Temporal Single Cell Analysis of Leukemia Microenvironment Identifies Taurine-Taurine Transporter Axis as a Key Regulator of Myeloid Leukemia.

Signals from the microenvironment are known to be critical for development, sustaining adult stem cells, and for oncogenic progression. While candidate niche-driven signals that can promote cancer progression have been identified 1-6 , concerted efforts to comprehensively map microenvironmental ligands for cancer stem cell specific surface receptors have been lacking. Here, we use temporal single cell RNA-sequencing to identify molecular cues from the bone marrow stromal niche that engage leukemia stem cells (LSC) during oncogenic progression. We integrate these data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map 7 to develop a temporal receptor-ligand interactome essential for disease progression. These analyses identify the taurine transporter (TauT)-taurine axis as a critical dependency of myeloid malignancies. We show that taurine production is restricted to the osteolineage population during cancer initiation and expansion. Inhibiting taurine synthesis in osteolineage cells impairs LSC growth and survival. Our experiments with the TauT genetic loss of function murine model indicate that its loss significantly impairs the progression of aggressive myeloid leukemias in vivo by downregulating glycolysis. Further, TauT inhibition using a small molecule strongly impairs the growth and survival of patient derived myeloid leukemia cells. Finally, we show that TauT inhibition can synergize with the clinically approved oxidative phosphorylation inhibitor venetoclax 8, 9 to block the growth of primary human leukemia cells. Given that aggressive myeloid leukemias continue to be refractory to current therapies and have poor prognosis, our work indicates targeting the taurine transporter may be of therapeutic significance. Collectively, our data establishes a temporal landscape of stromal signals during cancer progression and identifies taurine-taurine transporter signaling as an important new regulator of myeloid malignancies.

Interplay Between Skeletal and Hematopoietic Cells in the Bone Marrow Microenvironment in Homeostasis and Aging.

PURPOSE OF THE REVIEW: In this review, we discuss the most recent scientific advances on the reciprocal regulatory interactions between the skeletal and hematopoietic stem cell niche, focusing on immunomodulation and its interplay with the cell's mitochondrial function, and how this impacts osteoimmune health during aging and disease. RECENT FINDINGS: Osteoimmunology investigates interactions between cells that make up the skeletal stem cell niche and immune system. Much work has investigated the complexity of the bone marrow microenvironment with respect to the skeletal and hematopoietic stem cells that regulate skeletal formation and immune health respectively. It has now become clear that these cellular components cooperate to maintain homeostasis and that dysfunction in their interaction can lead to aging and disease. Having a deeper, mechanistic appreciation for osteoimmune regulation will lead to better research perspective and therapeutics with the potential to improve the aging process, skeletal and hematologic regeneration, and disease targeting.

Local Delivery of SBRT and IL12 by mRNA Technology Overcomes Immunosuppressive Barriers to Eliminate Pancreatic Cancer.

The immunosuppressive milieu in pancreatic cancer (PC) is a significant hurdle to treatments, resulting in survival statistics that have barely changed in 5 decades. Here we present a combination treatment consisting of stereotactic body radiation therapy (SBRT) and IL-12 mRNA lipid nanoparticles delivered directly to pancreatic murine tumors. This treatment was effective against primary and metastatic models, achieving cures in both settings. IL-12 protein concentrations were transient and localized primarily to the tumor. Depleting CD4 and CD8 T cells abrogated treatment efficacy, confirming they were essential to treatment response. Single cell RNA sequencing from SBRT/IL-12 mRNA treated tumors demonstrated not only a complete loss of T cell exhaustion, but also an abundance of highly proliferative and effector T cell subtypes. SBRT elicited T cell receptor clonal expansion, whereas IL-12 licensed these cells with effector function. This is the first report demonstrating the utility of SBRT and IL-12 mRNA in PC. Statement of significance: This study demonstrates the use of a novel combination treatment consisting of radiation and immunotherapy in murine pancreatic tumors. This treatment could effectively treat local and metastatic disease, suggesting it may have the potential to treat a cancer that has not seen a meaningful increase in survival in 5 decades.

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Vitamin D Receptor Antagonist MeTC7 Inhibits PD-L1.

Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.

Efferocytosis by bone marrow mesenchymal stromal cells disrupts osteoblastic differentiation via mitochondrial remodeling.

The efficient clearance of dead and dying cells, efferocytosis, is critical to maintain tissue homeostasis. In the bone marrow microenvironment (BMME), this role is primarily fulfilled by professional bone marrow macrophages, but recent work has shown that mesenchymal stromal cells (MSCs) act as a non-professional phagocyte within the BMME. However, little is known about the mechanism and impact of efferocytosis on MSCs and on their function. To investigate, we performed flow cytometric analysis of neutrophil uptake by ST2 cells, a murine bone marrow-derived stromal cell line, and in murine primary bone marrow-derived stromal cells. Transcriptional analysis showed that MSCs possess the necessary receptors and internal processing machinery to conduct efferocytosis, with Axl and Tyro3 serving as the main receptors, while MerTK was not expressed. Moreover, the expression of these receptors was modulated by efferocytic behavior, regardless of apoptotic target. MSCs derived from human bone marrow also demonstrated efferocytic behavior, showing that MSC efferocytosis is conserved. In all MSCs, efferocytosis impaired osteoblastic differentiation. Transcriptional analysis and functional assays identified downregulation in MSC mitochondrial function upon efferocytosis. Experimentally, efferocytosis induced mitochondrial fission in MSCs. Pharmacologic inhibition of mitochondrial fission in MSCs not only decreased efferocytic activity but also rescued osteoblastic differentiation, demonstrating that efferocytosis-mediated mitochondrial remodeling plays a critical role in regulating MSC differentiation. This work describes a novel function of MSCs as non-professional phagocytes within the BMME and demonstrates that efferocytosis by MSCs plays a key role in directing mitochondrial remodeling and MSC differentiation. Efferocytosis by MSCs may therefore be a novel mechanism of dysfunction and senescence. Since our data in human MSCs show that MSC efferocytosis is conserved, the consequences of MSC efferocytosis may impact the behavior of these cells in the human skeleton, including bone marrow remodeling and bone loss in the setting of aging, cancer and other diseases.

The bone marrow stroma in human myelodysplastic syndrome reveals alterations that regulate disease progression.

Myelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.

Mitochondrial Transfer to Host Cells from Ex Vivo Expanded Donor Hematopoietic Stem Cells.

Mitochondrial dysfunction is observed in various conditions, from metabolic syndromes to mitochondrial diseases. Moreover, mitochondrial DNA (mtDNA) transfer is an emerging mechanism that enables the restoration of mitochondrial function in damaged cells. Hence, developing a technology that facilitates the transfer of mtDNA can be a promising strategy for the treatment of these conditions. Here, we utilized an ex vivo culture of mouse hematopoietic stem cells (HSCs) and succeeded in expanding the HSCs efficiently. Upon transplantation, sufficient donor HSC engraftment was attained in-host. To assess the mitochondrial transfer via donor HSCs, we used mitochondrial-nuclear exchange (MNX) mice with nuclei from C57BL/6J and mitochondria from the C3H/HeN strain. Cells from MNX mice have C57BL/6J immunophenotype and C3H/HeN mtDNA, which is known to confer a higher stress resistance to mitochondria. Ex vivo expanded MNX HSCs were transplanted into irradiated C57BL/6J mice and the analyses were performed at six weeks post transplantation. We observed high engraftment of the donor cells in the bone marrow. We also found that HSCs from the MNX mice could transfer mtDNA to the host cells. This work highlights the utility of ex vivo expanded HSC to achieve the mitochondrial transfer from donor to host in the transplant setting.

Myelodysplastic syndromes disable human CD271+VCAM1+CD146+ niches supporting normal hematopoietic stem/progenitor cells.

Mesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+ (NGFR+/VCAM1+/MCAM+/Lin-; NVML) cells display stem cell characteristics, are compatible with murine BM-derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression of MDS.

The roles of bone remodeling in normal hematopoiesis and age-related hematological malignancies.

Prior research establishing that bone interacts in coordination with the bone marrow microenvironment (BMME) to regulate hematopoietic homeostasis was largely based on analyses of individual bone-associated cell populations. Recent advances in intravital imaging has suggested that the expansion of hematopoietic stem cells (HSCs) and acute myeloid leukemia cells is restricted to bone marrow microdomains during a distinct stage of bone remodeling. These findings indicate that dynamic bone remodeling likely imposes additional heterogeneity within the BMME to yield differential clonal responses. A holistic understanding of the role of bone remodeling in regulating the stem cell niche and how these interactions are altered in age-related hematological malignancies will be critical to the development of novel interventions. To advance this understanding, herein, we provide a synopsis of the cellular and molecular constituents that participate in bone turnover and their known connections to the hematopoietic compartment. Specifically, we elaborate on the coupling between bone remodeling and the BMME in homeostasis and age-related hematological malignancies and after treatment with bone-targeting approaches. We then discuss unresolved questions and ambiguities that remain in the field.

Immune Dysfunction, Cytokine Disruption, and Stromal Changes in Myelodysplastic Syndrome: A Review.

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow dysfunction and increased risk of transformation to leukemia. MDS represent complex and diverse diseases that evolve from malignant hematopoietic stem cells and involve not only the proliferation of malignant cells but also the dysfunction of normal bone marrow. Specifically, the marrow microenvironment-both hematopoietic and stromal components-is disrupted in MDS. While microenvironmental disruption has been described in human MDS and murine models of the disease, only a few current treatments target the microenvironment, including the immune system. In this review, we will examine current evidence supporting three key interdependent pillars of microenvironmental alteration in MDS-immune dysfunction, cytokine skewing, and stromal changes. Understanding the molecular changes seen in these diseases has been, and will continue to be, foundational to developing effective novel treatments that prevent disease progression and transformation to leukemia.

GM-CSF drives myelopoiesis, recruitment and polarisation of tumour-associated macrophages in cholangiocarcinoma and systemic blockade facilitates antitumour immunity.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is rising in incidence, and at present, there are limited effective systemic therapies. iCCA tumours are infiltrated by stromal cells, with high prevalence of suppressive myeloid populations including tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Here, we show that tumour-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) and the host bone marrow is central for monopoiesis and potentiation of TAMs, and abrogation of this signalling axis facilitates antitumour immunity in a novel model of iCCA. METHODS: Blood and tumours were analysed from iCCA patients and controls. Treatment and correlative studies were performed in mice with autochthonous and established orthotopic iCCA tumours treated with anti-GM-CSF monoclonal antibody. RESULTS: Systemic elevation in circulating myeloid cells correlates with poor prognosis in patients with iCCA, and patients who undergo resection have a worse overall survival if tumours are more infiltrated with CD68+ TAMs. Mice with spontaneous iCCA demonstrate significant elevation of monocytic myeloid cells in the tumour microenvironment and immune compartments, and tumours overexpress GM-CSF. Blockade of GM-CSF with a monoclonal antibody decreased tumour growth and spread. Mice bearing orthotopic tumours treated with anti-GM-CSF demonstrate repolarisation of immunosuppressive TAMs and MDSCs, facilitating T cell response and tumour regression. GM-CSF blockade dampened inflammatory gene networks in tumours and TAMs. Human tumours with decreased GM-CSF expression exhibit improved overall survival after resection. CONCLUSIONS: iCCA uses the GM-CSF-bone marrow axis to establish an immunosuppressive tumour microenvironment. Blockade of the GM-CSF axis promotes antitumour T cell immunity.

CCR5 maintains macrophages in the bone marrow and drives hematopoietic failure in a mouse model of severe aplastic anemia.

Severe aplastic anemia (SAA) is an acquired, T cell-driven bone marrow (BM) failure disease characterized by elevated interferon gamma (IFNγ), loss of hematopoietic stem cells (HSCs), and altered BM microenvironment, including dysfunctional macrophages (MΦs). T lymphocytes are therapeutic targets for treating SAA, however, the underlying mechanisms driving SAA development and how innate immune cells contribute to disease remain poorly understood. In a murine model of SAA, increased beta-chemokines correlated with disease and were partially dependent on IFNγ. IFNγ was required for increased expression of the chemokine receptor CCR5 on MΦs. CCR5 antagonism in murine SAA improved survival, correlating with increased platelets and significantly increased platelet-biased CD41hi HSCs. T cells are key drivers of disease, however, T cell-specific CCR5 expression and T cell-derived CCL5 were not necessary for disease. CCR5 antagonism reduced BM MΦs and diminished their expression of Tnf and Ccl5, correlating with reduced frequencies of IFNγ-secreting BM T cells. Mechanistically, CCR5 was intrinsically required for maintaining BM MΦs during SAA. Ccr5 expression was significantly increased in MΦs from aged mice and humans, relative to young counterparts. Our data identify CCR5 signaling as a key axis promoting the development of IFNγ-dependent BM failure, particularly relevant in aging where Ccr5 expression is elevated.

Reduction of leukemic burden via bone-targeted nanoparticle delivery of an inhibitor of C-chemokine (C-C motif) ligand 3 (CCL3) signaling.

Leukemias are challenging diseases to treat due, in part, to interactions between leukemia cells and the bone marrow microenvironment (BMME) that contribute significantly to disease progression. Studies have shown that leukemic cells secrete C-chemokine (C-C motif) ligand 3 (CCL3), to disrupt the BMME resulting in loss of hematopoiesis and support of leukemic cell survival and proliferation. In this study, a murine model of blast crisis chronic myelogenous leukemia (bcCML) that expresses the translocation products BCR/ABL and Nup98/HoxA9 was used to determine the role of CCL3 in BMME regulation. Leukemic cells derived from CCL3-/- mice were shown to minimally engraft in a normal BMME, thereby demonstrating that CCL3 signaling was necessary to recapitulate bcCML disease. Further analysis showed disruption in hematopoiesis within the BMME in the bcCML model. To rescue the altered BMME, therapeutic inhibition of CCL3 signaling was investigated using bone-targeted nanoparticles (NP) to deliver Maraviroc, an inhibitor of C-C chemokine receptor type 5 (CCR5), a CCL3 receptor. NP-mediated Maraviroc delivery partially restored the BMME, significantly reduced leukemic burden, and improved survival. Overall, our results demonstrate that inhibiting CCL3 via CCR5 antagonism is a potential therapeutic approach to restore normal hematopoiesis as well as reduce leukemic burden within the BMME.

Bone marrow mesenchymal stromal cells from acute myelogenous leukemia patients demonstrate adipogenic differentiation propensity with implications for leukemia cell support.

Bone marrow mesenchymal stromal cells (MSCs) constitute one of the important components of the hematopoietic microenvironmental niche. In vivo studies have shown that depletion of marrow MSCs resulted in reduction of hematopoietic stem cell content, and there is in vitro evidence that marrow MSCs are able to support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. How MSCs from leukemia marrow differ from normal counterparts and how they are influenced by the presence of leukemia stem and progenitor cells are still incompletely understood. In this work, we compared normal donor (ND) and acute myelogenous leukemia (AML) derived MSCs and found that AML-MSCs had increased adipogenic potential with improved ability to support survival of leukemia progenitor cells. To identify underlying changes, RNA-Seq analysis was performed. Gene ontology and pathway analysis revealed adipogenesis to be among the set of altered biological pathways dysregulated in AML-MSCs as compared with ND-MSCs. Expression of both SOX9 and EGR2 was decreased in AML-MSCs as compared with ND-MSCs. Increasing expression of SOX9 decreased adipogenic potential of AML-MSCs and decreased their ability to support AML progenitor cells. These findings suggest that AML-MSCs possess adipogenic potential which may enhance support of leukemia progenitor cells.

What is the role of the microenvironment in MDS?

Treating myelodysplastic syndromes (MDS) remains challenging. Hematopoiesis occurs within a heterogeneous, complex and dynamic microenvironment, and a multiplicity of mutations in hematopoietic stem and progenitor cells (HSPCs) lead to MDS. But is there a role for the microenvironment? Here we review experimental and conceptual arguments that support a role for the microenvironment, provide evidence for the disruption of the microenvironment in MDS, and explore microenvironmental signals that may provide a targetable and conserved vulnerability in MDS that transcend genetic heterogeneity.

Acute and late effects of combined internal and external radiation exposures on the hematopoietic system.

Purpose: Incidents, such as nuclear facility accidents and the release of a 'dirty bomb', might result in not only external irradiation of personnel, but additional internal exposures through concomitant inhalation and/or ingestion of radioactive particulates. The purpose of this study was to define the impact of such a combination of radiation injuries on the hematopoietic niche.Material and methods: To assess changes in the murine hematopoietic system, we used a combined exposure of total body irradiation (TBI, 6 Gy) followed immediately by an internal (intraperitoneal) administration of 100 µCi of soluble 137Cs. We then evaluated acute survival in combined versus single modality exposure groups, as well as assessing hematopoietic function at 12 and 26 week time points.Results: Acutely, the combination of external and internal exposures led to an unexpected delay in excretion of 137Cs, increasing the absorbed dose in the combined exposure group and leading to mortality from an acute hematopoietic syndrome. At 12 weeks, all exposure paradigms resulted in decreased numbers of phenotypic hematopoietic stem cells (HSCs), particularly the short-term HSCs (ST-HSC); long-term HSCs (LT-HSC) were depleted only in the internal and combined exposure groups. At 26 weeks, there was significant anemia in both the TBI alone and combined exposure groups. There were decreased numbers in both the LT- and ST-HSCs and decreased functionality, as measured by competitive repopulation, was seen in all radiation groups, with the greatest effects seen in the internal and combined exposure groups.Conclusions: Our data indicate that a combined injury of sublethal external irradiation with internal contamination induces significant and persistent changes in the hematopoietic system, as may have been predicted from the literature and our own group's findings. However, a novel observation was that the combined exposure led to an alteration in the excretion kinetics of the internal contamination, increasing the acute effects beyond those anticipated. As a result, we believe that a combined exposure poses a unique challenge to the medical community during both the acute and, possibly, delayed recovery stages.

Flaming and fanning: The Spectrum of inflammatory influences in myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) represent neoplasms derived from the expansion of mutated clonal hematopoietic cells which often demonstrate aberrant differentiation potential with resultant cytopenias and a propensity to evolve into acute myelogenous leukemia. While multiple mutations have been identified which may serve as drivers of the MDS clone, there is accumulating evidence that MDS clones and subclones are subject to modulation by the marrow microenvironment and its inflammatory milieu. There is also a strong link between autoimmune disorders and MDS. In this review, we examine the role of inflammatory cytokines, toll like receptors, pyroptosis, stromal cells, and cellular inflammatory mediators in MDS initiation, propagation, and progression. These contributions in a background of mutational, epigenetic, and aging changes in the marrow are also reviewed. Such inflammatory mediators may be subject to therapeutic agents which will enhance suppression of the MDS clone with potential to improve therapeutic outcomes in this disease which is usually incurable in aged patients not eligible for stem cell transplantation.