Volume 27, Issue 5, Pages 671-681 (May 2015) Treatment of Chronic Myelogenous Leukemia by Blocking Cytokine Alterations Found in Normal Stem and Progenitor Cells  Robert S. Welner, Giovanni Amabile, Deepak Bararia, Akos Czibere, Henry Yang, Hong Zhang, Lorena Lobo De Figueiredo Pontes, Min Ye, Elena Levantini, Annalisa Di Ruscio, Giovanni Martinelli, Daniel G. Tenen  Cancer Cell  Volume 27, Issue 5, Pages 671-681 (May 2015) DOI: 10.1016/j.ccell.2015.04.004 Copyright © 2015 Elsevier Inc. Terms and Conditions

Cancer Cell 2015 27, 671-681DOI: (10.1016/j.ccell.2015.04.004) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Experimental Design and Establishment of a Chimera Chronic Leukemia Model (A) Overview of the experimental design for generation of CML chimeras, 2 × 106 whole bone marrow (WBM) cells from either C57BL/6 mice (CD45.2; Control) or Scl-tTa-BCR-ABL (CD45.2; Transgenic) mice mixed with 2 × 106 C57BL/6 cells congenic for the CD45 locus (CD45.1; Normal) and transplanted into mice on Tet. Chimerism was established for 16 weeks before Tet withdrawal and induction of BCR-ABL in those cells carrying the two transgenes. Tissue cells were obtained 4 weeks after induction, and CD45.1+ cells from control transplants, leukemic-exposed cells (CD45.1+ cells from leukemic mice), or leukemic cells (CD45.2+ transgenic cells from leukemic mice) were analyzed. (B) Survival curves of control and transgenic chimeric mice after Tet withdrawal. (C) The CD45.1 chimerism results are based on the staining of lymphoid (B220/CD3) and myeloid (Mac1/Gr1) cells in PB versus CD45.2 (control or leukemic) over time; n = 4 per time point. Error bars represent mean ± SEM. (D) Upper panel: representative FACS profiles from four experiments characterizing the PB of control or leukemic mice showing the chimerism of CD45.1 versus CD45.2 from control or transgenic chimeric mice 4 weeks after Tet removal. Lower panel: FACS profiles of CD45.1 cells in the PB of control or leukemic mice at 4 weeks after Tet removal. Cells were stained for B220/CD3 and B220/Mac1-Gr1. Cells that stain positive for both fluorochromes are B-lineage cells (B220+), while those that stain only for B220/CD4 and CD8 are T-lineage cells (CD4/8), and those cells that are positive for only B220/Mac1-Gr1 are of the myeloid lineage (Mac1-Gr1). Leuk Exp, leukemic exposed. (E) Representative FACS profiles from the bone marrow gating for CD45.1+ B-lineage cells (B220+ CD43+/−) of either control or transgenic chimeric mice at 4 weeks after Tet withdrawal. Dot plots from leukemic cells (CD45.2) from the transgenic chimeric mice are shown for comparison (right). Similarly, bone marrow and spleens from control and transgenic chimeric mice were analyzed for the CD45.1+ myeloid-lineage cells (Mac1+ and Gr1+), and leukemic cells (CD45.2+) are shown for comparison, n = 5. See also Figure S1. Cancer Cell 2015 27, 671-681DOI: (10.1016/j.ccell.2015.04.004) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Normal Progenitor Profiles within the Leukemic Environment (A) Flow cytometry profiles for control and leukemic-exposed (Leuk Exp) progenitors (CD45.1+) of Lin− ckitHi Sca1+ (KSL) and Lin− ckitHi Sca1+ CD150+ CD48− (HSC), in either control or leukemic marrow at 4 weeks after Tet withdrawal, n = 5. (B) Numbers of control and leukemic-exposed (CD45.1+) progenitors were calculated per femur for Lin− ckitHi Sca1+ (KSL), Lin− ckitHi Sca1+ CD150+ CD48− (HSC), or Lin− ckitHi Sca1+ CD34+ Flt3− (ST-HSC) in either control or leukemic marrow, n = 5. Error bars represent mean ± SEM. (C) CD45.1+ HSCs (Lin− ckitHi Sca1+ CD150+ CD48−; HSC) or CD45.1+ KSLs (Lin− ckitHi Sca1+; progenitors) were sorted from either control or leukemic environments, and then cell cycle status was determined using Pyronin Y and Hoescht, n = 5. Error bars represent mean ± SD. (D) BrdU incorporation of CD45.1+ HSCs (KSL CD150+ CD48−) from control or leukemic mice was quantified, and lines indicate the mean; n = 5. (E) Annexin V staining of control or leukemic-exposed HSCs was used to determine the apoptosis within this subset of progenitors, and lines indicate the mean, n = 5. See also Figure S2. Cancer Cell 2015 27, 671-681DOI: (10.1016/j.ccell.2015.04.004) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Stem and Progenitor Cell Memory of Leukemic Exposure (A) CD45.1+ KSLs from either control or leukemic mice were sorted and placed in OP9 co-culture assay. Flow cytometry analysis of CD19 and Mac1 in these cultures after 14 days is shown; n = 4. Leuk Exp, leukemic exposed. (B) Quantification of B cells (CD19+) and myeloid-lineage cells (Mac1+) from OP9 co-cultures were plotted from CD45.1+ KSLs from either control or leukemic-exposed and CD45.2+ leukemic KSLs 14 days after initiation; n = 4. Error bars represent mean ± SEM. (C) Cartoon showing that control or leukemic-exposed CD45.1+ KSLs or HSCs (Lin− ckitHi Sca1+ CD150+ CD48−) were sorted from the primary chimeras and transplanted into sublethally irradiated C57BL/6 (CD45.2) hosts. KSL- transplanted mice were analyzed at 10 weeks post-transplant, while HSC-transplanted mice were analyzed after ≥16 weeks post-transplant. (D) CD45.2+ leukemic HSCs and CD45.1+ HSCs were sorted from control and/or leukemic mice and then labeled with CFSE (5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester). These cells were then transplanted into lethally irradiated mice for 12 hr, at which time the femurs were analyzed for the presence of CFSE and CD45.1+ cells. Plots show homing efficiency relative to the control HSCs; n = 5. Error bars represent mean ± SD. (E) Limiting dilution CRU (competitive repopulation unit) assay is shown. The indicated numbers of control or leukemic-exposed HSCs were transplanted along with 2 × 105 bone marrow cells of a competitor (CD45.2+) into lethally irradiated hosts (CD45.2+). Reconstitution was evaluated in the blood at 16 weeks post-transplantation (p = 0.0001). Mice with CD45.1+ chimerism <0.3% were considered as non-responders. (F) For serial transplantation, 2 × 106 sorted CD45.1+ bone marrow cells from either control or leukemic mice and appropriately adjusted per HSC numbers of CD45.1+ cells from leukemic mice were transplanted into lethally irradiated CD45.2 hosts. Rounds of transplantations with performed at 16-week intervals. The proportion of PB generated from the CD45.1+ cells in recipient mice was determined for each round of transplantation. Bar graphs indicate chimerism after each round of transplantation; n = 4. Error bars represent mean ± SD. (G) PB samples from either 10 weeks post-transplant for CD45.1+ KSLs or 16 weeks post-transplant for CD45.1+ HSCs transplants, respectively, were stained with CD45.1, CD45.2, and DAPI for viability. The bars show percentages of the donor-type cells (CD45.1) that expressed myeloid (Gr-1 and CD11b, filled), B-lineage (B220, striped), or T-lineage (CD4/8, open) markers in the blood of individual recipients. Cancer Cell 2015 27, 671-681DOI: (10.1016/j.ccell.2015.04.004) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Microarray Data Points to Cytokine Changes in the Progenitor Cells from the Leukemic Exposure (A) Hierarchical clustering with average linkage was used to generate the heatmap of lineage-specific transcription factors from the control and leukemic-exposed (Leuk Exp) progenitors. (B) Enrichment plots of GSEA using control, leukemic-exposed, and leukemic HSC and KSL expression data against a list of BCR-ABL-related gene changes. (C) We used an antibody-based protein array to measure cytokine changes in the sera from our chimera mice, both control and transgenic. Results are shown based on the normalization of the control sera. Error bars represent mean ± SD. (D) Enrichment plots of GSEA using control, leukemic-exposed, and leukemic HSC and KSL expression data against a list of IL-6-related gene changes. See also Figure S3. Cancer Cell 2015 27, 671-681DOI: (10.1016/j.ccell.2015.04.004) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Inhibition of IL-6 Signaling Protects Normal Progenitor Cells (A) Cartoon of the method used to determine leukemic-exposed KSLs skewing to the myeloid lineage was dependent on IL-6Rα signaling. CD45.2+ BCR-ABL leukemic cells were transplanted with CD45.2+ GFP+ MxCre+ IL-6RαΔ/Δ cells or CD45.2+ GFP+ MxCre− IL-6Rαflox/flox cells and kept on Tet for 16 weeks before the removal of Tet. Pepcb/BoyJ is the mouse name for CD45.1 B6 mice. lenti, lentivirus. (B) Contour plots from staining of PB gating on GFP+ and GFP− cells for B220/CD3 and B220/Mac1-Gr1. Top panels show the control chimera, while the bottom panels show the leukemic. GFP+ MxCre− cells (center panels) contain IL-6Rαflox/flox cells. GFP+ MxCre+ cells are excised for IL-6Rα (right panels); n = 4. (C) GFP+ KSL cells from control or leukemic mice were sorted and co-cultured on OP9 stromal cells for B-lineage differentiation. Flow cytometry plots show CD19 versus Mac1 staining from control and leukemic mice containing either IL-6Rαflox/flox or MxCre+ IL-6RαΔ/Δ cells. Quantification of the percentages of B-lineage cells and myeloid cells derived from these cultures are plotted; n = 4. Error bars represent mean ± SD. (D) Splenic numbers and morphology from IgG- and anti-IL-6-treated leukemic mice are shown. Mice were injected intravenously with anti-IL-6 diluted in PBS (0.2 mg/kg). Antibody-injected controls were similarly injected with IgG in PBS. Two weeks after removal from Tet, mice were injected ever other day for 6 weeks. Mice were then euthanized, and total cell numbers from the spleens of control, leukemic IgG-, and anti-IL-6-leukemic-treated mice were obtained; n = 5. Error bars represent mean ± SD. (E) Flow cytometry plots of CD45.1+ myeloid cells (Mac1+ Gr-1+) from the spleens of control or leukemic IgG- and anti-IL-6-treated mice. Spleens were stained for viable CD45.1, CD45.2, Mac1, and Gr1; n = 4. (F) Sorted control, leukemic-exposed, and leukemic KSLs were cultured in colony assays in the presence or absence of anti-IL-6 at two doses. Colonies were counted, and relative colony numbers were determined compared to untreated cells for each population; n = 3. Error bars represent mean ± SD. See also Figure S4. Cancer Cell 2015 27, 671-681DOI: (10.1016/j.ccell.2015.04.004) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Increased Proliferation and Skewed Differentiation of Normal Human CD34+ Progenitors when Mixed with Human CML Cells (A) Cytokine IL-6 mRNA levels from cultured patient bone marrow (healthy, chronic-phase CML, or mixed) were measured against GADPH and normalized to healthy controls; n = 5. Error bars represent mean ± SD. (B) Bar graph showing the yield per input of GFP+ cells when cultured with control or CML bone marrow with or without anti-IL-6. Bead-enriched CD34+ cells from human adult bone marrow were labeled with a lentiviral GFP reporter. These cells were then cultured with whole adult bone marrow from healthy patients (control) or those with chronic-phase CML (leukemic exposed) on stromal cells (MS5). Additionally, wells contained either IgG or anti-IL-6 as indicated (5 μg/ml). These cells were cultured for 96 hr with Flt3 ligand (Flt3L), thrombopoietin (TPO), and IL-3 and then counted and analyzed by flow cytometry for total GFP+ cells; n = 3. Error bars represent mean ± SD. (C) CD34+ GFP-marked cells were co-cultured with healthy bone marrow (Control) or chronic-phase CML (leukemic exposed; Leuk Exp) on stromal cells. These cultures were maintained with IL-7, stem cell factor (SCF), and Flt3L for 1 week and then an additional 3 weeks with only SCF and Flt3L. As indicated, some wells contained anti-IL-6. After the 4-week culture, GFP+ human cells were analyzed by flow cytometry for expression of CD19 and Mac1; n = 3. (D) GFP+ CD34+ cells from either control or leukemic-exposed cultures were stained for annexin V to determine the relative apoptosis in these conditions, and lines indicate the mean; n = 3. Error bars represent mean ± SEM. (E) Sorted GFP+ CD34+ cells were analyzed from control and CML cultures (with or without anti-IL-6) after staining with Pyronin Y and Hoescht to determine cell cycle status; n = 3. (F) Bone marrow from healthy, chronic-phase CML, or IM-resistant CML was cultured in Methocult4435 for colony formation with or without human anti-IL-6. Colony numbers are presented as relative to the healthy untreated control; n = 2. Error bars represent mean ± SD. Cancer Cell 2015 27, 671-681DOI: (10.1016/j.ccell.2015.04.004) Copyright © 2015 Elsevier Inc. Terms and Conditions