative PDCD4 protein expression 4 ATRA (1μM) Time (h)

22 ATRA fails to induce PDCD4 in differentiationresistant APL cells HL60 HL60R Time (h): PDCD4 β-actin ATRA (1 μm)

23 ATRA induces marked PDCD4 expression in primary APL cells isolated from APL patients APL patient # 1 - ATRA Control ATRA PDCD4 β- actin PDCD4 β- actin PDCD4 β-actin APL patient # 2 - ATRA APL patient # 3 - ATRA CD11c (PE) NT-72 h ATRA-72 h 37 %

24 Knockdown of PDCD4 by sirna inhibits ATRA-induced granulocytic differentiation of APL CD11c CD11b % CD11c Inhibition TR Control sirna P<0.05 PDCD4 sirna % CD11b Inhibition TR p<0.05 control sirna P<0.05 PDCD4 sirna

25 DAP5 (Death Associated protein 5)

26 ATRA induces DAP5 in APL and AML cells NB4 ATRA HL60 Time (h): 0 24 Control ATRA DAP5/p97 β-actin

27 ATRA-resistant APL (NB4.R1) cells do not express and fail to upregulate DAP5 NB4 NB4.R1 ATRA ATO ATRA ATO DAP5/p Actin

28 Downregulation of DAP5 partially inhibits ATRAinduced granulocytic differentiation of APL cells 35 % CD11b inhibition TR Control sirna #1 #2 #3 PDCD4 sirna

29 eif2α (Eukaryotic Initiatin Factor 2alpha)

30 ATRA induces phosphorylation (Ser51) of eif2α during the granulocytic differentiation of APL cells Time (h) p-eif2α eif2α ATRA (1 μm) ATRA (0.1 μm) Relative p-eif2α expression NB+ATRA (1 μm) p-eif2α/actin p-eif2α/eif2α β-actin ATO (0.4 μm) Time (h) Time (h) p-if2α eif2α β-actin Relative p-eif2α expression p-eif2α/actin p-eif2α/eif2α Time (h)

31 Knockdown of eif2α blocks ATRA-induced differentiation of APL cells % Differentiation (CD11b+) NT TR eif2α sirna PKR sirna Control sirna ATRA

32 ATRA inhibits translation initiation during terminal granulocytic differentiation in APL Time (h): ATRA (1 μm) PDCD4 DAP5 p(ser51)-if2α β-actin p-4eb-p1 β-actin Time PDCD4 DAP5 p-eif2α p-4e-bp1

33 ATRA-Induced translational control involves multiple mechanisms regulating cap-dependent and global translational control 60S 40S A) 60S 60S eif6 + eif6 40S eif3, eif1a p-eif2α eif1a eif3 40S eif1a eif2 eif3 + GTP 40S eif2 Met-tRNA i m et-tr NA i -GTP PDCD4 DAP5 4E-BP1 C) eif4g 60S B) PML m 7 G P1, P2 eif4ai eif4e eif4ai 4E 4A N terminus eif4f 60S rrna P0 P2-P1 PO 4 4A mnk1 C terminus m 7 G ATP ADP AUG m 7 G rem oval of initiation factors Met C n 80S m 7 G eif1a eif3 43S m 7 m et-tr N A i -GTP G eif2 AUG (A) ATP n (A) n ADP eif1a eif3 eif2 AUG 48S (A) n Met AUG m et-tr N A i -GTP (A) n eif2-gdp (A) n D) GTP-eEF1A-aminoacyl-tRNA aa stop codons "UAA, UAG, UGA" + tr N A GDP eef1a eef1b alpha eef1bbeta eef1bgamma (am inoacyl-trna) GTP eef1a

34 Summary ATRA induces translational suppression by PDCD4, DAP5, 4E-BP1 and p-eif2α during the granulocytic differentiation Induction of PDCD4 tumor suppressor protein is involved in granulocytic but not monocytic differentiation Inhibition of PDCD4, DAP5, eif2 block ATRA-induced granulocytic differentiation ATRA-resistant cells are not able to upregulate translational inhibitors (PDCD4 and DAP5) and cannot translocate them into nucleus PI3K/Akt/mTOR pathway negatively regulates PDCD4 and DAP5 expression in AML and solid tumors

35 Conclusions ATRA suppresses cap-dependent and global translational through multiple mechanisms during terminal granulocytic differentiation PDCD4, DAP5 and eif2α play a role in terminal differentiation program towards granulocytic lineage Lack of or altered of translational control may be involved in disease pathogenesis (leukomogenesis) and resistance/relapses in APL patients

36 Future Goals: 1. To determine the role of translational regulators (PDCD4,DAP5, eif2a) in response to therapy, resistance/relapse and survival in APL and AML patients 2. To identify downstream molecular targets of ATRA-induced differentiation in APL patients samples 3. To determine molecular mechanisms of ATRA resistance and relapses in APL patients using proteomics - NB4 cell lines (Sensitive vs. ATRA-resistant) - Patient APL cells (Before treatment vs. the relapse)

37 Grant support: U54 (PI-GBL, Co-Inv BO) Acknowledgements M.D. Anderson Cancer Center Gabriel Lopez-Berestein, M.D. Ugur Akar, M.D., Ph.D. Isabel Zorilla-Calancha, M.D. Magali Barrilla, M.D. Department of Leukemia Steven Kornblau, M.D. Elihu Estey, M.D. Department of Statistics Jianhua Hu, Ph.D. Los Alamos National Laboratory Xian Chen, Ph.D. Michael Harris, Ph.D. National Cancer Institute Nancy Colburn, Ph.D. University of Puerto Rico Cancer Center Maribel Tirado-Gomez, M.D. Ben Gurion University-Israel Michael Danilenko, Ph.D.

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