Deciphering HoxA9 function in multipotential progenitor biology and

Deciphering HoxA9 function in multipotential progenitor biology and B cell development Kay L. Medina, PhD Associate Professor Dept. of Immunology Mayo Clinic College of Medicine

Overview of adult hematopoiesis in the mouse MEP GMP CMP HSC STRC ELP ALP NKP ETP Mac pDC CDP GMLP Thymus Ery ILC2 BLP Meg PMN cDC PrePro-B Pro-B

Sequentially-acting regulatory circuits orchestrate B cell fate specification and commitment HoxA9 E2A HSC STRC Ikaros PU.1 Flt3 E2A B220 CD19- GMLP E2A, Flt3 ELP IL-7R c-Myb ALP EBF1 FOXO1 BLP IL-7R PrePax5 Pro-B B220 CD19 IL-7R Pro-B Pax5 DH-JH VH-DJH

Hoxa9 regulates Flt3 in lymphohematopoietic progenitors Gwin and Medina, JI, 2010

Inverse expression of HoxA9 and EBF1 in lymphoid/B cell precursors hoxa9 ebf1 CLP/BCP

HSC/MPP program Alternate lineage programs Hoxa9 Flt3 MyeloidLymphoid progenitor Hoxa9 Flt3 IL7R All-Lymphoid progenitor Hoxa9 Flt3 IL7 EBF1 B-Lymphoid progenitor IL7R EBF1 Pro-B cell es, microRNAs in ok m he c , s le cu le tors, signaling mo Transcription fac B cell program

hoxa9-/- flt3l-/- mice exhibit a severe lympho-hematopoietic block Gwin and Medina, JI, 2013

HoxA9 and Flt3 signaling function together to establish the multipotential progenitor pool competent to undergo lymphoid priming WT-RAG1-GFP Hoxa9-/- RAG1-GFP ELP Flt3L-/- RAG1-GFP ELP ELP Flt3 ELP Hoxa9-/-Flt3L-/- RAG1-GFP GFP GFP GFP GFP HSC STRC hoxa9-/- flt3l-/- GMLP

The role of HoxA9 in B cell fate specification. EBF1 FOXO1 GFI1b ebf1 foxo1 gfi1b

HoxA9-/- hematopoietic progenitors have impaired IL-7 signaling Lin- ckit Flt3 MFI IL-7R # cells x 105 B6 HoxA9 B6 HoxA9-/- IL-7R

Culture model to identify HoxA9 target genes critical for B cell development Lin- c-kit cells cultured for 17 days in SCF FL Tpo IL-6 WT HoxA9-/- c-kit B Sca-1 CD34 CD150 Flt3 HoxA9-/- MPPs B220 # cells x 106 WT MPPs IL-7R CD19 CD19

RNA-Seq Platform Day 17 MPP Harvest Switch 24 hr CLP IL7,SCF,F L 2 independent experiments Sequencing Sequencing WT MPP-1 WT MPP-2 HoxA9-/- MPP-1 HoxA9-/- MPP-2 WT CLP-1 WT CLP-2 HoxA9-/- CLP-1 HoxA9-/- CLP-2

Summary of RNA-Seq Platform Sample Total Reads Hoxa9ko-CLP-1 89,662,470 Hoxa9ko-CLP-2 86,812,194 Hoxa9ko-MPP-1 79,030,174 Hoxa9ko-MPP-2 102,001,936 WT-CLP-1 108,111,316 WT-CLP-2 118,748,182 WT-MPP-1 98,958,636 WT-MPP-2 99,110,526 Read length:50bp 40-50X depth of coverage/gene Differential expression cutoff: absolute fold change 1.4 False discovery rate of 0.1; p 0.05 Hoxa9-/- MPP Hoxa9-/- MPP 42 73 MPP 29 CLP 13 3 Hoxa9-/- CLP Hoxa9-/- CLP 16 102 MPP 56 CLP 17 86

Hoxa9-deficiency impairs lymphoid priming and downregulation of HSC/MPP and/or alternative lineage gene programs Hoxa9-/- MPPs Hoxa9-/- CLPs 64% HSC/MPP myeloid/DC 93% HSC/MPP myeloid/DC Shared upregulated Lcn2 – lipocalin 2 – neutrophil Mgam – maltase-glucoamylase - neutrophil Clec4b – ATP binding cassette - DC Adam23 – disintegrin and metallopeptidase - DC Pglyrp1 – peptidoglycan recognition protein - neutrophil Elane – elastase – macrophage/HSC/MPP Abca13 – ATP binding cassette - neutrophil Ehd3 – EH domain containing 3 – T cells Dio2 – deiodinase – HSC/MPP Mpo – myeloperoxidase - monocytes Vcam1 – vascular cell adhesion molecule 1 – HSC/MPP Dach1 – dachshund - neutrophil Ppic – peptidylprolyl isomerase C – HSC/MPP Rab38 – member of RAS family - macrophage Mgl2 – macrophage galactose N-acetyl-galactosamine specific Ikzf2 – ikaros zinc finger 2; Helios – HSC/T cells/T regs Gata2 – HSC/MPP Hoxa9-/- MPPs 54% Lymphoid Hoxa9-/- CLPs 63% Lymphoid Shared downregulated Rag1 – recombinase activating gene – lymphocytes Gfra1 – sperm stem cell self renewal - BCPs Rag2 - recombinase activating gene - lymphocytes Ppfia4 – protein tyrosine phosphatase – spl DC / Macs Slc15a2 – H /peptide transporter - BCPs P2rx3 – purinergic receptor P2X - BCPs Cdh17 – cadherin 17 - BCPs Cecr2 – cat eye syndrome - BCPs Gimap4 – GTPase - BCPs Lin28b - regulator adult to fetal HSC; let7 biogenesis - BCPs Cd27 – member of TNF receptor family – MPPs/BCPs Igfbp3 – insulin growth factor 2 binding protein - BCPs

Ectopic expression of EBF1 is sufficient to restore the generation of BCPs from HoxA9-/- MPPs WT MPPs HoxA9-/- MPPs B220 17 day MPP switched to SCF FL IL7 and cultured for an additional 6-8 days CD19 CD19 MigR1-GFP Mig-EBF-GFP Mac-1 Gated on GFP cells CD19 CD19 Hoxa9-/- d17 MPP in SCF FL IL7 6-8 days after retroviral transduction

Linking RNA-Seq and Microarray data to the IL-7R signaling pathway

Conclusions: We developed a short-term in vitro culture model to expand MPPs from WT and HoxA9-/- mice that will be informative in the identification, characterization, and validation of HoxA9 regulated genetic circuits in early hematopoiesis. RNA-Seq confirmed decreased lymphoid priming in MPPs and under B cell differentiation conditions due to HoxA9 deficiency independent of Flt3 signaling. HoxA9 deficiency impairs IL-7R expression and signaling – enforced expression of EBF1 can bypass the IL-7 signaling defect. Gfi1b Runx1 IL7R STAT5 E2A* FOXO1 EBF1 Pax5 HSC program Meis1 Hoxa9 c-Myb Flt3 PI3K MAPK Gfra1 Ctr9 Ccr9 Scdn4

Underway: 1. Identify novel HoxA9 target genes : compare results to microarray platform of genes upregulated in EBF1-/- multipotent cells that express endogenous Hoxa9 - underexpressed in Hoxa9-/- MPPs in RNA-Seq platform (transcriptional activation) - overexpressed in EBF1-/- MPPs in Affymetrix Array Chd17, CD27, CCR9, Ppfia4, Sorcs2, RAG2, GPR25, Jup, Igf2bp3 - overexpressed in Hoxa9-/- MPPs in RNA-Seq platform (transcriptional repressor) - underexpressed in EBF1-/- MPPs in Affymetrix Array Mgst1, Tgm2, Enpp4, Plekha8, Muc13, Cpne2, Tmem176a, Mreg, Adcy6, STAT4, Sulf2, SpiB 2. Identify novel EBF1 target genes : compare results to microarray platform of genes underexpressed in EBF1-/- multipotent cells to genes underexpressed in HoxA9-deficient MPPs or CLPs P2rx3, RAG1, Rgs8, Cecr2, Egfl7, Uaca, Lax1, Dntt, Slc15a2, Gimap4 3. Perform gain-of-function and loss-of-function experiments in WT or HoxA9-/MPPs to evaluate gene function in B cell development followed by molecular validation. 4. Determine if HoxA9 target genes are relevant to leukemogenesis.

Acknowledgements Medina Lab Kim Gwin Zhihui (Cherry) Xu, MD Zhixin (Jason) Hua, PhD Joe Dolence, PhD Mike Bell Elena Frank Mariya Shapiro Flow Cytometry Core Gene Expression Core Funding by NHLBI R01096108 Eagles Foundation Concern Foundation Dept of Immunology