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Although tyrosine kinase inhibitors are effective for treating chronic myeloid leukemia (CML), they often fail to eradicate the leukemia-initiating stem cells (LSCs), causing disease persistence and relapse. Evidence indicates that LSC persistence may be due to bone marrow (BM) niche protection. However, little is known about the underlying mechanisms. We here molecularly and functionally characterized BM niches in CML patients at diagnosis and revealed the altered niche composition and function in the CML patients. Long-term culture initiating cell (LTC-IC) assay showed that the mesenchymal stem cells from CML patients displayed an enhanced supporting capacity for normal and CML BM CD34+CD38- cells. Molecularly, RNA sequencing detected dysregulated cytokine and growth factor expression in CML patient BM cellular niches. Among them, CXCL14 was lost in the BM cellular niches in contrast to its expression in healthy BM. Restoring CXCL14 significantly inhibited CML LSC maintenance and enhanced their response to imatinib in vitro, and CML engraftment in vivo in NSG-SGM3 mice. Importantly, CXCL14 treatment dramatically inhibited CML engraftment in xenografted NSG-SGM3 mice, even to a greater degree than imatinib, and this inhibition persisted in patients with suboptimal TKI response. Mechanistically, CXCL14 upregulated inflammatory cytokine signaling but downregulated mTOR signaling and oxidative phosphorylation in CML LSCs. Together, we have discovered a suppressive role of CXCL14 in CML LSC growth. CXCL14 might offer a treatment option targeting CML LSCs.

Mutations of signal-induced proliferation-associated gene 1 ( SIPA1 ), a RAP1 GTPase-activating protein, were reported in patients with juvenile myelomonocytic leukemia, a childhood myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Sipa1 deficiency in mice leads to the development of age-dependent MPN. However, Sipa1 expression in bone marrow (BM) microenvironment and its effect on the pathogenesis of MPN remain unclear. We here report that Sipa1 is expressed in human and mouse BM stromal cells and downregulated in these cells from patients with MPN or MDS/MPN at diagnosis. By using the Sipa1 −/− MPN mouse model, we find that Sipa1 deletion causes phenotypic and functional alterations of BM mesenchymal stem and progenitor cells prior to the initiation of the MPN. Importantly, the altered Sipa1 −/− BM niche is required for the development of MDS/MPN following transplantation of normal hematopoietic cells. RNA sequencing reveals an enhanced inflammatory cytokine signaling and dysregulated Dicer1 , Kitl , Angptl1 , Cxcl12 , and Thpo in the Sipa1 −/− BM cellular niches. Our data suggest that Sipa1 expression in the BM niche is critical for maintaining BM niche homeostasis. Moreover, Sipa1 loss–induced BM niche alterations likely enable evolution of clonal hematopoiesis to the hematological malignancies. Therefore, restoring Sipa1 expression or modulating the altered signaling pathways involved might offer therapeutic potential for MPN.

Despite increasing evidence for the involvement of bone marrow (BM) hematopoietic stem cell niche in leukemogenesis, how BM mesenchymal stem and progenitor cells (MSPCs) contribute to leukemia niche formation and progression remains unclear. Using an MLL-AF9 acute myeloid leukemia (AML) mouse model, we demonstrate dynamic alterations of BM cellular niche components, including MSPCs and endothelial cells during AML development and its association with AML engraftment. Primary patient AML cells also induced similar niche alterations in xenografted mice. AML cell infiltration in BM causes an expansion of early B-cell factor 2+ (Ebf2+) MSPCs with reduced Cxcl12 expression and enhanced generation of more differentiated mesenchymal progenitor cells. Importantly, in vivo fate-mapping indicates that Ebf2+ MSPCs participated in AML niche formation. Ebf2+ cell deletion accelerated the AML development. These data suggest that native BM MSPCs may suppress AML. However, they can be remodeled by AML cells to form leukemic niche that might contribute to AML progression. AML induced dysregulation of hematopoietic niche factors like Angptl1, Cxcl12, Kitl, Il6, Nov, and Spp1 in AML BM MSPCs, which was associated with AML engraftment and partially appeared before the massive expansion of AML cells, indicating the possible involvement of the niche factors in AML progression. Our study demonstrates distinct dynamic features and roles of BM MSPCs during AML development.

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL(+)CD34(+)CD38(-) cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34(+)CD38(-) cells from 7 CML patients. The majority of the single leukemic BCR-ABL(+)CD34(+)CD38(-) cells expressed cysteinyl leukotriene receptors CYSLTI and CYSLT2. However, montelukast, an inhibitor of CYSLTI, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLTI signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients. (C) 2017 The Author(s). Published by Elsevier Inc.

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL

Although tyrosine kinase inhibitors (TKIs) have dramatically improved clinical outcome in chronic myeloid leukemia (CML), cure rarely occurs. This may be due to BCR-ABL-independent, aberrant signaling pathways, one of which leads to leukotriene (LT) formation. Well-recognized as inflammatory mediators, LT can also affect oncogenic mechanisms of several tumors. We have previously discovered elevated LT-synthesis and up-regulated cysteinyl-LT-inducing enzyme in CML. Here we report on dose-dependent inhibition of CML cell growth exerted by specific blockers of LT-signaling. Thus, the cysteinyl-LT1-receptor-antagonist montelukast significantly reduced the growth of K562, KCL22, and KU812 cells, as well as primary CD34

A novel Lin−CD34+CD38− integrin α2− bipotential megakaryocyte–erythrocyte progenitor population in the human bone marrow

Myelosuppression is a life-threatening complication of anti-cancer therapy including irradiation. Rapid and complete hematopoietic recovery after therapy-induced myelosuppression is required for a successful treatment outcome. This process relies on efficient regeneration of hematopoietic stem cells (HSCs) and is tightly controlled by bone marrow (BM) microenvironment consisting of mesenchymal stem/progenitor cells, endothelial cells as well as secreted factors including cytokines and extracellular matrix proteins (ECM). However, the extrinsic factors critical for promoting the hematopoietic recovery remain poorly understood. Laminins are heterotrimetric ECM composed of α, β, and γ chains. Laminin α4 chain (LAMA4) is an active component for laminin-411 and -421, which are located in vascular basement membrane. LAMA4 plays an important role for HSC homing after transplantation via interaction with laminin receptor integrin α6 (Qian H et al., Blood 2006). However, the role of LAMA4 in normal hematopoiesis and HSC reconstitution after irradiation-induced myelosuppression is not known. In this study, we first detected Lama4 gene expression in BM endothelial cells (CD31+), mesenchymal stem cells (MSC: CD45-Ter119-CD31-CD44-Sca1+CD51+), and mesenchymal progenitor cells (MPC: CD45-Ter119-CD31-CD44-Sca1-CD51+) in young adult mice. By using Lama4 deficient (Lama4-/-) mice, we analyzed the functional role of LAMA4 on hematopoietic activity at steady state. We found the lower number of platelets (PLTs) (p = 0.03), and neutrophils (Gr1+CD11b+) (p = 0.03) in the peripheral blood (PB) of Lama4-/- mice, but a higher frequency of common myeloid progenitor (Lin-Sca1-Kit+CD34+FcRlow) (p < 0.01) in the Lama4-/- BM at steady state, indicating that LAMA4 plays a role in the maintenance of physiological hematopoiesis. The important role of LAMA4 in hematopoietic recovery was demonstrated by delayed and incomplete recoveries of mature red blood cells, PLTs, and Gr1+CD11b+ cells in PB following sublethal irradiation (7Gy). The impaired recovery of erythropoiesis was also indicated by the higher values of mean corpuscular hemoglobin and mean corpuscular volume in PB as well as the higher frequency of megakaryocyte-erythrocyte progenitor (Lin-Sca1-Kit+CD34-FcR-) (p < 0.01) and colony-forming unit-erythrocyte (CFU-E) (p = 0.03) in the BM of the Lama4-/- mice at 6 weeks after irradiation, suggesting blocked erythrocyte maturation. In keeping with the refractory neutropenia, the frequency of colony-forming unit-granulocyte-macrophage (CFU-GM) was lower in the Lama4-/- BM compared to that in the age- and gender-matched wild type mice (p = 0.04). These data indicate that LAMA4 is critical for multiple hematopoietic lineage reconstitution post irradiation. To investigate the cellular and molecular mechanisms underlying the critical role of LAMA4 in hematopoietic recovery after the irradiation, we characterized the BM niche by colony assay, flow cytometry immunophenotyping, quantitative real time PCR (qPCR), and histological analysis. The number of colony-forming unit-fibroblast (CFU-F) was comparable between wild type and Lama4-/- in steady state. Interestingly, the proportion of BM MPCs, a population containing osteoblast progenitors, was significantly lower in the Lama4-/- mice compared to that in the wild type controls at steady state (p < 0.01). qPCR analysis showed downregulation of Il6 (p < 0.05) in the MSC and Angpt1 (p = 0.02) in the MPC of the Lama4-/-mice post irradiation. These data suggest that Lama4 deficiency alters BM stromal cell composition and gene expressions, which may be related to the impaired hematopoietic reconstitution. The recovery of BM vascular structure is essential for efficient reconstitution of hematopoiesis. We observed uniquely dilated blood vessels in Lama4-/- BM at 6-week post irradiation. This might be caused by the lower Angpt1 expression in Lama4-/- MPC since Angpt1/Tie2 signaling is required for vascular regeneration (Kopp HG et al., Blood 2005, Zhou BO et al., eLife 2015). The functional consequences of this phenotype are still under investigation. Altogether, LAMA4 is required for rapid and complete hematopoietic recovery post irradiation-induced myelosuppression. Therapeutic strategies to upregulate Lama4 may facilitate the recovery of hematopoiesis following HSC transplantation under preconditioning using irradiation. Disclosures No relevant conflicts of interest to declare.

Myelodysplastic Syndrome with Ring Sideroblast (MDS-RS), a clonal hematopoietic cell neoplasm, is low risk MDS, characterized by anemia, hyperplastic ineffective erythropoiesis and marrow ring sideroblasts. Mouse studies have shown that bone marrow niche, including endothelial cells, osteoblasts, adipocytes and mesenchymal stem cells (MSCs), contribute to progression of various hematological disorders. However, in vivo contribution of the different bone marrow stromal cells to the progression of MDS-RS in patients remains largely unknown. To investigate this, we have phenotypically, molecularly and functionally characterized the BM native stromal cell subsets including MSCs freshly isolated by multi-color fluorescence activated cell sorting (FACS) from bone marrow of MDS-RS patients and age-matched healthy donors. We found: 1) the MDS-RS MSCs, estimated by colony forming unit-fibroblast (CFU-F), shared similar immunophenotype with normal MSCs (CD45-CD235a-CD31-CD44-, most of which were CD271+CD146+CD106+); 2) the frequency of CFU-Fs was significantly increased in the phenotypically defined MSCs of MDS-RS bone marrow compared to that of age-matched healthy controls (p=0.005); 3) multi-lineage differentiation assay revealed impaired osteogenic differentiation potential, but enhanced adipogenic differentiation potential of MDS-RS MSCs; 4) FACS analysis showed increased frequency of the adhesion receptor integrin α4 (ITGA4) in the CD44- MSCs from MDS-RS bone marrow (p=0.013); 5) Correspondingly, RNA-sequencing of the freshly isolated bone marrow MSCs and endothelial cells revealed altered gene expression profile of these cells in MDS-RS patients. Among those, ITGA4, ITGA11, ITGAE and ITGB1 are upregulated in the MDS-RS MSCs, indicating potential abnormal adhesive interaction of the MSCs with hematopoietic stem cells in the patients. In addition, the cell cycling gene KI67 is upregulated whereas cell cycle negative regulators, like CDKN1A and CDKN1C are downregulated in the MDS-RS MSCs, which is consistent with their increased CFU-F activity. Interestingly, we detected abnormal expression of hematopoietic growth factors such as downregulation of ANGPTL4 in the MDS-RS MSCs and upregulation of ANGPT1 in the MDS-RS endothelial cells. The functional relationship between the stromal cell alterations and the abnormal hematopoiesis as well as the underlying molecular mechanisms are currently under investigation. Taken together, our data provide new evidence for phenotypic, functional and molecular alterations of bone marrow mesenchymal cells in MDS-RS patients. The molecular pathways mediating bone marrow niche alteration could be potential therapeutic targets for new treatment of MDS-RS. Disclosures No relevant conflicts of interest to declare.

It has been reported that bone marrow (BM) hematopoietic niche is critical for the maintenance of normal hematopoiesis and the development of hematopoietic disorders. However, little is known about how the different BM niche components contribute to the initiation and progression of acute myeloid leukemia (AML). To explore the role of BM stromal cells in the development of AML, we here characterized the BM stromal cells in AML mouse model induced by transplantation of mouse BM cellstransduced with MLL-AF9 oncogene. We subdivided the BM stromal cells (CD45-TER119-) into mesenchymal stem cells (MSCs, CD31-CD44-CD51+SCA1+), mesenchymal progenitor cells (MPCs, CD31-CD44-CD51+SCA1-), and endothelial cells (CD31+) in the mice after development of AML by multicolor fluorescent activated cell sorting (FACS) (Qian et al., Mol Cell Biol, 2013). We found significantly increased frequencies of BM MSCs (p < 0.0001), MPCs (p < 0.0001), and endothelial cells (p < 0.0001) in the AML mice, indicating an AML-induced alteration of BM stromal cell composition. The increased frequency of MSCs in AML mouse BM was confirmed by the increased number of colony foming unit-fibroblasts in the AML mice (p < 0.0001). Furthermore, in vitro multi-lineage differentiation assay revealed increased adipogenic and osteogenic differentiation capacities of the BM MSCs in the AML mice. Consistent with the increased osteogenic differentiation capacity of AML MSCs, Runx2 mRNA expression was increased in freshly sorted AML MSCs compared to that in the control mice. In addition, quantitative real time PCR on freshly sorted BM MPCs showed a dramatic downregulation of the genes important in hematopoietic stem cell regulation, including angiopoietin like 1 (Angptl1), C-X-C motif chemokine (Cxcl12), and kit ligand (Kitl) in the AML mice. The altered gene expression might contribute to the AML progression. To specifically determine the contribution of MSCs to AML development, we analyzed the MSC population expressing Early B cell factor 2 (Ebf2) (Qian et al., Mol Cell Biol, 2013) by establishing the AML in Ebf2-Egfp transgenic recipient mice. We detected an increased frequency of Ebf2+ cells in the recipient BM after development of AML (p = 0.0018). In addition, by using the triple transgenic Tg(Ebf2-Egfp x Ebf2-CreER x Rosa26-loxpStoploxp-Tomato) mouse model, where Ebf2+ MSCs and their progenies can be traced by tomato fluorescent protein after tamoxifen treatment, we found that the MSCs and MPCs generated from the Ebf2+ MSCs were also increased in the AML mouse BM (p < 0.05). To further investigate the functional contribution of Ebf2+ MSCs to the AML progression, we took advantage of the transgenic Tg(Ebf2-CreER x Rosa26-loxpStoploxp-DTA) mouse model allowing specific deletion of the Ebf2-expressing MSCs. Strikingly, we observed an accelerated initiation of AML, illustrated by the shorter survival of the MSC depleted recipients (Figure 1), compared to the non-depleted recipients, suggesting that Ebf2+ MSCs could suppress AML progression. Taken together, our data show that BM stromal cell composition and function are altered in the mice with AML and the BM MSCs may act to suppress the progression of the MLL-AF9 induced AML. These findings provide new evidence for the niche contribution to the development of AML. Disclosures No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) is a myeloproliferative stem cell neoplasm characterized by the presence of the BCR-ABL1 fusion gene. Although current treatment with tyrosine kinase inhibitors (TKI) has dramatically improved the prognosis of CML, these inhibitors do not eradicate leukemic stem cells (LSC) in most patients with the risk of recurrence of leukemia if TKI are stopped. In vitro studies have suggested that this might be attributable to protection of bone marrow (BM) stromal cells, such as osteoblasts, adipocytes, endothelial and mesenchymal stem cells (MSCs). However, how different BM stromal cells contribute to the persistence of LSC remains largely unknown. To investigate this issue we have compared freshly isolated BM stromal cell subsets including MSCs from newly diagnosed CML patients (n=10) with that from age-matched healthy donors (n=12). Distinct from the previous studies on culture-selected BM stromal cells, the naive stromal cells isolated by multi-color fluorescence activated cell sorting (FACS) were phenotypically, molecularly and functionally characterized in the present study. We observed: 1) Similar to the immunophenotype of normal MSCs (CD45-CD235a-CD31-CD44-, most of which were CD271+CD146+CD106+) (Qian et al., JBC, 2012), the CML MSCs, estimated by colony forming unit-fibroblast (CFU-F), were also enriched in the CD45-CD235a-CD31-CD44- cell fraction. 2) The frequency of CFU-Fs was significantly increased in CML BM compared to that in the age-matched healthy controls (p=0.005). 3) A decreased osteogenic, but enhanced adipogenic differentiation potential of CML MSC was revealed in multilineage differentiation assay. This suggests a skewed differentiation potential of the CML MSCs towards adipocytes, possibly related to an altered stromal cell composition in the patients; 4) An increased proportion of CD31+ endothelial cells was seen in CML BM stroma compared to controls (p=0.023) by FACS. 5) An upregulation of the adhesion receptor integrin α4/CD49D was seen in the CD44- MSCs from CML patients (p=0.0087). Conversely, a downregulation of transcripts of Angiopoietin 1, CXCL12, KIT ligand and LAMA4 in the patient MSCs was detected by Quantitative-PCR, indicating an altered hematopoiesis-supportive function of CML MSCs. 6) Importantly, no BCR-ABL fusion were found in the freshly sorted MSCs and mature stromal cells using Fluorescence In Situ Hybridization analysis, suggesting that these MSCs were not part of the leukemic clone. Taken together, our data provide evidence for phenotypic and functional alterations of BM mesenchymal cells in CML patients. The functional relationship between the stromal cell alterations and the growth of LSC as well as the underlying molecular mechanisms are currently under investigation. Disclosures Mustjoki: Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding.

Hematopoiesis is tightly regulated by bone marrow (BM) stromal cells through the production of soluble factors, matrix proteins, and intracellular signals initiated by cell-cell adhesive interactions. Despite the involvement of mesenchymal stem and progenitor cells in the maintenance of normal hematopoiesis and the development of hematological disorders have been reported (Mendez-Ferrer et al., 2010, Nature)(Raaijmakers et al., 2010, Nature), little is known how the different BM stromal cell fractions contribute to the processes. Signal-induced proliferation-associated gene 1 (Sipa1), a principal Rap1 GTPase activating protein, is expressed in hematopoietic cells (Ishida et al., 2003, Cancer Cell). Deletion of Sipa1 results in age-dependent development of myeloproliferative neoplasm (MPN) in mice, resembling human chronic myelogenous leukemia (Ishida et al., 2003, Cancer Cell). However, it is not known whether the deletion of Sipa1 affects BM cellular niche components, which could lead to the development of the MPN. Knowledge of this is important for better understanding the role of BM stromal cells in the initiation and progression of MPN. In the present study, we first analyzed Sipa1 expression by multicolor fluorescent activated cell sorting (FACS) using hematopoietic cell marker CD45 and TER119, mesenchymal stem cell (MSC) associated marker CD51 and SCA1, and endothelial cell marker CD31. We found that Sipa1 gene was also expressed in different BM stromal cell subsets including MSCs (CD45-TER119-CD31-CD44-CD51+SCA1+), more differentiated mesenchymal progenitor cells (CD45-TER119-CD31-CD44-CD51+SCA1-), and endothelial cells (CD45-TER119-CD31+). Among all the cell types, Sipa1 expression seemed to be higher in MSCs and endothelial cells than other cell types. We next studied whether the gene deficiency led to the alteration of BM stromal cell composition and function by phenotypically, functionally, and molecularly analyzing BM stromal cell subsets in young ( Taken together, our data suggested the BM stromal cell composition and function in the Sipa1-/- mice have already been altered before the initiation of MPN. Transplantations of normal hematopoietic stem cells into Sipa1 deficient BM have been initiated for examining if the Sipa1-/- BM niches play an instructive role in the initiation of MPN. Disclosures No relevant conflicts of interest to declare.

The Interleukin 12 (IL-12) family of cytokines is comprised of heterodimeric proteins including IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (IL27p28/Ebi3) and IL-35 (p35/Ebi3). IL-27p28 and IL-12p40 have recently been shown to antagonize IL-27 and IL-12 functions, respectively, suggesting that single chain IL-12 family members may act as dominant-negative inhibitors of IL-12 family heterodimeric cytokines. Here, we have genetically engineered IL-12p35 (rIL-12p35) and examined whether IL-12p35 has biological activities independent of its partner, IL-12p40 or EBI3. We show that rIL-12p35 blocked IL-6 mediated activation of STAT1 and STAT3 pathways of TCR-activated CD4+ T cells and inhibited the expansion of Th17 but not Th1 cells. Of particular significance, rIL-12p35 protected mice from autoimmune uveitis by inhibiting pathogenic Th17 cell expansion. Our study demonstrates that IL-12p35 can be used to modulate IL-6-mediated signaling pathways and treat a CNS autoimmune disease.