in MDS Amit Verma Albert Einstein College of Medicine

Transcription

1 Signal Transduction Inhibitors in MDS Amit Verma Albert Einstein College of Medicine

2 Ineffective Hematopoiesis in MDS Hypercellular marrows with decreased peripheral blood counts Increased Cell death (Apoptosis/Cell cycle arrest) Decreased roliferation / Differentiation ro Apoptotic / Myelosuppresive/ Inflammatory cytokines Tumor necrosis factor TNF mrna in MDS BMs TNF production by MDS Macrophages ro-apoptotic Type 1 receptors increased in low grade MDS and decreased in high grade cases and AML Anti-TNF therapies (Remicaid & Enbrel) show efficacy Transforming growth factor membrane bound TGF on progenitors conc. in serum Vascular Endothelial growth factor (VEGF) Secreted by malignant clone and ALI cells Expression in BM correlates with disease severity Higher expression of high affinity VEGFR1 Interferon (IFN g) Interleukin 1 Fibroblast Growth factor (FGF) Hepatocyte growth factor (HGF) Macrophage Inhibitory rotein (MI ) Thalidomide can degrade TNF mrna

3 Model of athogenesis of MDS Mutations / Genetic/ Epigenetic event Normal Clones Abnormal Clone + TNF-a IFN VEGF TGF Cytokine Signal Transduction Inhibitors Fas EARLY STAGE Stroma Apoptosis LATE STAGE Leukemia Apoptosis Methylation of tumor suppressor genes Navas et al, Blood 26, 18(13):417 Zhou et al Blood 28, 112(8):3434, Navas et al, Leuk and Lymph, 29

4 MA (Mitogen Activated rotein) Kinases Erk 1 / 2 (Extracellular Signal Regulated Kinase) JNK 1/2/3 Jun Amino Terminal Kinases p38 ERK5 Growth & Differentiation Stress Osmolarity, Oxidative, Apoptosis Inflammation, Growth, Cell Cycle, Cell Death ACTIVATED BY CYTOKINES Serum Induced Early Gene Expression IFN TNF TGF 38 MAK Hematopoietic stem cell apoptosis / cell cycle arrest Verma et al, JBC22, 277(1):7726, Verma et al J Immunol 22, 168 (12)

5 p38 MAK is overactivated in MDS A CONTROLS LOW GRADE MDS HIGH GRADE MDS B NO. OF CELLS / HOT FIELD N=9 <.5 N=9 N=6 Control Low Grade MDS High Grade MDS C G D 38 IHC STAININ NTENSITY / FIELD - IN.2.1 N=9 Control N=9 Low Grade MDS <.5 <.5 N=6 High Grade MDS

6 p38 MAK activation correlates with apoptosis in MDS

7 38 Inhibitors (SCIO- 469) can decrease apoptosis in MDS CD34+ progenitors ercentage of cells with 5q- Baseline 48hr+ Untx 48hr+ SCIO-469 MDS MDS

8 p38 MA kinase inhibitors can stimulate hematopoiesis in MDS Feature No. atients (%) Erythroid 11/47(23) Mj Major 6(13) A Randomized, Multicenter, Open-Label, Modified Dose- Ascension, arallel Study of the Safety, Tolerability, and Efficacy of Oral SCIO-469 in atients With Myelodysplastic Syndromes ClinicalTrials.gov Identifier: NCT latelet Major Neutrophil Major Time to Response (E,,N)(Days) Median Range 2/18 (11) 2(11) 1/31 (32) 6/19 72, 78, 72 (56-98), (56-1), (56-1) Week 16 Hematologic responses in patients with MDS on SCIO-469 treatment: Responses were seen in all three hematopoietic lineages. Responses were defined by IWG criteria for 56 consecutive days during 16 wk period. Ongoing: ARRY-614 in atients With Low or Intermediate-1 Risk MDS

9 TGF β TGF- signaling cascade Transphosphorylated TβR complex R III R II R I Smads 6/7 Smad2 Smad3 hosphorylated Smad 2/3 complex Smad TIF γ Inhibition of proliferation Erythroid differentiation

10 Smad2/3 is activated in MDS Bone Marrow Samples Fig 1

11 WHY IS TGF- SIGNALING ACTIVATED? Smad-7, a negative regulator of TBRI is reduced in a meta-analysis of gene expression studies of MDS CD34+ cells G.E) Log2 (G Controls MDS

12 Smad-7 protein is reduced in MDS Bone Marrows Controls MDS ad7 IHC Sma <.5, Fishers exact test

13 Smad-7 knockdown leads to increased TGF mediated transcription...that t can be inhibited by inhibition of TBRI 5 LTR CMV Smad7 shrna tgf IRES uro r WRE Amp r puc Sv4 ORI sinltr ion Normalized Sm mad7 Expressi Scr shrna Smad7 shrna Fold Ch hange Scr shrna Scr shrna Smad7 Smad7 + TGF + TGF shrna shrna + LY + TGF + TGF + LY Smad2/3 binding leads to luciferase expression

14 Knockdown of TBRI can abrogate the inhibitory effects of TGF and stimulate MDS hematopoiesis CMV tgf IRES uro r TBRI shrna WRE 5 LTR sinltr Amp r puc Sv4 ORI D % Co ontrol Erythroid Colonies F % Anti-TBRI-shRNA =.3 Control TGF-b 16+3% Scr-shRNA E Anti-TBRI shrna + TGF Scr shrna + TGF GF+ hase 6 Scr shrna No. of Colonies anti-tbri =.49 =.77 BFU- E CFU- GM BFU- E CFU- GM BFU- E CFU- GM BFU- E CFU- GM BFU- E CFU- GM BFU- E CFU- GM MDS 1 MDS 3 MDS 4 MDS 5 MDS 2 MEAN

15 A TGF LY-215 LY and SD-28, are small molecule TBRI inhibitors that can inhibit smad2 activation in hematopoeitic cells and reduce TGF mediated gene transcription in bone marrow stromal cells B C KG-1 TGF SD-28 K562 CFU- Erythroid TGF SD-28 p-smad2 smad2 p-smad2 smad2 p-smad2 smad2 D 5 Fold Luc ciferase Activ vity TGF-b TGF+SD Control TBR I- KR

16 Alb/TGF+ transgenic mice develop anemia and mimic human bone marrow failure A Alb/TGF+ 8wks old H&E Staining WT 8wks old Megakaryocytes Erythroid progenitors Reticulin staining 4x Fibrosis WBC (x1^3/cc) WT Alb/TGF+ Hgb (g/dl) Hct (%) lt (x 1^5/cc)

17 TBRI inhibitors can improve hematopoeisis in TGF overexpressing mice and can raise their hematocrit lacebo Co olonies/1 BM cells lacebo SD-28 3mg/kg/d p=.3 Erythroid p=.4 Myeloid -Sm mad2 IHC LY it se in Hemotocri er treatment Increas afte lacebo 1mg/kg/d =.4 2mg/kg/d SD-28 x 2 wks 3mg/kg/d SE IN T MEA AN INCREAS HEMATOCRIT 18 N=17 16 = N=17 LACEBO LY-215 TGF transgenic mice treated for 2 weeks

18 TBRI Inhibitors Stimulate Erythroid and Myeloid Colony Formation from MDS BM Mononuclear Cells % Control Colo onies BFU-E CFU-GM Control SD-28 1nM SD-22 2nM 1 Erythroid Control LY 2nM LY 5nM Myeloid Rationale for future clinical trial of TBRI inhibitor in low risk MDS

19 Acknowledgements Vermalab Li Zhou Yongkai Mo Yiting Yu Tushar Bhagat Davendra Sohal Einstein John Greally Ari Melnick Maria Figueroa athology Christine McMahon Howard Ratech G. Kong Samples UTSW, Dallas Simrit armar Kansas City VAMC Suman Kambhampati Moffit Cancer Center Alan List Cleveland Clinic Jaroslaw Maciejewski