Genetic analysis of human RNA binding motif protein 48 (RBM48) reveals an essential role in U12-type intron splicing.

U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for the evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome, predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA binding motif protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5' end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron-containing genes have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of minor intron-containing genes disrupted in both species. Mutations in the majority of these orthologous minor intron-containing genes have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.

Intratumoural haematopoietic stem and progenitor cell differentiation into M2 macrophages facilitates the regrowth of solid tumours after radiation therapy.

BACKGROUND: Bone-marrow-derived haematopoietic stem and progenitor cells (HSPCs) are a prominent part of the highly complex tumour microenvironment (TME) where they localise within tumours and maintain haematopoietic potency. Understanding the role HSPCs play in tumour growth and response to radiation therapy (RT) may lead to improved patient treatments and outcomes. METHODS: We used a mouse model of non-small cell lung carcinoma where tumours were exposed to RT regimens alone or in combination with GW2580, a pharmacological inhibitor of colony stimulating factor (CSF)-1 receptor. RT-PCR, western blotting and immunohistochemistry were used to quantify expression levels of factors that affect HSPC differentiation. DsRed+ HSPC intratumoural activity was tracked using flow cytometry and confocal microscopy. RESULTS: We demonstrated that CSF-1 is enhanced in the TME following exposure to RT. CSF-1 signaling induced intratumoural HSPC differentiation into M2 polarised tumour-associated macrophages (TAMs), aiding in post-RT tumour survival and regrowth. In contrast, hyperfractionated/pulsed radiation therapy (PRT) and GW2580 ablated this process resulting in improved tumour killing and mouse survival. CONCLUSIONS: Tumours coopt intratumoural HSPC fate determination via CSF-1 signaling to overcome the effects of RT. Thus, limiting intratumoural HSPC activity represents an attractive strategy for improving the clinical treatment of solid tumours.

Interleukin-8 blockade prevents activated endothelial cell mediated proliferation and chemoresistance of acute myeloid leukemia.

One of the greatest challenges in treating acute myeloid leukemia (AML) is chemotherapy refractory disease. Previously, we demonstrated a novel mechanism whereby AML-induced endothelial cell (EC) activation leads to subsequent leukemia cell adherence, quiescence and chemoresistance, identifying activated ECs as potential mediators of relapse. We now show mechanistically that EC activation induces the secretion of interleukin-8 (IL-8) leading to significant expansion of non-adherent AML cells and resistance to cytarabine (Ara-C). Through crystallography and computational modeling, we identified a pocket within IL-8 responsible for receptor binding, screened for small molecules that fit within this pocket, and blocked IL-8 induced proliferation and chemo-protection of AML cells with a hit compound. Results from this study show a new therapeutic strategy for targeting the sanctuary of an activated leukemia microenvironment.

Integrin α6 (CD49f), The Microenvironment and Cancer Stem Cells.

Cancer is a highly prevalent and potentially terminal disease that affects millions of individuals worldwide. Here, we review the literature exploring the intricacies of stem cells bearing tumorigenic characteristics and collect evidence demonstrating the importance of integrin α6 (ITGA6, also known as CD49f) in cancer stem cell (CSC) activity. ITGA6 is commonly used to identify CSC populations in various tissues and plays an important role sustaining the self-renewal of CSCs by interconnecting them with the tumorigenic microenvironment.

Effect of Irradiation on Tumor Microenvironment and Bone Marrow Cell Migration in a Preclinical Tumor Model.

PURPOSE: To characterize the tumor microenvironment after standard radiation therapy (SRT) and pulsed radiation therapy (PRT) in Lewis lung carcinoma (LLC) allografts. METHODS AND MATERIALS: Subcutaneous LLC tumors were established in C57BL/6 mice. Standard RT or PRT was given at 2 Gy/d for a total dose of 20 Gy using a 5 days on, 2 days off schedule to mimic clinical delivery. Radiation-induced tumor microenvironment changes were examined after treatment using flow cytometry and antibody-specific histopathology. Normal tissue effects were measured using noninvasive (18)F-fluorodeoxyglucose positron emission tomography/computed tomography after naïve animals were given whole-lung irradiation to 40 Gy in 4 weeks using the same 2-Gy/d regimens. RESULTS: Over the 2 weeks of therapy, PRT was more effective than SRT at reducing tumor growth rate (0.31 ± 0.02 mm(3)/d and 0.55 ± 0.04 mm(3)/d, respectively; P<.007). Histopathology showed a significant comparative reduction in the levels of Ki-67 (14.5% ± 3%), hypoxia (10% ± 3.5%), vascular endothelial growth factor (2.3% ± 1%), and stromal-derived factor-1α (2.5% ± 1.4%), as well as a concomitant decrease in CD45(+) bone marrow-derived cell (BMDC) migration (7.8% ± 2.2%) after PRT. The addition of AMD3100 also decreased CD45(+) BMDC migration in treated tumors (0.6% ± 0.1%). Higher vessel density was observed in treated tumors. No differences were observed in normal lung tissue after PRT or SRT. CONCLUSIONS: Pulsed RT-treated tumors exhibited slower growth and reduced hypoxia. Pulsed RT eliminated initiation of supportive mechanisms utilized by tumors in low oxygen microenvironments, including angiogenesis and recruitment of BMDCs.

Synergistic anti-leukemic interactions between panobinostat and MK-1775 in acute myeloid leukemia ex vivo.

MK-1775 is the first-in-class selective Wee1 inhibitor which has been demonstrated to synergize with CHK1 inhibitors in various malignancies. In this study, we report that the pan-histone deacetylase inhibitor (HDACI) panobinostat synergizes with MK-1775 in acute myeloid leukemia (AML), a malignancy which remains a clinical challenge and requires more effective therapies. Using both AML cell line models and primary patient samples, we demonstrated that panobinostat and MK-1775 synergistically induced proliferation arrest and cell death. We also demonstrated that panobinostat had equal anti-leukemic activities against primary AML blasts derived from patients either at initial diagnosis or at relapse. Interestingly, treatment with panobinostat alone or in combination with MK-1775 resulted in decreased Wee1 protein levels as well as downregulation of the CHK1 pathway. shRNA knockdown of CHK1 significantly sensitized AML cells to MK-1775 treatment, while knockdown of Wee1 significantly enhanced both MK-1775- and panobinostat-induced cell death. Our results demonstrate that panobinostat synergizes with MK-1775 in AML cells, at least in part through downregulation of CHK1 and/or Wee1, providing compelling evidence for the clinical development of the combination treatment in AML.

Functional integration of acute myeloid leukemia into the vascular niche.

Vascular endothelial cells are a critical component of the hematopoietic microenvironment that regulates blood cell production. Recent studies suggest the existence of functional cross-talk between hematologic malignancies and vascular endothelium. Here we show that human acute myeloid leukemia (AML) localizes to the vasculature in both patients and in a xenograft model. A significant number of vascular tissue-associated AML cells (V-AML) integrate into vasculature in vivo and can fuse with endothelial cells. V-AML cells acquire several endothelial cell-like characteristics, including the upregulation of CD105, a receptor associated with activated endothelium. Remarkably, endothelial-integrated V-AML shows an almost fourfold reduction in proliferative activity compared with non-vascular-associated AML. Primary AML cells can be induced to downregulate the expression of their hematopoietic markers in vitro and differentiate into phenotypically and functionally defined endothelial-like cells. After transplantation, these leukemia-derived endothelial cells are capable of giving rise to AML. These novel functional interactions between AML cells and normal endothelium along with the reversible endothelial cell potential of AML suggest that vascular endothelium may serve as a previously unrecognized reservoir for AML.

Hematopoietic stem and progenitor cell migration after hypofractionated radiation therapy in a murine model.

PURPOSE: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. METHODS AND MATERIALS: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. RESULTS: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133(+) HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b(+) counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. CONCLUSIONS: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors.

Leukemia Mediated Endothelial Cell Activation Modulates Leukemia Cell Susceptibility to Chemotherapy through a Positive Feedback Loop Mechanism.

In acute myeloid leukemia (AML), the chances of achieving disease-free survival are low. Studies have demonstrated a supportive role of endothelial cells (ECs) in normal hematopoiesis. Here we show that similar intercellular relationships exist in leukemia. We demonstrate that leukemia cells themselves initiate these interactions by directly modulating the behavior of resting ECs through the induction of EC activation. In this inflammatory state, activated ECs induce the adhesion of a sub-set of leukemia cells through the cell adhesion molecule E-selectin. These adherent leukemia cells are sequestered in a quiescent state and are unaffected by chemotherapy. The ability of adherent cells to later detach and again become proliferative following exposure to chemotherapy suggests a role of this process in relapse. Interestingly, differing leukemia subtypes modulate this process to varying degrees, which may explain the varied response of AML patients to chemotherapy and relapse rates. Finally, because leukemia cells themselves induce EC activation, we postulate a positive-feedback loop in leukemia that exists to support the growth and relapse of the disease. Together, the data defines a new mechanism describing how ECs and leukemia cells interact during leukemogenesis, which could be used to develop novel treatments for those with AML.

Acute myeloid leukemia targeting by myxoma virus in vivo depends on cell binding but not permissiveness to infection in vitro.

Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.

Leukemia regression by vascular disruption and antiangiogenic therapy.

Acute myelogenous leukemias (AMLs) and endothelial cells depend on each other for survival and proliferation. Monotherapy antivascular strategies such as targeting vascular endothelial growth factor (VEGF) has limited efficacy in treating AML. Thus, in search of a multitarget antivascular treatment strategy for AML, we tested a novel vascular disrupting agent, OXi4503, alone and in combination with the anti-VEGF antibody, bevacizumab. Using xenotransplant animal models, OXi4503 treatment of human AML chloromas led to vascular disruption in leukemia cores that displayed increased leukemia cell apoptosis. However, viable rims of leukemia cells remained and were richly vascular with increased VEGF-A expression. To target this peripheral reactive angiogenesis, bevacizumab was combined with OXi4503 and abrogated viable vascular rims, thereby leading to enhanced leukemia regression. In a systemic model of primary human AML, OXi4503 regressed leukemia engraftment alone and in combination with bevacizumab. Differences in blood vessel density alone could not account for the observed regression, suggesting that OXi4503 also exhibited direct cytotoxic effects on leukemia cells. In vitro analyses confirmed this targeted effect, which was mediated by the production of reactive oxygen species and resulted in apoptosis. Together, these data show that OXi4503 alone is capable of regressing AML by a multitargeted mechanism and that the addition of bevacizumab mitigates reactive angiogenesis.

Oncolytic viral purging of leukemic hematopoietic stem and progenitor cells with Myxoma virus.

High-dose chemotherapy and radiation followed by autologous blood and marrow transplantation (ABMT) has been used for the treatment of certain cancers that are refractory to standard therapeutic regimes. However, a major challenge with ABMT for patients with hematologic malignancies is disease relapse, mainly due to either contamination with cancerous hematopoietic stem and progenitor cells (HSPCs) within the autograft or the persistence of residual therapy-resistant disease niches within the patient. Oncolytic viruses represent a promising therapeutic approach to prevent cancer relapse by eliminating tumor-initiating cells that contaminate the autograft. Here we summarize an ex vivo "purging" strategy with oncolytic Myxoma virus (MYXV) to remove cancer-initiating cells from patient autografts prior to transplantation. MYXV, a novel oncolytic poxvirus with potent anti-cancer properties in a variety of in vivo tumor models, can specifically eliminate cancerous stem and progenitor cells from samples obtained from acute myelogenous leukemia (AML) patients, while sparing normal CD34+ hematopoietic stem and progenitor cells capable of rescuing hematopoiesis following high dose conditioning. We propose that a broader subset of patients with intractable hematologic malignancies who have failed standard therapy could become eligible for ABMT when the treatment schema is coupled with ex vivo oncolytic therapy.

The AC133+CD38-, but not the rhodamine-low, phenotype tracks LTC-IC and SRC function in human cord blood ex vivo expansion cultures.

Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture, better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs), we demonstrated that the rhodamine-low phenotype was lost, whereas AC133 expression was retained throughout culture. Furthermore, the AC133(+)CD38(-) subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number, and limiting dilution analysis in NOD/SCID mice, showed a 43-fold expansion of the AC133(+)CD38(-) subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation, respectively. Thus, AC133(+)CD38(-) is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures.

Bone marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger.

Adult bone marrow (BM) contributes to neovascularization in some but not all settings, and reasons for these discordant results have remained unexplored. We conducted novel comparative studies in which multiple neovascularization models were established in single mice to reduce variations in experimental methodology. In different combinations, BM contribution was detected in ischemic retinas and, to a lesser extent, Lewis lung carcinoma cells, whereas B16 melanomas showed little to no BM contribution. Using this spectrum of BM contribution, we demonstrate the necessity for site-specific expression of stromal-derived factor-1alpha (SDF-1alpha) and its mobilizing effects on BM. Blocking SDF-1alpha activity with neutralizing antibodies abrogated BM-derived neovascularization in lung cancer and retinopathy. Furthermore, secondary transplantation of single hematopoietic stem cells (HSCs) showed that HSCs are a long-term source of neovasculogenesis and that CD133(+)CXCR4(+) myeloid progenitor cells directly participate in new blood vessel formation in response to SDF-1alpha. The varied BM contribution seen in different model systems is suggestive of redundant mechanisms governing postnatal neovasculogenesis and provides an explanation for contradictory results observed in the field.

Cell-cell interaction networks regulate blood stem and progenitor cell fate.

Communication networks between cells and tissues are necessary for homeostasis in multicellular organisms. Intercellular (between cell) communication networks are particularly relevant in stem cell biology, as stem cell fate decisions (self-renewal, proliferation, lineage specification) are tightly regulated based on physiological demand. We have developed a novel mathematical model of blood stem cell development incorporating cell-level kinetic parameters as functions of secreted molecule-mediated intercellular networks. By relation to quantitative cellular assays, our model is capable of predictively simulating many disparate features of both normal and malignant hematopoiesis, relating internal parameters and microenvironmental variables to measurable cell fate outcomes. Through integrated in silico and experimental analyses, we show that blood stem and progenitor cell fate is regulated by cell-cell feedback, and can be controlled non-cell autonomously by dynamically perturbing intercellular signalling. We extend this concept by demonstrating that variability in the secretion rates of the intercellular regulators is sufficient to explain heterogeneity in culture outputs, and that loss of responsiveness to cell-cell feedback signalling is both necessary and sufficient to induce leukemic transformation in silico.

Marrow cell therapies for cardiovascular diseases.

The nascent field of regenerative medicine has taken root in cardiovascular disease. Preclinical data demonstrating hemangioblast potential of marrow cells and cardioprotective effects of growth factors served as the basis for numerous early phase clinical trials. With the first wave of safety and efficacy trials complete, much is still unknown regarding optimal cell dose and type, timing of injection, route of administration, mechanisms of action, and achievable response measures. The next generation of studies will aim to answers these questions and make way for cellular therapies that result in effective cardiac repair.

Clinically relevant expansion of hematopoietic stem cells with conserved function in a single-use, closed-system bioprocess.

The clinical potential of umbilical cord blood-derived stem and progenitor cells has been demonstrated in various animal and human transplantation studies. However, the need for increased numbers of appropriate umbilical cord blood-derived cells continues to limit the development and success of these therapies. Ex vivo expansion has been widely studied as a method to overcome this limitation. We describe the use of a clinically relevant single-use, closed-system bioprocess capable of generating greater numbers of hematopoietic stem and progenitor cells that maintain in vivo and in vitro developmental potential. In addition to expanded numbers of CD34+ cells, CD34(+)CD38(-) cells, colony-forming cells, and long-term culture-initiating cells, the bioprocess generated > or =3.3-fold more long-term nonobese diabetic/severe combined immunodeficient repopulating cells (quantitatively determined using limiting dilution analysis) than present at input. Interestingly, these cells were also capable of multilineage engraftment and were shown to maintain their engraftment potency on a per long-term nonobese diabetic/severe combined immunodeficient repopulating cell basis compared with input noncultured cells. The developmental capacity of bioprocess-generated cells was further demonstrated by their ability to repopulate secondary nonobese diabetic/severe combined immunodeficient recipients. In vitro lineage analysis confirmed that bioprocess-generated cells could differentiate into myeloid and natural killer, B, and T cell lymphoid lineages. This in-depth analysis describes a bioprocess that generates human hematopoietic stem and progenitor cells with conserved hematopoietic activity, establishes analysis criteria for in vitro hematopoietic stem cell expansion studies, and serves as a foundation to test the therapeutic utility of cultured hematopoietic stem cells in large animals and humans.

Dynamic changes in cellular and microenvironmental composition can be controlled to elicit in vitro human hematopoietic stem cell expansion.

OBJECTIVE: The absence of effective strategies for the ex vivo expansion of human hematopoietic stem cells (HSCs) limits the development of many cell-based therapies. Prior attempts to stimulate HSC expansion have focused on media supplementation using cytokines and growth factors. In these cultures, cellular and microenvironmental compositions change with time. In this study, the impact of controlling these dynamic changes on HSC output is determined. MATERIALS AND METHODS: Cord blood-derived lin(-) cells were cultured for 8 days in serum-free medium supplemented with stem cell factor, Flt3 ligand, and thrombopoietin. Functional, phenotypic, and molecular (gene and protein) analyses were used to characterize dynamic changes in cellular and microenvironmental composition. The effects of these changes and the mechanism behind their effects on HSC expansion were assessed using a selection/media exchange-based global culture manipulation (GCM) technique. RESULTS: We show that the direct secretion of negative regulators by culture-generated lin(+) cells, and the indirect stimulation of cells to secrete negative regulators by culture-conditioned media, limits in vitro HSC generation. The GCM strategy was able to abrogate these effects to produce elevated numbers of LTC-ICs (14.6-fold relative to input), migrating rapid NOD/SCID repopulating cells (12.1-fold), and long-term NOD/SCID repopulating cells (5.2-fold). CONCLUSIONS: Cellular and microenvironmental changes that occur during all in vitro HSC cultures can significantly affect HSC output through the direct or indirect secretion of negative regulators. This study provides insight into the mechanisms regulating HSC fate in vitro and describes a novel methodology to regulate overall in vitro microenvironmental dynamics to enable the generation of clinically relevant numbers of HSCs.