Thrombopoietin Affects the Behavior of Myelofibrosis Stem Cells

Abstract Recently the presence of myelofibrosis (MF) stem cells (MF-SC) in the spleens of MF patients which when transplanted into NOD/SCID/IL2R null (NSG) mice were capable of generating multiple hematopoietic lineages that belonged to the malignant clone has been observed. Although MF is characterized by marrow megakaryocyte (Mk) hyperplasia, limited numbers of human marrow Mks were, however, observed in these transplanted mice and evidence of both marrow and splenic fibrosis 9 months after the transplantation was lacking (Wang X, et al. J Clin Invest. 2012; 122:3888). Splenic MF CD34+ cells did retain the capacity to differentiate in vitro into CD41a+ and CD61+ Mks in the presence of thrombopoietin (TPO). We therefore investigated whether the administration of romiplostim, a novel human TPO peptide mimetic which lacks homology to endogenous TPO in MF humanized mice, might create appropriate environmental cues which affect the behavior of MF-SCs. Elevated levels of TPO (345±114ng/ml) were detected in MF plasmas (n=13) as compared to levels detected in normal plasma (10±4ng/ml, n=6, P=0.049), indicating the possibility that TPO affects MF-SCs and MF hematopoietic progenitor cells (HPC). Following the culture of CB (n=3) or PB MF CD34+ cells (n=4) for 1 and 2 wks in serum free expansion media (SFEM) supplemented with SCF (50ng/ml) + romiplostim (100ng/ml), the numbers of total cells, CD34+Lin- cells and assayable HPCs, including CFU-Mk, CFU-GM and BFU-E, CD41a+CD34-CD15- cells and CD15+CD34-CD41a- cells generated were greater (CB) or similar (MF) to the number generated in the cultures containing SCF + TPO (100ng/ml). Moreover, similar proportions of colonies (CFU-GM+BFU-E) generated in cultures supplemented with SCF+TPO or SCF+ romiplostim (100ng/ml, 1000ng/ml) were JAK2V617F-positive. These findings suggest that both romiplostim and TPO are capable of promoting MF-SC and HPC proliferation in vitro. To assess the effects of romiplostim on human (h) platelet production, CB CD34+ cells (5×105) were transplanted via the tail vein into eight- to nine-week-old sublethally irradiated (240 cGy) NSG mice. One week after transplantation, mice were treated with water or 10, 100 or 1000 µg/kg of romiplostim. Both the percentage and number of hCD41a+ platelets in the PB increased 4 days following the treatment with 10ug/kg romiplostim, and peaked at day 8 with a 2.58±0.29 fold increase in the percentage (P<0.05) and 3.51±1.32 fold increase in the absolute number (P<0.05) of hCD41a+ platelets being observed. The number of human platelets in the PB, were reduced to the levels detected in mice not receiving romiplostim by day 15. MF splenic CD34+ cells (5-10×105) were next transplanted into romiplostim (10ug/kg or 30ug/kg) treated or control NSG mice. Two months after the transplantation, a 1.83±0.62 fold increase in the number of hCD41a+ cells was observed in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not treated with romiplostim. Moreover, a 7.13±2.13 fold elevation in the percentage of hCD45+ cells was detected in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not receiving romiplostim. Moreover, enhanced hCD45+ cell chimerism was achieved in both the marrow (55.6%, 7.2%) and spleen (47.7%, 1.5%) of mice receiving splenic CD34+ cells from 2 MF patients and romiplostim as compared with mice not receiving romiplostim (marrow: 30.4%, 5.6%; spleen: 23.6%, 0.9%), respectively. The splenic MF CD34+ cells receiving or not receiving romiplostim had a similar differentiation patterns in the marrow and spleen, however, the numbers of hCD33+, hCD19+, hCD3+, hCD41a+ and hCD15+ cells detected in mice treated with romiplostim were dramatically greater than those detected in mice not receiving romiplostim. Greater numbers of hCD34+ cells were detected in the BM (7.1%; 3.5%) of recipient mice receiving romiplostim as that detected in mice not receiving romiplostim (6.6%; 0.6%). Furthermore, a similar degree of hCD45+ cell and hCD34+ cell chimerism was observed in both the marrow and spleen of mice receiving splenic MF CD34+ cells and romiplostim 4 months after the transplantation. These findings suggest that the administration of thrombopietin agonist has a profound effect on the behavior of MF-SCs and that the elevated levels of TPO documented in MF patients likely has important effects on the biology of MF-SCs. Disclosures Wang: The MPN Research Foundation (MPNRF) and the Leukemia & Lymphoma Society (LLS) : Research Funding.

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A Thrombopoietin Antagonist Is Capable of Affecting Stem and Progenitor Cells of Patients with Myelofibrosis

Blood ◽

10.1182/blood.v124.21.819.819 ◽

2014 ◽

Vol 124 (21) ◽

pp. 819-819

Author(s):

Xiaoli Wang ◽

David Haylock ◽

Cing Siang Hu ◽

Goar Mosoyan ◽

Dave Winkler ◽

...

Keyword(s):

Progenitor Cells ◽

Myeloid Cells ◽

Absolute Number ◽

Free Expansion ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Cd34 Cells ◽

The Absolute ◽

Expansion Media

Abstract Aberrant thrombopoietin (TPO)/MPL signaling has been hypothesized to contribute to the pathogenesis of myelofibrosis (MF) (Kaushansky K. J Clin Invest. 2005; 115: 3339; Moliterno AR, et al. N Engl J Med.1998; 338:572). Agents that would be capable of inhibiting this signaling pathway are possible novel therapeutic agents that might be effective for MF treatment. A peptide antagonist of TPO, LCP4, has been created which is highly antagonistic to CB CD34+ cell proliferation and differentiation induced by TPO. In this report we examined the effect of LCP4 on the proliferation of MF CD34+ cells and their differentiation to megakaryocytes (Mk). Elevated levels of TPO (345±114ng/ml, n=13) were detected in MF plasmas as compared to that detected in normal plasma (10±4ng/ml, n=6, P=0.049), indicating the possibility that TPO affects MF hematopoietic stem cells (HSC) and progenitor cells (HPC). MF splenic CD34+ cells (2.5×104/mL) were incubated in serum free expansion media (SFEM) alone, with 50 ng/ml SCF+ varying doses of TPO (0, 10, 30,100 ng/mL) or 50 ng/ml SCF +varying doses of TPO+ varying concentrations of LCP4 (0, 10, 50, 100, 500, 1000nM) for 1 or 2 wks. Cells were then enumerated and stained with CD34, lineage cocktail, CD15 and CD41a mAbs. When splenic MF CD34+ cells were cultured in the presence of SCF and varying concentrations of TPO, 100ng/ml TPO resulted in the generation of the greatest numbers of CD34+Lin-, CD41a+CD34-CD15- and CD15+CD34-CD41a- cells, suggesting that TPO promotes the proliferation of MF HSCs/HPCs and the production of MF MKs and myeloid cells. By contrast, after 1 wk of treatment of MF CD34+ cells (n=8; 5 MF spleens and 3 MF PB) with 100nM and 500nM LCP4, the numbers of MF total cells, CD34+Lin- (MF HSCs/HPCs), CD34+CD41a+ (immature Mks), CD41a+CD34-CD15- (mature Mks), CD15+CD34-CD41a- cells (myeloid cells) as well as hematopoietic colonies (HC), including CFU-Mk, CFU-GM, BFU-E/CFU-E, CFU-GEMM, were all significantly reduced (P all <0.05), as compared with cells cultured with SCF and TPO alone (Table 1). Two days of treatment of MF splenic CD34+ with 100nM LCP4 led to a greater degree of apoptosis (15.4% ± 3.5%) as compared with cells treated with cytokines alone (6.75% ± 1.7%, P =0.05; n=4). These findings suggest that LCP4 is able to inhibit the proliferation of MF HSCs/HPCs and the generation of immature and mature Mks as well as myeloid cells in a dose dependent fashion. Furthermore, the treatment of JAK2V617F-positive MF CD34+ cells from 4 JAK2V617F-positive MF patients (JAK2V617F allele burden: 78-90%) with 100nM LCP4 for 7 days reduced the percentage of JAK2V617F-positive HCs by 9-20% and JAK2V617F homozygous HCs by 1-20%. Since LCP4 treatment of MF CD34+ cells resulted in a reduction in the number of HPCs, LCP4 treatment reduced the absolute numbers of JAK2V617F-positive HCs generated by 53.0±4.0% (P<0.0002) and the absolute number of JAK2V617F homozygous HCs by 50.3±4.3% (P<0.0003). These data suggest that LCP4 treatment is able to impair the in vitro generation of MF HPCs and thereby leads to a depletion but not elimination of the number of malignant HPCs. We next examined the effect of LCP4 on MF HSCs by transplanting NSG mice with cells generated after splenic MF CD34+ cells were cultured in the presence of cytokines alone or cytokines plus LCP4 for 1 week. Two months after the transplantation, hCD45+ cells were detected in the BM of recipient mice receiving splenic MF CD34+ cells treated with cytokines alone and were reduced by 28% in mice receiving grafts treated with LCP4. MF CD34+ cells treated with or not treated with LCP4 had similar multilineage differentiation patterns (myeloid, lymphoid and erythroid). These data suggest that TPO antagonist therapy is capable of depleting MF HSCs and HPCs and that therapeutic strategy utilizing such strategies might serve as novel approaches to treating MF. Abstract 819. Table 1 Inhibitory effects of LCP4 on MF HSC/HPC proliferation and generation of Mks and myeloid cells Treatment Cells or HCs Generated (% of Cytokines Alone) Total Cells CD34+ Lin- Cells CD34+CD41a+ Cells CD41a+CD34-CD15- Cells CD15+CD34-CD41a- Cells CFU-Mk CFU-GM BFU-E /CFU-E CFU-GEMM SCF+TPO 100±0 100±0 100±0 100±0 100±0 100±0 100±0 100±0 100±0 SCF+TPO+LCP4 (100nM) 61.3±4.6 61.6±10.3 57.3±11.4 54.9±8.7 68.9±4.5 54.4±8.0 54.0±8.7 52.6±15.1 2.1±2.1 SCF+TPO+LCP4 (500nM) 53.3±8.1 46.8±7.0 59.1±17.1 42.8±8.0 62.5±7.3 43.6±10.0 44.6±5.7 50.5±12.9 0±0 Disclosures Wang: The MPN Research Foundation (MPNRF) and the Leukemia & Lymphoma Society (LLS): Research Funding.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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Functional ABCG2 Is Expressed on CML Stem Cells and Its Inhibition Selectively Depletes CML CD34+ Cells.

Blood ◽

10.1182/blood.v104.11.716.716 ◽

2004 ◽

Vol 104 (11) ◽

pp. 716-716

Author(s):

Joanne C. Mountford ◽

Diane Gilmour ◽

Susan M. Graham ◽

Niove E. Jordanides ◽

Siobhan McMillan ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Efflux Pump ◽

Residual Disease ◽

Chronic Phase ◽

Cell Number ◽

Similar Degree ◽

Cd34 Cells ◽

And Function

Abstract We have previously described a population of deeply, but reversibly, quiescent stem cells (qSC) found in patients with chronic phase (CP) CML at diagnosis. In vitro studies have proven this population to be highly insensitive to imatinib mesylate (IM; Gleevec, STI571) induced killing, and more worryingly shown that qSC are accumulated after CML CD34+ cells are treated with IM. As it is likely that CML qSC closely resemble normal HSC, we hypothesise that they too may express the stem cell-associated ABCG2 and have therefore examined the expression and function of this drug efflux pump on CML cells. In agreement with other studies we show the interaction between ABCG2 and IM. Using ABCG2 over-expressing cells (AML6.2 and HL60-BCRP) we found that ≥0.5μM IM reduced efflux of the ABCG2 substrate BODIPY-Prazosin by a similar degree as the inhibitor fumitremorgin C (FTC; 10μM). We have now examined expression and function of ABCG2 on primary CML cells taken from patients in chronic phase (CP) and prior to any treatment. Quantitative Taqman analysis of 8 CD34+ enriched (≥90%+) CML samples revealed that the level of expression is 2.46 fold higher than that in normal mobilised CD34+ cells (n=8 CML, n=4 normal). In addition, we undertook microarray analysis of normal or CML CP CD34+ cells fractionated according to cell cycle using Hoechst-Pyronin (G0, G1 and G2/S/M). These analyses (n=3 normal, n=5 CML) show that at all stages of the cycle CML cells express more ABCG2 than normal cells and that G0 CML cells express 2.48 fold more than those in G1 , confirming both the over-expression in CML and relationship to the most primitive subset of cells. Using the antibody BXP21 we found that 8 of 9 samples contain ABCG2+ve cells (5 of 9 ≥60% of cells ABCG2+). We also examined the function of ABCG2 on CML CD34+ cells by performing efflux assays, 4 of 6 showed efflux that was inhibited by 10μM FTC or ≥0.5μM IM, and this efflux capacity correlated with BXP21 staining. We therefore considered whether the combination of IM therapy and ABCG2 inhibition would overcome the accumulation of CML qSCs we have previously reported after treatment with IM. Using CFSE to track cell division we treated CD34+ enriched CML samples with 5μM IM +/− FTC or with 10μM FTC alone for 3 days. In comparison to untreated controls 5μM IM reduced the total number of cells to 31.9±9.2 % and the number of CD34+ cells to 43.2±17.6%. However, the non-cycling qSC significantly increased to 318±75.8% of control. In contrast, the ABCG2 inhibitor FTC did not effect a reduction in total cells (99.5±11.9%) but gave a significant reduction of CD34+ cells (58.6±8.4%; p=0.02) and no accumulation of qSC (104.6±33.8%) when used alone. We saw no cumulative effect when IM and FTC were given concurrently. These data suggest strongly that FTC may be used to deplete CD34+ ‘stem cells’ from CML, as the total cell number is unchanged it is likely that this depletion is by the induction of differentiation. We propose that the expression of ABCG2 may be clinically significant in CP CML and that inhibition of this pump may result in a ‘stem cell targeted therapy’ that could be followed by IM treatment to reduce the tumor load. Such reduction of CML stem cells would result in elimination of minimal residual disease and effect a lasting remission.

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Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽

10.1182/blood.v120.21.4295.4295 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4295-4295

Author(s):

Jae-Hung Shieh ◽

Tsann-Long Su ◽

Jason Shieh ◽

Malcolm A.S. Moore

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Culture System ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Cd34 Cells ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

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The Role of Cbx Proteins in Human Benign and Malignant Hematopoiesis

Blood ◽

10.1182/blood.v128.22.2651.2651 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2651-2651

Author(s):

Johannes Jung ◽

Hein Schepers ◽

Seka Lazare ◽

Sonja Buisman ◽

Ellen Weersing ◽

...

Keyword(s):

Stem Cells ◽

Leukemic Cell ◽

Fold Increase ◽

Leukemic Cells ◽

Self Renewal ◽

Nsg Mice ◽

Leukemic Cell Lines ◽

Human Cd34

Abstract Introduction: Hematopoietic stem cells (HSCs) need to properly balance self-renewal and differentiation to prevent malignant transformation or HSC exhaustion. We recently showed that the presence of specific Cbx proteins in the Polycomb Repressive Complex 1 (PRC1) plays a crucial role in balancing self-renewal and differentiation of murine HSCs. Whereas Cbx7 induces self-renewal, Cbx4 and Cbx8 instead induce differentiation. In this project, we investigate the role of various CBX Polycomb proteins in human normal and malignant hematopoiesis. We aim to identify genes which are controlled by CBX7 in normal and malignant human hematopoiesis. Methods: We overexpressed CBX2, -4, -6, -7 and -8 in human CD34+HSPCs. We assessed functional consequences by measuring cobblestone area-forming cell (CAFC), colony-forming unit (CFU) frequencies, and performed stem cell xenotransplantation studies in NSG mice. To identify genes which are controlled by CBX7 or CBX8, we performed RNA- and Chip-Seq in CD34+ HSPCs. To explore the role of CBX7 in leukemic cells, we performed short hairpin RNA-mediated knockdown of protein expression in leukemic cell lines. Results: Overexpression of CBX7 and CBX8 in human CD34+ HSPCs led to a significant 5-10-fold increase of week 5 CAFC (fold increase: 9,62 for CBX7; 4,92 for CBX8) and 1,5-fold increase CFU-frequencies (fold increase: 1,32 for CBX7; 1,41 for CBX8) after 14 days. In contrast, overexpression of CBX4 and CBX2 led to equal or decreased CAFC- and CFU-frequencies. Transplantation of CBX7 overexpressing human CD34+ HSPCs in NSG mice led to higher long-term engraftment in comparison to the empty vector control (p<0,05 after 18 weeks). Even after one week of in vitro culture of CBX7-overexpressing CD34+cells significant engraftment levels were obtained. Conversely, downregulation of CBX7 in various leukemic cell lines resulted in markedly decreased proliferation and strongly induced differentiation. Expression analysis showed that genes that are upregulated upon CBX7 overexpression are preferentially expressed in primitive stem cells, and are repressed in more differentiated cell types. Conclusions: Our study indicates that CBX7 and CBX8 regulate self-renewal of human HSPCs. Furthermore, our data show that repression of CBX7 markedly inhibits proliferation of leukemic cells and can release the differentiation block of leukemic cells in vitro. Collectively our results indicate that targeting CBX7 may be a viable strategy to induce terminal differentiation of leukemic cells. Disclosures No relevant conflicts of interest to declare.

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Identification of Small Molecules Selectively Targeting Leukemic Stem Cells within Their Stromal Niche

Blood ◽

10.1182/blood.v120.21.413.413 ◽

2012 ◽

Vol 120 (21) ◽

pp. 413-413

Author(s):

Alissa R. Kahn ◽

Kimberly A. Hartwell ◽

Peter G. Miller ◽

Benjamin L. Ebert ◽

Todd R. Golub ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Cord Blood ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Minimal Toxicity ◽

Nsg Mice ◽

Cd34 Cells

Abstract Abstract 413 Current therapies for acute myeloid leukemia (AML) are highly toxic, yet the relapse rate remains high. New therapies are needed to improve cure rates while decreasing toxicity. Because therapies may be affected by the tumor niche, we aimed to test new compounds on leukemic stem cells (LSCs) within their stromal microenvironment. A niche-based high throughput screen identified candidate small molecules potentially toxic to MLL-AF9 murine leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell et al, Blood 118, Abs 760, 2011.) Three such compounds, including a selective serotonin receptor antagonist highly specific for the 5-HT1B receptor, SB-216641, and two antihelminthics, parbendazole and methiazole, were found to be effective and selected for studies on human leukemias. We first examined SB-216641, studying the effects of this compound on 7 human primary AML samples. We began by assessing the compound's effect on LSCs using the week 5 cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. CD34+ cells were isolated with immunomagnetic beads. The leukemic cells were pulse treated for 18 hours and washed prior to placement on MS-5 murine stroma. We performed serial drug dilutions using the CAFC assay with the human primary samples as well as with HSCs derived from cord blood. All human leukemic samples formed cobblestone areas in the control setting (46-200 CAFCs/106 cells plated). IC50 for the human primary leukemia CAFCs was 630 nm, and at 10 μM all LSCs were killed while normal human HSCs had 100% survival. A combination of the AML cell line HL60 transduced with GFP-luciferase and normal cord blood CD34+ cells (1:200) were then pre-incubated overnight with SB-216641 at 5 and 10 μM and injected into Nod Scid IL2R-gamma null (NSG) mice. The control mice had leukemic engraftment by luciferase imaging and flow cytometry and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. Primary patient AML samples were also pre-incubated overnight with SB-216641 at 10 μM and injected into NSG mice. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had no engraftment following exposure to SB-216641. Finally, an in vivo study was completed on NSG mice injected intraperitoneally with 20 mg/kg/day beginning on day 1 or day 8 after inoculation with HL60 (500 cells). The mice were imaged at 2 and 3 week time points and both treatment groups had significantly less leukemia on imaging than the control group with minimal toxicity noted. Another specific 5-HT1B receptor antagonist, SB-224289, was found to have similar activity to SB-216641 against leukemic cells and to spare HSCs in preliminary studies. Similar CAFC studies with serial dilutions on primary AML samples were performed on the two anti-helminthic agents. IC50 for parbendazole was 1.25 μM and for methiazole 5 μM. As shown by luciferase imaging and flow cytometry, when injected with combined HL60 and cord blood pre-incubated overnight at 5 and 10 μM with each compound as described above, the control mice engrafted with leukemia and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. NSG mice were then injected with primary AML pretreated overnight with parbendazole at 10 μM. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had significantly lower engraftment following exposure to parbendazole (p = 0.01). Two new avenues of leukemia therapy were discovered warranting further investigation. SB-216641, an agent with a completely novel receptor target in leukemia therapy, has shown both in vitro success in human leukemia as well as preliminary success in vivo with minimal toxicity. We aim to move forward with this agent while also testing parbendazole in vivo, as this compound is already known to have good pharmacokinetics and minimal toxicity in animals. The high toxicity to LSCs and sparing of normal HSCs give both these agents an attractive profile for future clinical trials. Disclosures: Ebert: Genoptix: Consultancy; Celgene: Consultancy.

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Selective Targeting of CML Progenitor/Stem Cells by the Class 1 Histone Deacetylase (HDAC) Inhibitor MS275 in Combination with Imatinib.

Blood ◽

10.1182/blood.v120.21.2791.2791 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2791-2791

Author(s):

Su Chu ◽

YinWei Ho ◽

Ravi Bhatia

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Hdac Inhibitor ◽

Hdac Inhibitors ◽

Normal Cells ◽

Nsg Mice ◽

Cd34 Cells ◽

Selective Targeting ◽

Class 1

Abstract Abstract 2791 BCR-ABL tyrosine kinase inhibitors (TKI) effectively inhibit CML stem/progenitor cell proliferation and induce remission in CML patients, but do not completely eliminate primitive leukemia stem cells, which persist as potential sources of relapse. There is considerable interest in identifying additional therapeutic strategies to selectively induce apoptosis in CML stem and progenitor cells. We have shown that the pan-HDAC inhibitor Pabinostat, that targets Class 1, Class 2 and Class 4 HDACs, can eliminate CML stem cells in vitro and in vivo when combined with Imatinib (Cancer Cell 17:427, 2010). However pan-HDAC inhibitors can have significant toxicity to normal stem cells, which limits their clinical utility. Here we used siRNA knockdown to determine the role of individual HDACs in proliferation and apoptosis of CML compared with normal CD34+ cells. CD34+ cells were transfected with siRNAs to 11 individual HDAC enzymes and control non-specific siRNA, using an Amaxa nucleofector system. 50–80% knockdown of target gene expression was achieved. Knockdown of individual Class 1, 2 and 4 HDACs resulted in increased apoptosis of CML CD34+ cells, which was increased in combination with IM. On the other hand, normal CD34+ cells demonstrated increased apoptosis in response to knockdown of Class 2 and 4 HDACs, but not Class 1 HDACs. These results suggested that inhibition of Class I HDACs may allow selective targeting of CML stem/progenitor cells while sparing normal cells, and led us to evaluate the effects of MS275, an orally available HDAC Class I specific inhibitor currently being evaluated in clinical trials in solid tumors, against CML and normal stem/progenitor cells. MS275 (1μM) exposure increased acetylated Histone H3 and histone H4 levels in CML CD34+ cells, as analyzed by Western blots and flow cytometry. Treatment of CML CD34+CD38- cells and CD34+CD38+ cells with MS-275 resulted in significantly increased apoptosis compared with their normal counterparts (17.93±1.1% for CML vs. 7.9±2.3% for normal CD34+CD38- cells, p=0.01; 20.1±4.6% for CML vs. 9±1.9% for normal CD34+CD38+ cells, p=0.05). Combination of MS275 with IM resulted in significant enhancement of apoptosis of CML but not normal cells (72±11.1% for CML vs. 6.3±1.87% for normal CD34+CD38- cells, p=0.02; 41.3±9.8% for CML vs. 8.45±1.75% for normal CD34+CD38+ cells, p=0.04). Quiescent CML stem cells are especially resistant to TKI-induced apoptosis. The combination of MS275 and IM resulted in significantly increased apoptosis of non-dividing CML but not normal CD34+38- cells (72.1±14.3% for CML vs. 10.6±2.0% for normal cells, n=3). Cell cycle analysis by Ki67 and DAPI labeling demonstrated significant increase in G0, and decrease in S/G2/M phase, in CML but not normal CD34+ cells treated with MS275, alone or combination with IM. Consistent with these findings, we observed downregulation of the anti-apoptotic protein Mcl1 and up-regulation of the cell cycle inhibitor p21 in MS-275 treated CML CD34+ cells. MS275 treatment also significantly reduced colony forming cell growth from CML but nor normal CD34+CD38+ and CD34+CD38- cells in methylcellulose progenitor culture. To further evaluate the effects of MS275 on normal stem cells, normal CD34+ cells were treated with MS275, IM or the combination of MS275 and IM for 72 hours in vitro, followed by injection into sub-lethally irradiated (300 cGy) NSG mice and evaluation of human cells engraftment after 12 weeks. These studies showed that treatment with MS275 did not significantly affect engraftment of normal stem cells in NSG mice. In conclusion, the selective Class1 HDAC inhibitor MS-275 significantly increases apoptosis in CML primitive and committed progenitors, including non-proliferating cells, which is further significantly enhanced in combination with IM. In contrast to pan-HDAC inhibitors, MS275 treatment does not affect survival or growth of normal stem cells. These results indicate that use of inhibitors specific to Class 1 HDAC enzymes could avoid toxicity to normal HSC associated with pan-HDAC inhibitors, allowing more selective and effective targeting of CML LSC. Disclosures: No relevant conflicts of interest to declare.

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Human and rhesus macaque hematopoietic stem cells cannot be purified based only on SLAM family markers

Blood ◽

10.1182/blood-2009-03-212803 ◽

2011 ◽

Vol 117 (5) ◽

pp. 1550-1554 ◽

Cited By ~ 37

Author(s):

Andre Larochelle ◽

Michael Savona ◽

Michael Wiggins ◽

Stephanie Anderson ◽

Brian Ichwan ◽

...

Keyword(s):

Stem Cells ◽

Rhesus Macaque ◽

Rhesus Macaques ◽

Surface Markers ◽

Cell Surface Markers ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Cd34 Cells ◽

Slam Family

Abstract Various combinations of antibodies directed to cell surface markers have been used to isolate human and rhesus macaque hematopoietic stem cells (HSCs). These protocols result in poor enrichment or require multiple complex steps. Recently, a simple phenotype for HSCs based on cell surface markers from the signaling lymphocyte activation molecule (SLAM) family of receptors has been reported in the mouse. We examined the possibility of using the SLAM markers to facilitate the isolation of highly enriched populations of HSCs in humans and rhesus macaques. We isolated SLAM (CD150+CD48−) and non-SLAM (not CD150+CD48−) cells from human umbilical cord blood CD34+ cells as well as from human and rhesus macaque mobilized peripheral blood CD34+ cells and compared their ability to form colonies in vitro and reconstitute immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 γc receptornull, NSG) mice. We found that the CD34+ SLAM population contributed equally or less to colony formation in vitro and to long-term reconstitution in NSG mice compared with the CD34+ non-SLAM population. Thus, SLAM family markers do not permit the same degree of HSC enrichment in humans and rhesus macaques as in mice.

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Crispr Engineering in CD34+ Progenitors Reveals Cis-Acting Regulatory Regions Mediating 3D Interactions and Stem Cell Fate Decisions

Blood ◽

10.1182/blood.v128.22.1466.1466 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1466-1466

Author(s):

Mira Jeong ◽

Yong Lei ◽

Ivan Bochkov ◽

Muhammad S Shamim ◽

Anna G Guzman ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

T Cells ◽

Hoxa Cluster ◽

Self Renewal ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract DNA methylation is a critical regulator of cis-regulatory elements that can impact the distribution of epigenetic regulators and transcription factors. We have mapped DNA methylation changes in human CD34+ cells and downstream progeny to detect changes in DNA methylation with differentiation. Some large regions of low DNA methylation emerge with differentiation, which we call dynamic (d-) canyons. In order to determine whether these correlate with changes in the 3D genome, we generated 6 high-resolution Hi-C contact maps from CD34+CD38- cells, Adult bone marrow CD34+ cells, 7 days cultured CD34+ cells, CD71+CD36+CD235+ Erythroid cells, CD4+ T cells and AML patient blast cells. We generated ~ 1 billion mapped reads in order to create each 3D map of the genome at kilobase resolution. We identified several sites of d-canyons which correlate with changes in Hi-C loops. To study the functional role of d-canyons, we performed CRISPR/CAS9 mediated genome engineering targeting three sites of interest in CD34+ progenitors. We designed sgRNAs to delete several d-canyon regions that exhibited cell-type specific methylation and dynamic H3K27ac marking. First, we targeted a novel putative regulatory region of the RUNX1 gene, a critical master regulator of hematopoiesis that is frequently mutated in human leukemia. From genome-wide DNA methylation profiling, we identified a d-canyon located in the first intron of RUNX1, which overlaped with a H3K27ac peak in human CD34+CD38- hematopoietic progenitor cells. DNA methylation in this region is further depleted in T cells and increased in AML cells, suggesting a role for regulating RUNX1 in specific cell types. To study its function, we designed 2 sgRNAs to delete a 1.8 kb d-canyon. CRISPR/Cas9-mediated deletion of this region resulted in ablation of RUNX1 expression in cord blood hematopoietic stem cells and a significant decrease of engraftment activity in NSG mice along with an increase of erythroid colony forming ability in in vitro assays. In addition, we identified d-canyons upstream of the master regulators GATA2 and in the HOXA cluster. We deleted 1.7kb d-canyon upstream of GATA2 gene, finding that it resulted in increased self-renewal of CD34+CD38- cells in NSG xenografts. In contrast, when we deleted one a d-canyon located ~2Mb upstream of the HOXA cluster, within a HiC loop signal present only in CD34+CD38- cells, we observed in decreased CD34+CD38- cell self-renewal but a significant increase of CD34+CD38+ differentiated progenitors in an in vitro culture system, as well as in NSG mice. Colony forming assay showed decreased colony size and numbers. Finally, we identified a d-canyon associated with TCF3, also known as E2A, a transcription factor involved in B and T cell lineage differentiation. We designed 4 sgRNAs to delete 1.5kb d-canyon edge regions within the second intron of TCF3. Removal of this region resulted in a significant decrease of CD19+ B cells, but an increase of CD3+ T cells in NSG mice. Taken together, these results suggest the functional importance of d-canyons for orchestrating genome architecture and fate decisions of hematopoietic stem cells. These findings advance our understanding of the relationship between DNA methylation changes and loop interactions, providing new insights into the potential impact of potential aberrant DNA methylation and chromatin structure. Disclosures No relevant conflicts of interest to declare.

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A Stemness Screen Reveals C3ORF54/INKA1 As a Gate-Keeper of Human Stem Cell Latency

Blood ◽

10.1182/blood-2018-99-115065 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 325-325

Author(s):

Kerstin B. Kaufmann ◽

Laura Garcia Prat ◽

Shin-Ichiro Takayanagi ◽

Jessica McLeod ◽

Olga I. Gan ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Fold Increase ◽

Steady Increase ◽

Post Transplantation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract The controversy generated from recent murine studies as to whether hematopoietic stem cells (HSC) contribute to steady-state hematopoiesis emphasizes how limited our knowledge is of the mechanisms governing HSC self-renewal, activation and latency; a problem most acute in the study of human HSC and leukemia stem cells (LSC). Many hallmark stem cell properties are shared by HSC and LSC and therefore a better understanding of stemness regulation is crucial to improved HSC therapies and leukemia treatments targeting LSC. Our previous work on LSC subsets from >80 AML patient samples revealed that HSC and LSC share a transcriptional network that represent the core elements of stemness (Eppert, Nature Med 2011; Ng, Nature 2016). Hence, to identify the key regulators of LSC/HSC self-renewal and persistence we selected 64 candidate genes based on expression in functionally validated LSC vs. non-LSC fractions and assessed their potential to enhance self-renewal in a competitive in vivo screen. Here, we transduced cord blood CD34+CD38- cells with 64 barcoded lentiviral vectors to assemble 16 pools, each consisting of 8 individual gene-transduced populations, for transplantation into NSG mice. Strikingly, individual overexpression (OE) of 5 high scoring candidates revealed delayed repopulation kinetics of human HSC/progenitor cells (HSPC): gene-marking of human CD45+ and lin-CD34+ cells was reduced relative to input and control at 4w post transplantation, whereas by 20w engraftment of marked cells reached or exceeded input levels. For one of these candidates, C3ORF54/INKA1, we found that OE did not alter lineage composition neither in in vitro nor in vivo assays but increased the proportion of primitive CD34+ cells at 20w in vivo; moreover, secondary transplantation revealed a 4.5-fold increase in HSC frequency. Of note, serial transplantation from earlier time points (2w, 4w) revealed superior engraftment and hence greater self-renewal capacity upon INKA1-OE. Since we observed a 4-fold increase of phenotypic multipotent progenitors (MPP) relative to HSC within the CD34+ compartment (20w) we assessed whether INKA1-OE acts selectively on either cell population. The observation of latency in engraftment was recapitulated with sorted INKA1-OE HSC but not MPP. Likewise, liquid culture of HSPC and CFU-C assays on sorted HSC showed an initial delay in activation and colony formation upon INKA1-OE that was completely restored by extended culture and secondary CFU-C, respectively. INKA1-OE MPP showed a slight increase in total colony count in primary CFU-C and increased CDK6 levels in contrast to reduced CDK6 levels in INKA1-OE HSC emphasizing opposing effects of INKA1 on cell cycle entry and progression in either population. Taken together, this suggests that INKA1-OE preserves self-renewal capacity by retaining HSC preferentially in a latent state, however, upon transition to MPP leads to enhanced activation. Whilst INKA1 has been described as an inhibitor of p21(Cdc42/Rac)-activated kinase 4 (PAK4), no role for PAK4 is described in hematopoiesis. Nonetheless, its regulator Cdc42 is implicated in aging of murine HSPC by affecting H4K16 acetylation (H4K16ac) levels and polarity and has recently been described to regulate AML cell polarity and division symmetry. In our experiments immunostaining of HSPC subsets cultured in vitro and from xenografts indicates that INKA1-OE differentially affects epigenetics of these subsets linking H4K16ac to the regulation of stem cell latency. In AML, transcriptional upregulation of INKA1 in LSC vs. non-LSC fractions and at relapse in paired diagnosis-relapse analysis (Shlush, Nature 2017) implicates INKA1 as a regulator of LSC self-renewal and persistence. Indeed, INKA1-OE in cells derived from a primary human AML sample (8227) with a phenotypic and functional hierarchy (Lechman, Cancer Cell 2016) revealed a strong latency phenotype: In vitro and in vivo we observed label retention along with a steady increase in percentage of CD34+ cells, transient differentiation block, reduced growth rate, G0 accumulation and global reduction of H4K16ac. In summary, our data implicates INKA1 as a gate-keeper of stem cell latency in normal human hematopoiesis and leukemia. Studying the detailed pathways involved will shed light upon the mechanisms involved in HSC activation and latency induction and will help to harness these for novel therapeutic approaches. Disclosures Takayanagi: Kyowa Hakko Kirin Co., Ltd.: Employment.

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