Expansion Of Myeloid Cells Research Articles

In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development

Cytokines are essential regulators of hematopoiesis, acting in an instructive or permissive way. Fms-like tyrosine kinase 3 ligand (FLT3L) is an important cytokine for the development of several hematopoietic populations. Its receptor (FLT3) is expressed on both myeloid and lymphoid progenitors and deletion of either the receptor or its ligand leads to defective developmental potential of hematopoietic progenitors. In vivo administration of FLT3L promotes expansion of progenitors with combined myeloid and lymphoid potential. To investigate further the role of this cytokine in hematopoietic development, we generated transgenic mice expressing high levels of human FLT3L. These transgenic mice displayed a dramatic expansion of dendritic and myeloid cells, leading to splenomegaly and blood leukocytosis. Bone marrow myeloid and lymphoid progenitors were significantly increased in numbers but retained their developmental potential. Furthermore, the transgenic mice developed anemia together with a reduction in platelet numbers. FLT3L was shown to rapidly reduce the earliest erythroid progenitors when injected into wild-type mice, indicating a direct negative role of the cytokine on erythropoiesis. We conclude that FLT3L acts on multipotent progenitors in an instructive way, inducing their development into myeloid/lymphoid lineages while suppressing their megakaryocyte/erythrocyte potential.

C/EBPα: critical at the origin of leukemic transformation

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by clonal expansion of myeloid progenitor cells. A major mechanistic theme in AML biology is the extensive collaboration among fusion oncoproteins, transcription factors, and chromatin regulators to initiate and sustain a transformed cellular state. A new study in this issue describes how the C/EBPα transcription factor is crucial for the initiation of AML induced by MLL fusion oncoproteins, but is entirely dispensable for the maintenance of established disease. These observations provide a unique glimpse into the pioneer round of regulatory events that are critical at the origin of AML formation. Furthermore, this study implies the existence of oncogene-induced positive feedback loops capable of bypassing the continuous need for certain regulators to propagate disease.

Myeloid-derived suppressor cells in the development of lung cancer.

Myeloid-derived suppressor cells (MDSC) are widely implicated in immune suppression associated with tumor progression and chronic inflammation. However, very little is known about their possible role in tumor development. Here, we evaluated the role of MDSC in two experimental models of lung cancer: inflammation-associated lung cancer caused by chemical carcinogen urethane in combination with exposure to cigarette smoke; and a transgenic CC10Tg model not associated with inflammation. Exposure of mice to cigarette smoke alone resulted in significant accumulation in various organs of cells with typical MDSC phenotype (Gr-1(+)CD11b(+)). However, these cells lacked immunosuppressive activity and could not be defined as MDSC. When cigarette smoke was combined with a single dose of urethane, it led to the development of tumor lesions in lungs within 4 months. By that time, Gr-1(+)CD11b(+) cells accumulated in the spleen and lung and had potent immunosuppressive activity, and thus could be defined as MDSC. In the CC10Tg model, accumulation of immunosuppressive MDSC was observed only at 4 months of age, after the appearance of tumor lesions in the lungs. Accumulation of MDSC in both models was abrogated in S100A9 knockout mice. This resulted in a dramatic improvement in survival of mice in both models. Thus, cigarette smoke results in the expansion of immature myeloid cells lacking suppressive activity. Accumulation of bona fide MDSC in both models was observed only after the development of tumor lesions. However, MDSC played a major role in tumor progression and survival, which suggests that their targeting may provide clinical benefits in lung cancer.

PU.1 promotes cell cycle exit in the murine myeloid lineage associated with downregulation of E2F1

Acute myeloid leukemia (AML) is characterized by increased proliferation and reduced differentiation of myeloid lineage cells. AML is frequently associated with mutations or chromosomal rearrangements involving transcription factors. PU.1 (encoded by Sfpi1) is an E26 transformation-specific family transcription factor that is required for myeloid differentiation. Reduced PU.1 levels, caused by either mutation or repression, are associated with human AML and are sufficient to cause AML in mice. The objective of this study was to determine whether reduced PU.1 expression induces deregulation of the cell cycle in the myeloid lineage. Our results showed that immature myeloid cells expressing reduced PU.1 levels (Sfpi1(BN/BN) myeloid cells) proliferated indefinitely in cell culture and expanded invivo. Transplantation of Sfpi1(BN/BN) cells induced AML in recipient mice. Cultured Sfpi1(BN/BN) cells expressed elevated messenger RNA transcript and protein levels of E2F1, an important regulator of cell cycle entry. Restoration of PU.1 expression in Sfpi1(BN/BN) myeloid cells blocked proliferation, induced differentiation, and reduced E2F1 expression. Taken together, these data show that PU.1 controls cell cycle exit in the myeloid lineage associated with downregulation of E2F1 expression.

Anti-Obese Hormone Adiponectin Regulates Emergency Hematopoiesis and Antibacterial Response Through Downregulation Of Socs3 In Hematopoietic Progenitor Cells

Anti-obesity countermeasures attract rising attention because obesity induces multiple health complications such as metabolic syndrome, predisposition to cancer, heart diseases, stroke, infections, etc. One of the key features of obesity is decreased adiponectin, a critical anti-diabetic hormone secreted by adipocytes to modulate a number of metabolic processes including glucose regulation and fatty acid oxidation. Hematopoietic stem and progenitor cells (HSPCs) expand in size against infections, especially bacterial infections through immune response. In spite of the emerging clinical and basic reports of adiponectin, precise mechanisms of adiponectin for infectious control, especially for hematopoietic response, are largely unknown.As adipocytes are one of the major cellular components of bone marrow microenvironment which is critical for the proliferation and maintenance of hematopoietic cells, we hypothesized that obesity-related dysfunction of adipocytes in bone marrow might cause compromised hematopoietic response leading to increased susceptibility to infection. With the use of high-fat-fed mice, a well-known obesity model, we revealed that obesity had no apparent impact on steady-state hematopoiesis except for a slightly hypercellular bone marrow. Upon G-CSF administration to mimic harmful infection in vivo, high-fat-fed mice showed impaired expansion of HSPCs and myeloid cells in the bone marrow, along with the reduction of myeloid cells in peripheral circulation, i.e. diminished “emergency hematopoiesis”. In a Listeria monocytogenes peritonitis model, high-fat-fed mice also confirmed reduced expansion of HSPCs and myeloid cells in the bone marrow, attenuated leukocytosis and infiltration of leucocytes into the peritoneal cavity, resulting in a delayed bacterial clearance in vivo. Interestingly, the concentration of adiponectin was significantly reduced in the bone marrow of high-fat-fed mice compared to control mice, possibly reflecting a high relevance of adiponectin to hematopoietic response. To assess the exact role of adiponectin in emergency hematopoiesis, we utilized adiponectin-deficient (adipo-/-) mice. Genetic ablation of adiponectin caused no change in steady-state hematopoiesis, whereas G-CSF administration in adipo-/- mice showed attenuated HSPC expansion in the bone marrow, consistent with a series of high-fat-fed mice. Reciprocal transplants with adipo+/+ and adipo -/- mice demonstrated that adiponectin from bone marrow environment of the recipient, not from the donor hematopoietic cells, is essential for the efficient expansion of HSPCs in response to G-CSF. Furthermore, adipo-/- mice showed reduced hematopoietic response against Listeria monocytogenes infection, similar to the phenotype of high-fat-fed mice. Importantly, we confirmed that intravenous administration of recombinant adiponectin restored the responsiveness to G-CSF and the bacterial clearance on Listeria infection, both in adipo-/- and high-fat-fed mice, which affirm the importance of adiponectin treatment against infection. In HSPCs and myeloid cells from adipo-/- mice or high-fat-fed mice, phosphorylation of Stat3, a key component for emergency hematopoiesis, was suppressed, underscoring a role of adiponectin in emergency hematopoiesis at the molecular level. We revealed that those cells aberrantly expressed Socs3, a major inhibitory factor of Stat3, and that was reverted by intravenous administration of adiponectin, indicating that adiponectin potentiated G-CSF-dependent Stat3 phosphorylation in vitro and in vivo. These data revealed that adiponectin regulates hematopoietic response against infections, highlighting adiponectin as a legitimate target against infectious diseases in obese patients. From our findings breakthrough drugs to increase adiponectin are highly warranted. Disclosures:No relevant conflicts of interest to declare.

Recovery Of Myeloid Derived Suppressor Cell Subsets Following Allogeneic Hematopoietic Stem/Progenitor Cell Transplantation

Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that expand during many inflammatory conditions and malignancies. MDSCs may play an important role following allogeneic hematopoietic stem/progenitor cell transplant (HSCT). MDSCs suppress T-cell, B-cell and dendritic cell responses by a number of mechanisms, including promoting regulatory T cell expansion and producing soluble mediators such as Arginase 1 (Arg-1) and iNOS. MDSCs are divided into two subsets: monocytic (M-MDSCs) and granulocytic (G-MDSCs). MDSC morphology and function differ in various tissues under different inflammatory conditions. In a murine asthma model, M-MDSCs inhibit airway inflammation, but the other subset of MDSCs exacerbated airway inflammation. In a sepsis model, MDSCs exaggerated inflammation in the early stage, but suppressed inflammation in the later stage of sepsis. As the early post-transplant period is characterized by the rapid expansion of immature myeloid cells, we postulated this time period may also be a time when MDSCs might play a major role in modulating immune recovery post-transplant, and aid in the development of immune regulatory networks potentially important in the pathophysiology of graft-versus-host disease (GVHD). In nine patients undergoing allogeneic HSCT, peripheral blood was drawn on the day prior to the start of conditioning, days +4-5, +7-9, +14-16, +21-23, +27-29 and +80-100 post HSCT. White blood cells were quantified, red cell depleted using HetaSep (Stem Cell Technologies), then stained with fluorescence-labelled antibodies against CD45, CD15, CD14, HLA-DR, CD33 and CD66b and analyzed by flow cytometry for MDSC subsets. The soluble mediators iNOS and Arg-1were evaluated by intracellular staining for iNOS and Arg-1 and analyzed by flow cytometry. Four of the nine patients developed acute GVHD (II-IV) and/or extensive chronic GVHD. Early recovery of CD33+CD14+HLA-DR-/low M-MDSCs and CD33+CD15+CD66b+ G-MDSCs was seen post-transplant. Compared to healthy donors, the percentage of M- and G-MDSCs was increased by 3 weeks post-transplant. Interestingly, the patients who went on to develop GVHD had lower percentage and number of M-MDSCs, but inversely had higher numbers of G-MDSCs by day +27-29 and day +80-100 post-transplant (Fig. 1). When compared with healthy donors, the expression of Arg-1 in G-MDSCs, a measure of activation of MDSCs, was increased in patients pre- and post-HSCT, especially at day +80-100; while there was no difference seen iNOS expression in G-MDSCs (Fig 2). The expression of Arg-1 and iNOS in M-MDSCs was increased pre-transplant but fell by day +80-100 post HSCT (Fig 2). Taken together, our pilot data indicates that both M- and G- MDSCs recover early post HSCT and may contribute to the pathophysiology of GVHD. Patients with lower numbers of M-MDSCs and higher numbers of G-MDSCs at earlier time points post-transplant might be at greater risk for developing GVHD. Disclosures: No relevant conflicts of interest to declare.

IL3-Receptor Signaling Is Dispensable For The Generation and Maintenance Of Jak2V617F-Induced Myeloproliferative Neoplasm (MPN

Myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and primary myelofibrosis are clonal diseases driven by acquired somatic mutations in hematopoietic stem cells (HSCs), the most common of which is JAK2V617F. JAK2V617F leads to cytokine hypersensitivity and activation of JAK-STAT signaling in the presence of an erythropoietin (EPO), thrombopoietin (TPO) or interleukin-3 (IL3) cytokine receptor scaffold (Nature 2005; 434:1144-8, Leukemia 2008; 22:1828-40). Physiological Jak2V617F expression in long-term hematopoietic stem cells (LT-HSCs; lineagelowcKit+Sca1+CD150+CD48-) is necessary and sufficient to generate MPN, and these LT-HSCs contain the sole reservoir of Jak2V617F MPN-initiating stem cells (Cancer Cell 2010; 17:584-96; Blood 2012; 120:166-72). Although Jak2V617F-mediated EPO hypersensitivity drives erythroid expansion and in vitro EPO-independent colony formation, the EPO receptor is not expressed on Jak2V617F HSC populations (Blood 2012; 120:166-72). This suggests that hypersensitivity to IL3 and/or TPO signaling is responsible for driving LT-HSC proliferation and survival in vivo.To determine the respective contributions of IL3 and TPO signaling to Jak2V617F-induced MPN, pStat5 was measured in HSC and progenitor populations from E2ACre+Jak2V617F+/- knockin mice (hereafter Jak2VF) after stimulation with rmIL3 or rmTPO. Committed myeloid progenitors showed robust pStat5 stimulation with rmIL3, but only low level stimulation with rmTPO (TPO 460.25 ± 25.02 MFI vs. IL3 598.25 ± 69.05 MFI, p<0.05, Vehicle 362.25 ± 76.66 MFI). In contrast, LT-HSCs showed strong induction of pStat5 signaling with rmTPO stimulation but less so with rmIL3 stimulation (TPO 571 ± 47.36 MFI vs. IL3 487.5 ± 53.69 MFI, p=0.05, Vehicle 249.5 ± 47.63 MFI). Concordant with these results, TPO signaling appears essential for the generation of Jak2V617F-induced MPN (ASH 2012 Abstract 427). To determine the contribution of IL3 receptor signaling in Jak2V617F-induced MPN, we crossed Jak2VF knockin mice with mice lacking the common beta subunit of the IL3 receptor that is responsible for signal transduction of IL3, IL5 and GM-CSF (Jak2VFIL3Rb-/-). Jak2VFIL3Rb-/- mice developed MPN with similar latency and mortality to Jak2VF controls. There were no differences in peripheral blood white cell count (16.51 ± 1.5×109/L vs. 14.89 ± 2.4×109/L, p=0.39, n=3), haematocrit (67.97 ± 7.85×109/L vs. 61.97 ± 5.73×109/L, p=0.35, n=3) or extramedullary erythropoiesis (spleen weight, 665 ± 107mg vs. 541 ± 106mg, p=0.22, n=3) between Jak2VFIL3Rb-/- and Jak2VF mice respectively. In competitive bone marrow transplantation assays, all recipients of Jak2VFIL3Rb-/- or Jak2VF bone marrow developed MPN with similar diagnostic parameters such as elevated white cell count (14.29 ± 3.41×109/L vs. 18.38 ± 5.78×109/L, p=0.11, n=8), hematocrit (63.4 ± 16.5% vs. 50.5 ± 13%, p=0.16, n=8) and splenomegaly (529 ± 86mg vs. 465 ± 119mg, p=0.23, n=8) in Jak2VFIL3Rb-/- and Jak2VF mice respectively. Jak2VFIL3Rb-/- bone marrow cells initially showed reduced short-term (4 weeks) engraftment and white cell count in recipients compared to the Jak2V617F group (p<0.0005). However after 16 weeks post-transplant there was no difference in chimerism between recipients of Jak2VFIL3Rb-/- or JakVF cells. IL3 was unable to stimulate pStat5 in Jak2VFIL3Rb-/- LT-HSCs or progenitors, but was preserved in Jak2VF LT-HSCs and progenitors.These data show that IL3Rb signaling is dispensable for Jak2V617F-induced MPN and LT-HSC function, however may regulate short-term myeloid progenitor cell expansion. TPO signaling appears preferentially important for Jak2VF LT-HSC pStat5 induction, whereas IL3 is more important for pStat5 induction in progenitor cells. These findings will help to inform strategies aimed at targeting long term Jak2V617F-initiating HSC populations. Disclosures:No relevant conflicts of interest to declare.

Upregulation Of IL1RAP On Human Progenitor/Stem Cells Induces Features Of a Myeloproliferative Disorder In Mice

Myeloid malignancies, including chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML), display upregulation of IL1RAP at the cell surface of the primitive (CD34+CD38-) leukemic cells (Järås et al. PNAS, 2010; Barreyro et al. Blood, 2012; Askmyr et al. Blood 2013). IL1RAP is an essential co-receptor for the interleukin-1 (IL1R1) and the interleukin-33-receptors (IL33R, ST2) to convey signals upon binding by their ligands IL1 and IL33, respectively. IL1RAP has previously mainly been studied in the context of inflammation and little is known about its role in normal hematopoiesis and if it's upregulation in malignant hematopoiesis is of pathogenetic importance.In this study, we first characterized normal hematopoiesis in Il1rap(-/- ) mice, and discovered that these mice displayed a reduction in myeloid cells in the peripheral blood (9.8x10^5 vs. 1.68x10^6 myeloid cells per mL, p=0.0076), suggesting that Il1rap is involved myelopoiesis. To test whether Il1rap regulates normal hematopoietic stem cells (HSC), we performed competitive stem cell transplantations, and demonstrated that Il1rap-/- HSC gave rise to equal donor contribution as Il1rap+/+ HSC, even in secondary transplantations, suggesting that Il1rap is dispensable for normal HSC function.As IL1RAP is upregulated in myeloid neoplasms but it has been unclear whether this upregulation is functionally involved in disease biology, we next explored if IL1RAP upregulation alone is sufficient to cause features of a myeloid neoplasm. To this end, we retrovirally expressed IL1RAP along with GFP in cord blood (CB) CD34+ cells and transplanted the cells to NSG mice. At 200 days post transplantation, a striking myeloid lineage skewing (67% N=6, vs. 34% N=3, CD33+ among GFP+ cells in bone marrow, p=0.031), accompanied by a reduction of CD19+ cells (21% vs. 53%, p=0.041), was observed in mice that had received IL1RAP overexpressing (IL1RAP+) cells. In addition, mice that had received IL1RAP+ cells displayed enlarged spleens (102 mg vs. 71mg, p=0.034) and showed a myeloid cell expansion when compared to MIG control mice (10% vs. 3.7% CD33+ among GFP+ cells, p=0.035).Having demonstrated that IL1RAP is involved in steady-state myelopoiesis and that IL1RAP upregulation leads to myeloid lineage skewing, we next investigated if primary CML primitive cells display a different sensitivity for cytokines (IL1B and IL33) that signal through IL1RAP-associated receptors. CB and CML cells were stimulated with either single cytokines or cytokines in combination with SCF, in serum free liquid cultures. Whereas IL1B did not affect the in vitro proliferation of normal CB CD34+ cells, primary CML CD34+ cells, and particularly CML CD34+CD38- cells, showed a strong response to IL1B stimulation with more than 10-fold higher cell counts compared to unstimulated CML CD34+CD38- cells, following 7 days of culture (5.6x103 vs. 7.5x104 cells p= 0.006), while IL33 had minor effects (5.6x103 vs. 8.5x103 p=0.040), suggesting that IL1RAP upregulation may render primitive malignant myeloid cells hypersensitive to IL1B stimulation.In summary, these findings demonstrate that IL1RAP is involved in steady state myelopoiesis and that enforced IL1RAP expression, in cord blood CD34+ cells, alone is sufficient to induce features of a myeloproliferative disorder in mice. Primitive CML cells with upregulation of IL1RAP at the cell surface were more sensitive to IL1B compared to corresponding normal cells, which were unaffected. Collectively, these observations suggest that IL1RAP upregulation may contribute to the pathogenesis of myeloid neoplasms. Disclosures:Richter:Cantargia: Consultancy, Equity Ownership. Järås:Cantargia: Equity Ownership. Fioretos:Cantargia AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b+Gr-1+ myeloid cells without compromising CD8+ T cell immune responses.

BackgroundAdministration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo.MethodsWe evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.ResultsWe find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.ConclusionsOur findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.

The Peripheral Myeloid Expansion Driven by Murine Cancer Progression Is Reversed by Radiation Therapy of the Tumor

Expansion of myeloid-lineage leukocytes in tumor-bearing mice has been proposed as a cause of systemic immunosuppression. We demonstrate that radiation therapy of tumors leads to a decline in myeloid cell numbers in the blood and a decrease in spleen size. The frequency of myeloid cells does not decline to the level seen in tumor-free mice: we demonstrate that metastatic disease can prevent myeloid cell numbers from returning to baseline, and that tumor recurrence from residual disease correlates with re-expansion of myeloid lineage cells. Radiation therapy results in increased proliferation of T cells in the spleen and while T cell responses to foreign antigens are not altered by tumor burden or myeloid cell expansion, responses to tumor-associated antigens are increased after radiation therapy. These data demonstrate that myeloid cell numbers are directly linked to primary tumor burden, that this population contracts following radiation therapy, and that radiation therapy may open a therapeutic window for immunotherapy of residual disease.

STAT3 mutations identified in human hematologic neoplasms induce myeloid malignancies in a mouse bone marrow transplantation model

STAT3 protein phosphorylation is a frequent event in various hematologic malignancies and solid tumors. Acquired STAT3 mutations have been recently identified in 40% of patients with T-cell large granular lymphocytic leukemia, a rare T-cell disorder. In this study, we investigated the mutational status of STAT3 in a large series of patients with lymphoid and myeloid diseases. STAT3 mutations were identified in 1.6% (4 of 258) of patients with T-cell neoplasms, in 2.5% (2 of 79) of patients with diffuse large B-cell lymphoma but in no other B-cell lymphoma patients (0 of 104) or patients with myeloid malignancies (0 of 96). Functional in vitro assays indicated that the STAT3Y640F mutation leads to a constitutive phosphorylation of the protein. STA21, a STAT3 small molecule inhibitor, inhibited the proliferation of two distinct STAT3 mutated cell lines. Using a mouse bone marrow transplantation assay, we observed that STAT3Y640F expression leads to the development of myeloproliferative neoplasms with expansion of either myeloid cells or megakaryocytes. Together, these data indicate that the STAT3Y640F mutation leads to constitutive activation of STAT3, induces malignant hematopoiesis in vivo, and may represent a novel therapeutic target in some lymphoid malignancies.

Temozolomide-Mediated DNA Methylation in Human Myeloid Precursor Cells: Differential Involvement of Intrinsic and Extrinsic Apoptotic Pathways

An understanding of how hematopoietic cells respond to therapy that causes myelosuppression will help develop approaches to prevent this potentially life-threatening toxicity. The goal of this study was to determine how human myeloid precursor cells respond to temozolomide (TMZ)-induced DNA damage. We developed an ex vivo primary human myeloid precursor cells model system to investigate the involvement of cell-death pathways using a known myelosuppressive regimen of O(6)-benzylguanine (6BG) and TMZ. Exposure to 6BG/TMZ led to increases in p53, p21, γ-H2AX, and mitochondrial DNA damage. Increases in mitochondrial membrane depolarization correlated with increased caspase-9 and -3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), and increased cell death. During myeloid precursor cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on approximately 100% of the cells following exposure to 6BG/TMZ. Although c-flipshort, an endogenous inhibitor of FAS-mediated signaling, was decreased in 6BG/TMZ-treated versus control, 6BG-, or TMZ alone-treated cells, there were no changes in caspase-8 activity. In addition, there were no changes in the extent of cell death in myeloid precursor cells exposed to 6BG/TMZ in the presence of neutralizing or agonistic anti-FAS antibodies, indicating that FAS-mediated signaling was not operative. In human myeloid precursor cells, 6BG/TMZ-initiated apoptosis occurred by intrinsic, mitochondrial-mediated and not extrinsic, FAS-mediated apoptosis. Human myeloid precursor cells represent a clinically relevant model system for gaining insight into how hematopoietic cells respond to chemotherapeutics and offer an approach for selecting effective chemotherapeutic regimens with limited hematopoietic toxicity.

Abstract 4979: Multidimensional crosstalk regulates myeloid cell hyperplasia, immune escape, and tumor progression.

Abstract Myeloid-derived suppressor cells (MDSCs) encompass a heterogeneous class of cells in different maturation states that proliferate and accumulate in tumors, secondary lymphoid organs and some epithelial organs during tumor growth. Tumor secretion of growth factors and chemokines regulate myeloid cell proliferation, apoptosis, differentiation, circulation and biodistribution both locally and systemically. There is a reciprocal interaction that occurs between tumor and myeloid cells, which can facilitate tumor growth and metastasis and myeloid cell proliferation and survival via unique and tumor specific arrays of growth factors and chemokines. The tumor microenvironment, a complex, highly dynamic environment composed of host-derived MDSCs, endothelial cells, and fibroblasts; contributes to the microenvironmental crosstalk regulating host immunity and tumor progression. However, the macroenvironment is also a critical to myeloid cell expansion, tumor invasion and immune escape and tumor progression. The primary site of MDSC expansion and immune suppression is splenic extramedullary hematopoiesis, which is regulated by growth factors secreted by the distant primary tumor cells. Similarly, regulation of tumor infiltration by myeloid cells is by tumor secretion of CCL2; however, this occurs at the macroenvironmental level as CCL2/CCR2 blockade inhibits medullary myeloid cell mobilization and recruitment from secondary lymphoid organs and not at the tumor. Further, regulatory crosstalk is bidirectional with tumor-secreted factors differentially upregulating myeloid and lymphoid cell growth factors and enzymes (G-CSF, CXCL1, CCL2, arginase) by medullary myeloid cells and splenic lymphocytes. In tumor bearing, mice the tumor-secreted growth factors support hematopoietic stem/progenitor cell proliferation, limit their apoptosis and inhibit T-cell function. Conversely, leukocyte-secreted cytokines limit immune and hematopoietic growth factor transcript levels by tumor cells (G-CSF, IL-23, CXCL1 and NOS-2) with associated effects on tumor malignancy and leukocyte proliferation and differentiation. In summary, tumors form both local and systemic, bidirectional, regulatory loop(s) between metastatic tumor cells and MDSCs that regulates their proliferation and differentiation supporting tumor metastasis and systemic and regional T-cell suppression. Citation Format: Ibrahim H. Younos, Alicia J. Dafferner, Holly Britton, James E. Talmadge. Multidimensional crosstalk regulates myeloid cell hyperplasia, immune escape, and tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2013-4979

The Soluble Interleukin-6 Receptor Is a Mediator of Hematopoietic and Skeletal Actions of Parathyroid Hormone

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6(+/+) and IL-6(-/-) mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6(-/-) compared with IL-6(+/+) bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6(-/-) earlier than IL-6(+/+) marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6(-/-) marrow. In the skeletal system, although intermittent PTH administration to IL-6(+/+) and IL-6(-/-) mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.

PhotoImmunoNanoTherapy Reveals an Anticancer Role for Sphingosine Kinase 2 and Dihydrosphingosine-1-Phosphate

Tumor-associated inflammation mediates the development of a systemic immunosuppressive milieu that is a major obstacle to effective treatment of cancer. Inflammation has been shown to promote the systemic expansion of immature myeloid cells which have been shown to exert immunosuppressive activity in laboratory models of cancer as well as cancer patients. Consequentially, significant effort is underway toward the development of therapies that decrease tumor-associated inflammation and immunosuppressive cells. The current study demonstrated that a previously described deep tissue imaging modality, which utilized indocyanine green-loaded calcium phosphosilicate nanoparticles (ICG-CPSNPs), could be utilized as an immunoregulatory agent. The theranostic application of ICG-CPSNPs as photosensitizers for photodynamic therapy was shown to block tumor growth in murine models of breast cancer, pancreatic cancer, and metastatic osteosarcoma by decreasing inflammation-expanded immature myeloid cells. Therefore, this therapeutic modality was termed PhotoImmunoNanoTherapy. As phosphorylated sphingolipid metabolites have been shown to have immunomodulatory roles, it was hypothesized that the reduction of immature myeloid cells by PhotoImmunoNanoTherapy was dependent upon bioactive sphingolipids. Mechanistically, PhotoImmunoNanoTherapy induced a sphingosine kinase 2-dependent increase in sphingosine-1-phosphate and dihydrosphingosine-1-phosphate. Furthermore, dihydrosphingosine-1-phosphate was shown to selectively abrogate myeloid lineage cells while concomitantly allowing the expansion of lymphocytes that exerted an antitumor effect. Collectively, these findings revealed that PhotoImmunoNanoTherapy, utilizing the novel nontoxic theranostic agent ICG-CPSNP, can decrease tumor-associated inflammation and immature myeloid cells in a sphingosine kinase 2-dependent manner. These findings further defined a novel myeloid regulatory role for dihydrosphingosine-1-phosphate. PhotoImmunoNanoTherapy holds the potential to be a revolutionary treatment for cancers with inflammatory and immunosuppressive phenotypes.

Pancreatic Tumors and Immature Immunosuppressive Myeloid Cells in Blood and Spleen: Role of Inhibitory Co-Stimulatory Molecules PDL1 and CTLA4. An In Vivo and In Vitro Study

BackgroundBlood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied in vivo circulating and splenic T lymphocyte and IMC subsets in patients with benign and malignant pancreatic diseases. We ascertained in vitro whether pancreatic adenocarcinoma (PDAC)-associated IMC subsets are induced by tumor-derived soluble factors and whether they are immunosuppressive focusing on the inhibitory co-stimulatory molecules PDL1 and CTLA4.Methodology and Principal Findings103 pancreatic and/or splenic surgical patients were enrolled including 52 PDAC, 10 borderline and 10 neuroendocrine tumors (NETs). Lymphocytes and IMCs were analysed by flow cytometry in blood, in spleen and in three PDAC cell conditioned (CM) or non conditioned PBMC. PDL1 and CTLA4 were studied in 30 splenic samples, in control and conditioned PBMC. IMCs were FACS sorted and co-coltured with allogenic T lymphocytes. In PDAC a reduction was found in circulating CD8+ lymphocytes (p = 0.004) and dendritic cells (p = 0.01), which were reduced in vitro by one PDAC CM (Capan1; p = 0.03). Blood myeloid derived suppressive cells (MDSCs) CD33+CD14−HLA-DR− were increased in PDAC (p = 0.022) and were induced in vitro by BxPC3 CM. Splenic dendritic cells had a higher PDL1 expression (p = 0.007), while CD33+CD14+HLA-DR− IMCs had a lower CTLA4 expression (p = 0.029) in PDAC patients. In vitro S100A8/A9 complex, one of the possible inflammatory mediators of immune suppression in PDAC, induced PDL1 (p = 0.018) and reduced CTLA4 expression (p = 0.028) among IMCs. IMCs not expressing CTLA4 were demonstrated to be immune suppressive.ConclusionIn PDAC circulating dendritic and cytotoxic T cells are reduced, while MDSCs are increased and this might favour tumoral growth and progression. The reduced CTLA4 expression found among splenic IMCs of PDAC patients was demonstrated to characterize an immune suppressive phenotype and to be consequent to the direct exposure of myeloid cells to pancreatic cancer derived products, S100A8/A9 complex in particular.

Abstract P4-04-02: Tumor-initiated peripheral myeloid cell expansion is reversed by radiation therapy

Abstract Myeloid lineage cells, particularly monocytes and macrophages, have an important role in the development and progression of cancer. Within established tumors, macrophages are critical for angiogenesis, invasion, metastasis, immunosuppression and response to therapy. In addition, both transplantable and transgenic mouse models of mammary cancer are associated with myeloid expansions detectible in the tumor, spleen and peripheral blood, and these myeloid cells are able to suppress T cell activation in vitro. Reducing the tumor burden through chemotherapy and resection has been shown to control the myeloid expansion. However, chemotherapies that reduce tumor burden also have direct effects on myeloid cells, and surgical trauma has been shown to mobilize myeloid cells. Radiation therapy is the third major option to treat cancer, but the consequence of radiation therapy to systemic myeloid populations has not been described. We applied tumor-specific radiation therapy to test the hypothesis that the frequency of myeloid lineage cells correlates with the primary tumor burden. We demonstrate in the MMTV-PyMT model of mammary cancer that myeloid expansion in the blood closely correlates with myeloid involvement in the tumor and with tumor progression. We demonstrate that radiation therapy of 4T1 mammary tumors leads to a decline in myeloid cell numbers in the blood and a decrease in spleen size. The frequency of myeloid cells does not decline to the level seen in tumor-free mice: we demonstrate that metastatic disease can prevent myeloid cell numbers from returning to baseline, and that tumor recurrence from residual disease correlates with re-expansion of myeloid lineage cells. Despite these changes in the frequency of myeloid lineage cells, T cell numbers in the blood and spleen remains constant. However, radiation therapy improves the myeloid cell:CD8+ T cell ratio and results in increased proliferation of T cells in the spleen. Finally, we demonstrate that T cell responses to foreign antigens are not altered by tumor burden or myeloid cell expansion, while T cell responses to tumor-associated antigens are increased after radiation therapy of the tumor. These data demonstrate that myeloid cell numbers are directly linked to primary tumor burden, that this population contracts following radiation therapy, and that radiation therapy may open a therapeutic window for immunotherapy of residual disease. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-02.

Loss of Krüppel-Like Factor 4 (KLF4) Impairs the Self-Renewal of Leukemic Stem Cells

Abstract 911The identification of pathways that differentially regulate the self-renewal in normal hematopoietic stem cells (HSC) and leukemic stem cells (LSC) is critical for effective treatment of relapsed leukemia without affecting normal hematopoiesis. Krüppel-like factor 4 (KLF4) is a bi-functional transcription factor that can either activate or repress transcription and thus function as an oncogene or a tumor suppressor depending on the cellular context. We have previously shown that loss of KLF4 leads to increased self-renewal of HSC in serial bone marrow transplantation. In this work, we investigated whether KLF4 also regulates self-renewal in LSCs using a mouse model of somatic deletion of KLF4 in hematopoietic cells (Klf4fl/fl Vav-iCre+) and the BCR-ABL-induced chronic myeloid leukemia (CML) model. Mice transplanted with wild type bone marrow (BM) cells transduced with retrovirus carrying BCR-ABL (p210) (n=18) showed an expansion of myeloid cells (GFP+ Gr1+) with a median survival of 19 days. In contrast, mice transplanted with KLF4-deficient BM cells transduced with BCR-ABL retrovirus (n=12) exhibited prolonged survival with a median survival of 27 days (p<0.0011). Progression of leukemia was monitored in peripheral blood, BM and the spleen by flow cytometry. Mice transplanted with KLF4-deficient cells showed expansion of myeloid leukemic cells (GFP+ Gr1+) in the first two weeks after BM transplant, which was followed by a progressive loss of myeloid cells and an expansion of B cells (GFP+ B220+). In control group, 90% of leukemic mice succumbed from CML whereas more than 50% of KLF4-deficient leukemic mice developed mixed CML and B-ALL leukemia. These results suggest that loss of KLF4 impairs the maintenance of BCR-ABL-induced CML, while allowing expansion of BCR-ABL positive B cells (GFP+ B220+). Since KLF4 deletion prevented continuous expansion of myeloid leukemic cells (GFP+ Gr1+), we postulated that KLF4 may be critical to LSC maintenance. Thus, we analyzed the frequency of LSCs (GFP+ Lin–c-Kit+ Sca-1+and GFP+ Lin– c-Kit+ Sca-1+Flt3–) in BM and the spleen. We found that LSCs were significantly reduced in recipients of BCR-ABL-transduced KLF4-deficient BM cells 18 days after transplantation (n=6, p <0.001). These studies demonstrate that KLF4 has different roles in the maintainenance of LSCs and normal HSCs and that inactivation of KLF4 provides a therapeutic strategy for eradicating LSCs without damaging normal HSC. Disclosures:No relevant conflicts of interest to declare.

Nrf2 Deficiency Leads to Reduced Hematopoietic Stem Cell Self-Renewal and Increased Sensitivity to Genotoxic Stressors Through Impaired P53 Function

Abstract 397Nrf2 is a basic region-leucine-zipper transcription factor that regulates expression of numerous genes required for oxidative stress response, drug and toxin detoxification, protein ubiquitination, and proteasomal degradation of damaged proteins. Therefore Nrf2 represents a major cellular defense mechanism against oxidative and toxic stresses. We have shown that CD34+ hematopoietic stem/progenitor cells (HSPC) drawn from peripheral blood stem cell (PBSC) autografts of lymphoma patients who develop therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) after autologous hematopoietic cell transplantation (aHCT) demonstrate significantly reduced expression of Nrf2-mediated stress response pathway genes compared to cells from patients who do not develop t-MDS/AML (Cancer Cell 20, 591–605). We have extended these findings to study the role of Nrf2 in HSPC regulation and the response to genotoxic chemotherapeutic agents by using an Nrf2 knockout (KO) mouse model. Nrf2 KO mice demonstrated impaired bone marrow (BM) long-term hematopoietic stem cell (LT-HSC) function, manifested by significantly reduced competitive engraftment of BM cells in recipient mice at 16 weeks compared to wild-type (WT) controls (42.5±2.8% vs. 57.2±3.3% peripheral blood engraftment for KO and WT donors respectively, p<0.01). An even more significant defect in engraftment of KO cells was seen after transplantation to secondary recipients (16.4±1.7% vs. 61.9±1.5% peripheral blood engraftment at 8 weeks for KO and WT donors respectively, p<0.01). Nrf2 KO mice also demonstrated increased sensitivity to ENU treatment compared to WT mice. ENU (100mg/kg) treatment resulted in severe anemia at 7 months in 100% of KO mice compared with 36% of WT mice (p<0.001). Furthermore, anemia developed at a median of 5 months in Nrf2 KO mice compared to 10 months in WT controls. Pathological examination of BM from anemic mice revealed reduced erythropoiesis with impaired erythroid maturation and expansion of myeloid cells, consistent with myeloid dysplasia. Q-RT-PCR analysis demonstrated significantly reduced expression of Nrf2 target genes, including Hmo-1, Nqo-1, Gclc and Gpx1, in BM c-kit+ HSPC from KO mice compared to WT mice (p<0.05). Interestingly, expression of several P53 target genes including Bax, Puma, Cdkn1a and Necdin were also significantly reduced in BM c-kit+ cells from Nrf2 KO compared to WT mice. We also observed reduced P53 target gene expression in Nrf2 KO c-Kit cells compared with WT cells 4 hours after treatment with ENU (p<0.05), indicating impaired P53 response in Nrf2 deficient HSPC. To examine the P53 response to DNA damaging agents in LT-HSC, we exposed Nrf2 KO and WT mice to 2Gy irradiation and selected BM LT-HSC (Lin-Sca-1+c-kit+Flt-3-CD150+CD48-) using flow cytometry 12 hours after irradiation. Q-RT-PCR analysis showed significantly reduced expression of P53 target genes Bax, Puma and Necdin (p<0.05), and a trend towards reduced expression of Cdkn1a (p=0.08), in Nrf2 KO compared to WT LT-HSC. Immunofluorescence microscopy showed significantly reduced nuclear and increased cytoplasmic localization of P53 in Nrf2 KO compared to WT LT-HSC. Western blotting analysis showed increased levels of ubquitinated proteins in BM c-kit+ HSPC from Nrf2 KO compared to WT mice, and immunoprecipitation followed by Western blotting for P53 revealed increased high molecular weight bands indicative of ubiquitinated P53 in Nrf2 KO HSPC. Nrf2 is a key regulator of protein ubiquitination and proteasomal degradation, and these results suggest that abnormal cytoplasmic accumulation of ubiquitinated P53 may contribute to altered P53 function in Nrf2 deficient HSPC. In conclusion, our results show that Nrf2 deficiency results in impaired HSC self-renewal capacity under physiological conditions, and significantly increased sensitivity to genotoxic stress, potentially explained by altered P53 protein modulation in Nrf2 deficient HSPC. These observations suggest that Nrf2 deficiency could contribute to development of t-MDS/AML following exposure to genotoxic agents, and support further evaluation of Nrf2 as a potential target of chemopreventive efforts. Disclosures:No relevant conflicts of interest to declare.

C/EBPβ Is Upregulated Through STAT5 and Accelerates Exhaustion of Leukemic Stem Cells During BCR-ABL-Mediated Myeloid Expansion

Abstract 907Enhanced proliferation and differentiation of myeloid cells are features common to emergency granulopoiesis and chronic phase of chronic myeloid leukemia (CP-CML). We have previously shown that C/EBPβ, a transcription factor regulating emergency granulopoiesis, is upregulated in hematopoietic stem/progenitors in CP-CML and that C/EBPβ promotes BCR-ABL-mediated myeloid cell expansion (ASH annual meeting abstract. 2011; 118: 3747). However, the molecular mechanisms involved in the upregulation of C/EBPβ and the effects of C/EBPβ on the CML stem cells remained to be elucidated. Here we show that STAT5 is involved in the BCR-ABL-mediated C/EBPβ upregulation and that C/EBPβ accelerates the exhaustion of CML stem cells.In order to investigate the regulation of C/EBPβ in CP-CML, BCR-ABL gene was retrovirally introduced into EML cells, a mouse hematopoietic stem cell line, to make EML-BCR-ABL. The expression of C/EBPβ was upregulated at mRNA and protein level in EML-BCR-ABL when compared to EML cells transduced with a control vector. The upregulation of C/EBPβ was significantly repressed by adding imatinib mesylate, suggesting that the downstream signaling pathway of BCR-ABL is directly involved in the process. Neither a PI3K inhibitor nor a MEK inhibitor affected the levels of C/EBPβ in EML-BCR-ABL, but a STAT5 inhibitor reversed the upregulation of C/EBPβ in EML-BCR-ABL. Retroviral transduction of dominant-negative STAT5 mutant also impaired the upregulation of C/EBPβ in EML-BCR-ABL. Transduction of constitutively-active STAT5 mutant into parental EML cells significantly upregulated C/EBPβ expression. These results suggest that BCR-ABL upregulates C/EBPβ expression at least in part through activation of STAT5.To clarify the role of C/EBPβ in the regulation of CML stem cells, BCR-ABL-transduced bone marrow (BM) cells from C/EBPβ knockout (KO) mice or wild type (WT) mice were serially transplanted into irradiated recipient mice. In the first round of transplantation, all the recipient mice developed myeloproliferative status and the mice transplanted with BCR-ABL-transduced C/EBPβ KO BM cells survived longer than the mice transplanted with BCR-ABL-transduced WT BM cells. Interestingly, the frequency of c-kit+ cells within BCR-ABL+ cells was higher in the BM of the mice transplanted with BCR-ABL-transduced C/EBPβ KO cells than in the BM transplanted with BCR-ABL-transduced WT cells (Figure). When 2 × 106 BCR-ABL+ BM cells from primary recipient mice were transplanted into sublethally irradiated secondary recipients, all the mice developed a myeloproliferative status. Transplantation of 1 × 106 BCR-ABL+ BM cells from primary recipient mice, four out of the five recipient mice that transplanted with BCR-ABL-transduced C/EBPβ KO BM cells developed a myeloproliferative status and only one out of the four recipient mice that transplanted with BCR-ABL-transduced WT BM cells developed a myeloproliferative status. Transplantation of less than 0.5 × 106 BCR-ABL+ BM cells engrafted none of the secondary recipient mice. The frequencies of leukemia-initiating cells in BM of primary recipient mice that transplanted with BCR-ABL-transduced WT cells or BCR-ABL-transduced C/EBPβ KO cells were 1 in 1,404,129 and 1 in 683,773, respectively. These findings suggest that C/EBPβ promotes exhaustion of CML stem cells.In conclusion, these results suggest that C/EBPβ is upregulated by BCR-ABL at least in part through STAT5 and that C/EBPβ has the ability to exhaust CML stem cells. We are currently investigating the molecular mechanisms which protect CML stem cells from C/EBPβ-mediated exhaustion for complete eradication of CML stem cells in the future. [Display omitted] Disclosures:No relevant conflicts of interest to declare.