Pathogenetic Features of Hematopoiesis in MDS: Focus on Aging

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1 Pathogenetic Features of Hematopoiesis in MDS: Focus on Aging Irving L. Weissman, MD Stanford University School of Medicine Director, Institute for Stem Cell Biology and Regenerative Medicine Director, Stanford Ludwig Center for Cancer Stem Cell Research and Medicine Professor of Pathology and Developmental Biology Stanford, California

2 DISCLOSURE I have no relevant financial relationships to disclose.

3 Blood-Forming Stem Cells 1. Development 2. Maintenance 3. Malignant transformation IN VIVO VERITAS Blood forming stem cells make blood, and only blood Ludwig Center at Stanford University

4 Hematopoietic Hierarchy Mouse Human Lin - c-kit + Sca1 + IL7Ra - CD34 - Slamf1 + Tie2 + Lin - CD34 + CD38 - CD9 + CD45RA - HSC Long-term Self-renewal Lin - c-kit + Sca1 + IL7Ra - CD34 + Slamf1 + Tie2 + Lin - c-kit + Sca1 + IL7Ra - CD34 + Slamf1 - Tie2 + Multipotent Progenitors Lin - CD34 + CD38 - CD9 - CD45RA - Lin - c-kit + Sca1 + IL7Ra - CD34 + Slamf1 - Tie2 - Lin - c-kit + Sca1 -/lo IL7Ra - CD34 + FcgR low Slamf1 - Tie2 + Lin - CD34 + CD38 + IL3Ra low CD45RA - CMP CLP Lin - c-kit lo Sca1 lo IL7Ra + Flk2 hi Lin - CD34 + CD38 + CD1 + Lin - c-kit + Sca1 -/lo IL7Ra - CD34 + FcgR low Slamf1 + Tie2 + Lin - c-kit + Sca1 -/lo IL7Ra - CD34 + FcgR low Slamf1 - Tie2 - Lin - c-kit + Sca1 - IL7Ra - CD34 - FcgR - Slamf1 + Lin - CD34 + CD38 + IL3Ra CD45RA - Lin - c-kit + Sca1 - IL7Ra - CD34 - FcgR + MEP PreMEP GMP PreGMP Oligopotent Progenitors Lin - CD34 + CD38 + IL3Ra CD45RA + All isolated MkP EP GP MacP Pro-DC Pro-B Pro-T Pro-NK Lineage restricted Progenitors Akashi, Kondo, Traver, Seita, Miyamoto, Bryder, Spangrude, Ikuta, Morrison, et al Platelets Erythrocytes Granulocytes Macrophages Dendritic cells B-cells T-cells NK-cells Mature effector cells

5 A Model of HSC Ontogeny Cell intrinsic changes Young adult Mid-age Old age Lymphoid gene expression Myeloid gene expression Leukemia genes Self-renewal Myeloid potential Myeloid genes Leukemia genes Lymphoid potential Homing Lymphoid genes 1) The cell-intrinsic functional and molecular properties of aging LT-HSC places the cellular and molecular rationale for the decreased immuno-competence that accompanies aging at the level of the LT-HSC. 2) Age-dependent upregulation of genes frequently activated as protooncogenes in leukemogenesis (17/32 highest.) 3)..and quiescent aged HSC build up DNA DS breaks, repaired upon G to G1 to S transition Rossi, Bryder, Weissman,Seita, Beerman 4) Is this maturation of all HSC, or clonal selection of some HSC? )

6 Clonal Selection or epigenetic programming of HSC with Aging Normal HSC 2 mo Normal HSC 2 years Hypothesis and data: Rossi, Eaves, Muller-Sieburg, Nakauchi, Bryder, Beerman, and Weissman

7 Leukemic cells in AML patients LT STEM CELLS MPP CD34 + CD38 - Thy + Lin - CD34 + CD38 - Thy - Lin - Leukemia Stem Cells [LSC] 5-4% AML-1/ETO + Normal colonies Leukemic blast colonies Miyamoto, Weissman Akashi, PNAS 2: 97: 6924

8 W e Cell of origin progression to leukemia Jak2

9 CML Fialkow: clonal disorder in G,M.E,B cells; Rowley/Nowell bcr-abl translocation; fusion protein in chronic, myeloproliferative phase; LSC proposed to be HSC or MPP; Jamieson and Weissman HSC. No increase in CD34+38lo9+Lin- frequency: competitive advantage vs nl HSC Myeloid blast crisis is at the stage of GMP, and overexpress activated β-catenin;axin inhibits them. 4/7 pts overexpress β-catenin by mis-splicing GSK3 the other inhibitor of β-catenin Jamieson and Weissman, 24, 29

10 CD123 Figure 3. Aberrant GSK3 Expression by Blast Crisis CML Progenitors <PE-A>: CD A. Progenitor FACS Analysis B. Q-PCR of GSK3 Expression C. GSK3 FACS Normal Progenitors CMP MEP <PE-A>: CD <FITC-A>: CD45RA CD45RA GMP CML BC Progenitors CMP MEP GMP <FITC-A>: CD45RA D. Confocal Analysis of -catenin Expression Normal GMP CML GMP % of Max BP CML AP CML CP CML GSK3 <Alexa 45-A>: GSK3b E. GSK3 cdna Sequencing Analysis

11 Identification of Somatic Mutations by Exome Sequencing AML Gene Annotation Normal Tissue (T Cells) Next generation exome sequencing N Mutations In AML AML FACS HSC SU7 PXDN V616I KALRN S44P TET2 Y1649stop TET2 T1884A TMEM8B nt G471A NCRNA2 nt G354A TMEM2 A143T ZRANB1 nt G4659A SCN4B H227N GABARAPL1 nt C1583T DOCK9 A1475V PLAG2G4D P246A CACNA1H R169stop CTCF R339Q GZF1 nt G3835C PRPF6 R527H CXorf36 I225L CXorf66 G321S FLT ITD! Max Jan Thomas Snyder Ryan Corces- Zimmerman Steve Quake Ravi Majeti Irv Weissman Science Translatinal Medicine 212 4(149): 149ra18.

12 Analysis of Single HSC to Identify Pre-Leukemic Clones SKP2 ELP2 PDZD3 FLT3 Leukemic Clonal Expansion TET2 E1357stop TET2 D1384V, SMC1A FLT3, NPM1 Leukemic Clonal Expansion Max Jan Thomas Snyder Ryan Corces- Zimmerman Steve Quake Ravi Majeti Normal HSC Stages of preleukemia progression in HSC clone--- LSC at progenitor stage

13 If you want to know which genes are likely oncogenes, ask the cancer. Ryan corces Zimmerman, Ravi Majeti, and IW Now 21 AMLs PNAS 214

14 Lessons for leukemia and perhaps all cancers 1 In AML, progression is in a blood stem cell clone, while the leukemia stem cells is at the progenitor stage: preleukemic clones of HSC compete with nl HSC 2 There are no leukemias we have found that are leukemias of HSC 3 If this is true for leukemia, it is probably true for all cancers in tissues that regenerate from tissue stem cells

15 CD47 was discovered as a marker of aging RBC by Oldenborg. We found it on m/h AML LSC Hypothesis: Increased expression of CD47 on myeloid leukemia cells contributes to pathogenesis by facilitating evasion of phagocytosis --- SIRPa + Receptor Don t eat me CD47 Eat me Stimulus AML LSC Macrophage Programmed cell removal Net Result: No Phagocytosis Prediction: Increased expression of CD47 on human AML is associated with a worse clinical outcome Traver and IW 1998; Jaiswal, Majeti, Chao,and IW 28.

16 TET47 MOLM 24h TET MOLM 24h 2X TET47 MOLM 24h 2X TET MOLM 24h 4X 4X Chao, Jaiswal, Majeti, Weissman

17 Jaiswal et al 29 Cell. 138(2): l TetCD4 7 MOLM Tet MOLM < A P < C A < A P < C A < A P < C A - A liv < e P liv E < e - P C liv E < e - P C liv E < e - P C liv E < y e - P 7 C liv E - < e - A P C > liv E < : e - P E v E v E v E v E v E v E v < A P < C A - P A R-femur R-tibia R-humerus L-femur L-tibia Injection site < A P < C A - A < A P < C A - A < A P < C A - A e e e liv T E < e P liv E < e - P C liv E < e - P C liv E < e - P C liv E < y e - P 7 C liv E - < y e - A P7 C > liv E- < y : e - A P7 E T E T E T T E M T T E O M T T L EO M M T T L EO MM T v E v E v e E n v t e E n v C t e o E n u v C t n e o E t n u vc : t n eo hucd45

18 Anti-CD47 Antibodies Enable Phagocytosis of AML LSC Human Macrophages NBM1 NBM2 NBM3 NBM4 SU1 SU8 SU9 SU14 SU16 SU18 X SU28 SU32 SU35 Mark Chao, Majeti et al

19 Anti-CD47 Antibody Depletes AML in the Bone Marrow IgG Control Anti-CD47 Anti-CD47 Mark Chao, Majeti et al Cell 29 Cell. 138(2):

20 Volkmer, Willingham, Chin, and IW Inject Tumor Cells Into Peritoneal Cavity Initiate Treatment Evaluate Tumor Growth With Bioluminescence Imaging 2 Weeks 2.5 Months

21 MITRA, JIANG, CHESHIER, RAVEH, IW Inject Tumor Cells Into Left Hemisphere 2 Weeks Initiate Treatment Monitor Tumor Growth With Bioluminescence imaging 8 Weeks Evaluate Tumor Formation

22 Investigation and Targeting of CD47 in Human Cancers Breast Ovarian Bladder Pancreatic Colon Prostate Lung Kidney Leiomyosarcoma Head & Neck Melanoma Monotherapy trials Glioblastoma Medulloblastoma Oligodendroglioma Hepatocellular Carcinoma Gastric Cancer Multiple Myeloma Chronic Myeloid Leukemia Acute Myeloid Leukemia Non-Hodgkin s Lymphoma T-Acute Lymphoblastic Leukemia B-Acute Lymphoblastic Leukemia In all cases tested with metastatic tumors, the metastases are eliminated.

23 Precancer cells express calreticulin, and emergent cancer clones overcome this with CD47 Increased expression of CD47 on myeloid leukemia cells contributes to pathogenesis by facilitating evasion of phagocytosis --- SIRPa Don t eat me CD47 lrl + Receptor Eat me Stimulus AML LSC Macrophage Lrp 1 calreticulin Chao, Jaiswal, Weissman-Tsukamoto, Majeti, and IW Science Transl Med 21. 2(63)

24 Toll like receptor agonist incubation of macrophages increases anti-cd47 mediated phagocytosis of cancer cells Mingye Feng, Volkmer, Weissman

25 TLR agonists synergize with anti-cd47 to eliminate a prostate cancer xenograft in vivo

26 BTK is expressed in many blood lineage cells, including macrophages

27 Phagocytosis of tumor cells is inhibited by the BTK inhibitor, ibrutinib Mingye Feng, Volkmer, Weissman

28 Mingye Feng, Volkmer, Weissman Anti calreticulin blocking antibody inhibits phagocytosis when incubated with macrophages, but not with tumor cells

29 Precancer cells express calreticulin, and emergent cancer clones overcome this with CD47 Increased expression of CD47 on myeloid leukemia cells contributes to pathogenesis by facilitating evasion of phagocytosis --- SIRPa Don t eat me CD47 lrl + Receptor Eat me Stimulus AML LSC Macrophage Lrp 1 calreticulin Calreticulin is an ER protein that is secreted following TLR to BTK signalling; BTK phosphorylates calreticulin, which in the ER is leaved to separate from KDEL domain. py-calr binds to lrp-1 or tumor cell. Mingye Feng,J.Volkmer, and Weissman: PNAS 215

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32 hcd45 Chimerism / 5 HSC HSC / CD34+ Frequency A HSC frequency is similar in MDS and age-matched controls 4% N.S. 3% 2% B MDS clone dominates HSC pool 7(-) nuclei Normal nuclei SU1 SU2 1% 1% % 1% % Normal age-matched MDS C Xenotransplantation chimerism D Clonal dominance post-xenotransplantation 1% 7(-) nuclei normal nuclei NBM N.S. 7(-) MDS SU3 2 4 % of total SU1-NSG1 SU1-NSG2 SU2-NSG1 SU2-NSG2 SU3-NSG1 SU3-NSG2 2 4 % of total MDS is a pre-aml disease of older patients in which a cytopenia Precedes leukemia. MDS HSC outcompete normal HSC in MDS patients and in transplanted NSG mice Wendy Pang, John Pluvinage, Chris Park, IW, et al

33 Phagocytic Inex Relative MFI Phagocytic Inex Relative MFI Relative MFI % of maximum A CRT not increased on MDS HSC B Increased cell-surface CRT on MDS CD34+CD38+ cells 6 N.S Normal HSC MDS HSC * NBM MDS Normal MDS CRT Expression C Increased CRT on MDS GMP D Increased phagocytosis of MDS CD34+CD38+ cells Normal + PBS 1 ** 1 *** MDS + PBS Normal GMP MDS GMP Normal MDS Calreticulin expressed on MDS progenitors, but not HSC, causes programmed cell removal Wendy Pang, John Pluvinage, Chris Park, IW, et al E CRT-blocking peptide abrogates phagocytosis of MDS CD34+CD38+ cells Normal + CRT-blocking 4 **** 3 2 MDS + CRT-blocking 1 Normal MDS

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35 Wendy Pang, Weissman, Schrier

36 Conclusions MDS HSC outcompete normal HSC in patient and xenotransplants The MDS-initiating cell resides in the HSC compartment High CRT predisposes MDS myeloid [GMP, MkP, EP] progenitors for programmed cell removal Increased CD47 expression is a crucial step in the progression from MDS to AML Calreticulin and LRP1 are potential therapeutic targets Wendy Pang, John Pluvinage, Chris Park, IW, et al

37 BCL2 blocks apoptosis, but not programmed cell removal of neutrophiles: Lagasse and Weissman JEM 1994 Apoptotic alive

38 PCDeath and PCRemoval PCD is accompanied by PCR; blocking PCD with bcl2 does NOT block PCR [Lagasse and Weissman 1994, JEM]. PCR prevents inflammation. All cancers defeat PCD: p53, bcl2, bax,nfkb, etc All cancers defeat PCR: calreticulin, Ph-serine, asialoglycoprotein are eat me signals countered by the CD47 don t eat me signal. Stimuli that induce PCD and/or PCR develop cell competition/selection in pre-cancer lineages that can result in cancer clones

39 Supported grants from NCI, NHLBI, Calif Inst Reg Medicine, Lacob Foundation, Siebel SCI Lokey Stem Cell Institute at Stanford Ludwig Center at Stanford Institute for Stem Cell Biology and Regenerative Medicine

40 Wendy Pang, Weissman, Schrier

41 Wendy Pang, Weissman, Schrier