Nurse-like Cells Research Articles

Extracellular vesicles from type-2 macrophages increase the survival of chronic lymphocytic leukemia cells ex vivo

The resistance of Chronic Lymphocytic Leukemia (CLL) B-cells to cell death is mainly attributed to interactions within their microenvironment, where they interact with various types of cells. Within this microenvironment, CLL-B-cells produce and bind cytokines, growth factors, and extracellular vesicles (EVs). In the present study, EVs purified from nurse-like cells and M2-polarized THP1 cell (M2-THP1) cultures were added to CLL-B-cells cultures. EVs were rapidly internalized by B-cells, leading to a decrease in apoptosis (P = 0.0162 and 0.0469, respectively) and an increased proliferation (P = 0.0335 and 0.0109). Additionally, they induced an increase in the resistance of CLL-B-cells to Ibrutinib, the Bruton kinase inhibitor in vitro (P = 0.0344). A transcriptomic analysis showed an increase in the expression of anti-apoptotic gene BCL-2 (P = 0.0286) but not MCL-1 and an increase in the expression of proliferation-inducing gene APRIL (P = 0.0286) following treatment with EVs. Meanwhile, an analysis of apoptotic protein markers revealed increased amounts of IGFBP-2 (P = 0.0338), CD40 (P = 0.0338), p53 (P = 0.0219) and BCL-2 (P = 0.0338). Finally, exploration of EVs protein content by mass spectrometry revealed they carry various proteins involved in known oncogenic pathways and the RNAseq analysis of CLL-B-cells treated or not with NLCs EVs show various differentially expressed genes.

Active Hexose-Correlated Compound Shows Direct and Indirect Effects against Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a disease characterized by the accumulation of mature CD19+CD5+CD23+ B cells in the bloodstream and in lymphoid organs. It usually affects people over 70 years of age, which limits the options for treatments. The disease is typically well-managed, but to date is still incurable. Hence, the need for novel therapeutic strategies remains. Nurse-like cells (NLCs) are major components of the microenvironment for CLL, supporting tumor cell survival, proliferation, and even drug resistance. They are of myeloid lineage, guided toward differentiating into their tumor-supportive role by the CLL cells themselves. As such, they are analogous to tumor-associated macrophages and represent a major therapeutic target. Previously, it was found that a mushroom extract, Active Hexose-Correlated Compound (AHCC), promoted the death of acute myeloid leukemia cells while preserving normal monocytes. Given these findings, it was asked whether AHCC might have a similar effect on the abnormally differentiated myeloid-lineage NLCs in CLL. CLL-patient PBMCs were treated with AHCC, and it was found that AHCC treatment showed a direct toxic effect against isolated CLL cells. In addition, it significantly reduced the number of tumor-supportive NLCs and altered their phenotype. The effects of AHCC were then tested in the Eµ-TCL1 mouse model of CLL and the MllPTD/WT Flt3ITD/WT model of AML. Results showed that AHCC not only reduced tumor load and increased survival in the CLL and AML models, but it also enhanced antitumor antibody treatment in the CLL model. These results suggest that AHCC has direct and indirect effects against CLL and that it may be of benefit when combined with existing treatments.

A CX3CR1 (fractalkine receptor) Small Molecular Antagonist (KAND567) Suppressed the Growth Promoting Effect of Monocytes on Chronic Lymphocytic Leukemia Cells

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia worldwide. Novel treatment strategies are needed to improve the prognosis. Non-malignant cells of the tumor microenvironment (TME) are of importance for the growth of tumor cells. Monocytes expressing various chemokine receptors as CX3CR1 (the fractalkine receptor (FKNR)) have been shown to promote the growth of tumor cells in vitro and in vivo. In CLL, monocyte derived nurse-like cells (NLC) support the growth of CLL cells, promoting leukemic cell proliferation and survival by secretion of chemokines/cytokines. In this study, we studied the role of autologous monocytes on the survival of CLL cells and the effects of a small molecule antagonist of CX3CR1 (KAND567/FKNRa). KAND567 is in clinical trials for diseases with an activated fractalkine system as part of the pathobiology including covid-19 infected patients as well as patients with acute myocardial infarction and ovarian carcinoma. Methods: Primary CLL cells and B cells (CD19 +) of healthy donors as well as autologous monocytes (CD14 +) from CLL patients (n=22) and healthy control donors (n=11) were cultured alone or in co-cultures (isolated B cells together with autologous monocytes) with the FKNRa (250 and 1000 nM) for up to 120 h. Apoptosis of CD19 + cells and monocytes was analysed by Annexin V/PI staining, Western blotting and the IncuCyte® Live Cell Imaging System. The NBT assay was also applied to check morphological changes of monocytes. Plasma concentration of fractalkine (CX3CL1) was determined by ELISA. Results and discussion: CLL patients had a significant (p=0.0001) increase in the plasma concentration of CX3CL1 compared to healthy controls. CLL cells as well as normal B cells did not express surface CX3CR1. However, monocytes from both CLL patients and healthy donors expressed high levels of surface CX3CR1. A significantly higher proportion of pro-inflammatory intermediate (CD14 +/CD16 +) and non-classical (CD14 -/CD16 +) monocytes (p<0.0001 and <0.01 resp.) as well as intermediate and non-classical CX3CR1 + monocytes were observed in patients with active CLL compared to healthy donors (p<0.0001 and <0.01 resp.). Moreover, CX3CR1 + intermediate and non-classical monocytes were significantly higher in CLL patients with active disease compared to those with early-stage disease (p<0.0001 and <0.001 resp.). A growth supportive effect of autologous monocytes was observed on survival of both CLL cells and healthy donor B cells in 120 h cell co-culture experiments (p<0.001 and <0.01 resp.). FKNRa reduced the number of alive CLL cells in a dose-dependent manner in co-cultures with autologous monocytes (p<0.05). No effect was seen on CD19 + B cells of healthy donors co-cultured with autologous monocytes. FKNRa did not induce apoptosis of CLL cells alone. Furthermore, FKNRa also inhibited transformation of CLL monocytes to NLC (p<0.05) which was not the case for normal monocytes. Conclusion: The CX3CR1/CX3CL1 (fractalkine receptor/ligand)) axis appears to be activated in CLLand might be of importance for the NLC-driven growth of CLL cells. This is the first report of a small molecule antagonist of CX3CR1 reducing the growth supportive effect of autologous CLL monocytes on leukemic CLL cells. Disruption of growth promotion induced by monocytes may prevent leukemic cell proliferation and survival. Inhibition of autologous monocytes in the TME might represent a new therapeutic strategy in CLL, complementary to treatment with drugs that directly target the tumor cells.

Targeting CD24 “Don't Eat Me” Signal Restores Phagocytic Activity of Nurse-like Cells from Patients with Chronic Lymphocytic Leukemia

Introduction Chronic Lymphocytic Leukemia (CLL) is an indolent B-cell lymphoproliferative disease that carries the risk of evolving into a life-threatening aggressive histology, known as Richter's syndrome. Despite the advent of new agents such as ibrutinib and venetoclax that have dramatically changed the clinical fate of CLL, young patients still experience refractory and resistant disease, with few therapeutic options available over time. Therefore, the need for new strategies is pivotal for this chronic and insidious condition. Drug resistance might be triggered by the tumor microenvironment (TME), which provides survival signals to CLL tumor cells. Their interaction is mainly established with monocyte-derived nurse-like cells (NLCs). NLCs resemble the immunosuppressive M2-like tumor-associated macrophages (TAMs) and can create a profitable connection with CLL in terms of increasing proliferation and survival (Filip, Blood Cells Mol Dis. 2013). The possibility of targeting the TME by blocking the “Don't Eat Me” signal (DEMs) CD47 has provided restoration of phagocytosis within TME and interesting clinical results in heavily pretreated cases of lymphoma (Advani, NEJM. 2019). The use of agents targeting the DEMs within TME could be a promising approach in CLL as well. CD24 is another DEMs that was first validated in a preclinical model of solid cancer (Barkal, Nature. 2019). It was subsequently found to be upregulated in Mantle-cell lymphoma (MCL) and involved in DEMs as well, since CD24 blockade provided an increase in phagocytosis of MCL cells in vitro (Aroldi, Blood. 2021). This effect was particularly evident in association with anti-CD47 monoclonal antibody (mAb) and Rituximab (Aroldi, Blood. 2022). Although MCL has different pathogenesis compared to CLL, these two entities share some features, particularly regarding the B-cell of origin and the vital interaction with non-cancerous cells within TME. In this study, we explore the possibility of targeting CD24 in human primary samples of CLL (huCLL), redirecting the activity of NLCs against CLL cells using anti-CD24 mAb alone or in combination with other agents (anti-CD47, Rituximab). Methods We analyzed a microarray dataset of patients with CLL to check for any correlation between CD24 expression and survival (GSE22762). We also checked the CD24 antigen expression of huCLL, isolated and differentiated monocytes into M2-like TAMs in vitro, as well as conducted co-culture assays using flow cytometry. These methods were previously described (Aroldi, Blood. 2021; Aroldi, Blood. 2022). Unstimulated autologous CD14+ cell population - defined as precursors of NLCs - were isolated from patients with CLL and used for co-culture analysis without any previous cytokine stimulation for differentiation. Results In a cohort of CLL patients, higher CD24 levels correlated with a lower survival rate compared to lower expression ones (Figure 1). We also checked the CD24 surface in an extended cohort of huCLL and compared it to MEC-1 and PCL-12. huCLL showed higher CD24 surface levels than MEC-1 and PCL-12 and demonstrated better phagocytosis when co-cultured with M2-like TAMs and anti-CD24 mAb (Figure 2A). We then isolated precursors of circulating NLCs from 5 huCLL patients and found higher levels of Siglec-10 (CD24 ligand) than in healthy controls. In this setting, blocking CD24 redirected the phagocytic activity of NLCs against huCLL, showing higher phagocytic rate in cases of multiple combinations (anti-CD24, anti-CD47, anti-CD20, Figure 2B). Conclusions CLL could be another blood cancer subtype sensitive to CD24/Siglec-10 DEMs blocking agents, and CD24 expression negatively impacts survival. Anti-CD24 mAb, in particular, showed the ability to redirect patient-derived NLCs' phagocytic activity to cognate huCLL cancer cells, resulting in higher clearance in cases of multiple combination. Boosting the autologous innate immune system might, therefore, provide further therapeutic options in the setting of CD24 + CLL, particularly for those patients who have failed several lines of treatment and have limited options available.

Effects of blocking CD24 and CD47 'don't eat me' signals in combination with rituximab in mantle-cell lymphoma and chronic lymphocytic leukaemia.

Mantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) with a poor prognosis, at high risk of relapse after conventional treatment. MCL-associated tumour microenvironment (TME) is characterized by M2-like tumour-associated macrophages (TAMs), able to interact with cancer cells, providing tumour survival and resistance to immuno-chemotherapy. Likewise, monocyte-derived nurse-like cells (NLCs) present M2-like profile and provide proliferation signals to chronic lymphocytic leukaemia (CLL), a B-cell malignancy sharing with MCL some biological and phenotypic features. Antibodies against TAMs targeted CD47, a 'don't eat me' signal (DEMs) able to quench phagocytosis by TAMs within TME, with clinical effectiveness when combined with Rituximab in pretreated NHL. Recently, CD24 was found as valid DEMs in solid cancer. Since CD24 is expressed during B-cell differentiation, we investigated and identified consistent CD24 in MCL, CLL and primary human samples. Phagocytosis increased when M2-like macrophages were co-cultured with cancer cells, particularly in the case of paired DEMs blockade (i.e. anti-CD24 + anti-CD47) combined with Rituximab. Similarly, unstimulated CLL patients-derived NLCs provided increased phagocytosis when DEMs blockade occurred. Since high levels of CD24 were associated with worse survival in both MCL and CLL, anti-CD24-induced phagocytosis could be considered for future clinical use, particularly in association with other agents such as Rituximab.

An agent-based model of monocyte differentiation into tumour-associated macrophages in chronic lymphocytic leukemia

Monocyte-derived macrophages help maintain tissue homeostasis and defend the organism against pathogens. In tumors, recent studies have uncovered complex macrophage populations, including tumor-associated macrophages, which support tumorigenesis through cancer hallmarks such as immunosuppression, angiogenesis, or matrix remodeling. In the case of chronic lymphocytic leukemia, these macrophages are known as nurse-like cells (NLCs) and they protect leukemic cells from spontaneous apoptosis, contributing to their chemoresistance. We propose an agent-based model of monocyte differentiation into NLCs upon contact with leukemic B cells invitro. We performed patient-specific model optimization using cultures of peripheral blood mononuclear cells from patients. Using our model, we were able to reproduce the temporal survival dynamics of cancer cells in a patient-specific manner and to identify patient groups related to distinct macrophage phenotypes. Our results show a potentially important role of phagocytosis in the polarization process of NLCs and in promoting cancer cells' enhanced survival.

CCL3 as Possible Negative Prognostic Factor in Chronic Lymphocytic Leukemia

Introduction: Both microenvironmental signals from surrounding cells and changes in the genome of leukemic cells play essential role in the development of chronic lymphocytic leukemia. Nurse-like cells (NLCs) are one of the important elements of the microenvironment of CLL cells. The key role in the interactions of leukemic cells with NLCs is played by chemokines, which may interfere with the programmed cell death process in the leukemic lymphocytes. The aim of our study was analysis of selected microenvironmental factors having a potential impact on the leukemic cells survival, as well as their association with clinical, cytogenetic, and molecular parameters. For this study, we selected three types of molecules which can modulate microenvironment: chemokines IL-8 and CCL3 (which are classically secreted to extracellular matrix), soluble forms of adhesion molecules JAG1 and CD163, and secreted form of endogenous protein BIRC5. We assessed their expression in the serum of CLL patients as well as in medium of long-term NLCs cultures. Methods: Long-term cell culture was prepared from mononuclear cells derived from the blood of 34 patients with CLL. Number of NLCs cells was evaluated, under a light inverted microscope. The concentration of IL-8, CCL3, sBIRC5, sCD163, and sJAG1 in culture medium and serum was assessed by enzyme-linked immunosorbent assays. Results: There were significant differences in the concentration of IL-8, sBIRC5, CCL3, sCD163, and sJAG1 between the patient’s blood serum and the culture medium. The concentrations of IL-8, CCL3, and JAG1 were higher in the culture medium, which confirmed the role of the microenvironment in the production of these proteins. In addition, the concentration of CCL3 chemokine in both patient’s blood serum and in the culture medium correlated with the number of NLCs and with known prognostic factors in the course of CLL, e.g., Rai stage, WBC, expression of ZAP-70, CD38, and CD5/19. Conclusion: The microenvironment of CLL cells, which includes NLCs, plays an important role in the pathogenesis of CLL. The CCL3 chemokine seems to be a good factor representing microenvironment of CLL cells. Chronic lymphocytic leukemia is a complex and very heterogeneous disease; therefore, its progress should be considered both in the context of genetic changes and the interaction with microenvironmental cells.

EMT-like Activation in CLL Provides Novel Therapeutic Target

Introduction: There is growing interest in the role of Epithelial to Mesenchymal Transition (EMT) regulators in immune cell development, but the functional role of EMT in B-cell biology and transformation remains obscured. The outcomes of the "classical" EMT activation of epithelial cells are known; committed migratory cells do not divide and become resistant to apoptotic stimuli. Importantly, EMT supports migratory cells' "stem cell-like" properties, allowing differentiation into more than one cell type and/or terminal differentiation and senescence. Chronic lymphocytic leukaemia (CLL) cells engage with antigen and microenvironment stimuli in tissue sites that lead to proliferation, followed by their egress from the tissue sites into the peripheral blood. This lifecycle implies functional changes altering CLL cell movement. Here we show EMT-like activation in CLL that correlates with disease progression and transformation. We identified a targetable candidate and show that it can be inhibited by small molecule ligands. Results: Our analysis of available expression datasets showed the expression of various EMT factors in healthy B cells. The single-cell analysis of lymph nodes confirms strong EMT-like activation in healthy B-cells (GSE159929), confirming the idea that genes involved in classical EMT would be expressed differently depending on the stage of B-cell differentiation. The following GENEVESTIGATOR analysis of B cell malignancies highlighted high EMT-like activation in CLL cases. However, differential expression (DE) analysis of peripheral blood (PB) samples from 44 CLL patients and 24 healthy B-cell donors identified six EMT factors aberrantly expressed in CLL cells compared to healthy B cells indicating significant changes in CLL EMT-like activation. At the transcription level, ZEB1 expression was higher and ZEB2 expression lower in PB CLL cells. The main stimulatory events in CLL cells appear restricted to affected tissue sites, primarily in lymph nodes (LN) where CLL cells migrate to reactivate and proliferate. Further, EMT factor protein expression was confirmed in CLL LN tissue microarrays. Strikingly, transformation into a more aggressive form (Richter syndrome) strongly correlates with an increase in ZEB2 but not ZEB1 protein expression. Interestingly, analysis of the published transcriptome data set of CLL cells co-cultured with nurse-like cells shows an increase in ZEB2 and a decrease in ZEB1 expression after 14 days (GSE13811). An apparent reliance of the EMT-like activation in CLL on the environmental stimuli has been demonstrated in CLL cells stimulated with BCR activation (anti-IgM) or mitogen (PMA), implying protection from cell death and activation of migration. Additionally, we observed higher S100A4 expression in CLL cells compared to mature B-cells from healthy individuals. Previously it has been shown that this calcium-dependent protein can control cell movement by direct interaction with non-muscle myosin and via regulation of stress fibre formation, thus facilitating cellular protrusions and increasing cell migration and invasion. Our analysis has shown the S100A4 positive correlation with CLL markers of poor prognosis, Zap70 and CD38. The S100A4 knockdown with siRNA dramatically impeded CLL migration in trans-well assay (Figure 1). Biochemical inhibitor screening for disruption of S100A4 interaction with myosin II identified several promising leads active in the low micromolar range in both biochemical and cell assays. Conclusions: Our findings improve the understanding of CLL pathobiology. Normally, healthy naïve B cells give rise to multiple B-cell subtypes with specific functions and locations. EMT-like activation likely contributes to B-cell development at different stages of differentiation and B-cell malignant transformation. Our investigation shows that the EMT-like activation is dysregulated in CLL cells compared to healthy B cells. The EMT expression signature changes upon B-cell receptor stimulation. These results suggest that targeting some of the aberrantly expressed EMT components such as S100A4 in CLL can be exploited, prolonging remission by, for example, impeding CLL returning to the tissue sites after an egress due to the treatment with Ibrutinib depriving these cells of vital microenvironmental support. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Disruption of Nurse-like Cell Differentiation as a Therapeutic Strategy for Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, but, despite advances in treatment, many patients still experience relapse. CLL cells depend on interactions with supportive cells, and nurse-like cells (NLCs) are the major such cell type. However, little is known about how NLCs develop. Here, we performed DNA methylation analysis of CLL patient-derived NLCs using the 850K Illumina array, comparing CD14+ cells at day 1 (monocytes) versus day 14 (NLCs). We found a strong loss of methylation in AP-1 transcription factor binding sites, which may be driven by MAPK signaling. Testing of individual MAPK pathways (MEK, p38, and JNK) revealed a strong dependence on MEK/ERK for NLC development, because treatment of patient samples with the MEK inhibitor trametinib dramatically reduced NLC development in vitro. Using the adoptive transfer Eµ-TCL1 mouse model of CLL, we found that MEK inhibition slowed CLL progression, leading to lower WBC counts and to significantly longer survival time. There were also lower numbers of mouse macrophages, particularly within the M2-like population. In summary, NLC development depends on MEK signaling, and inhibition of MEK leads to increased survival time in vivo. Hence, targeting the MEK/ERK pathway may be an effective treatment strategy for CLL.

Abstract LB050: Targeting cells in the microenvironment as a novel therapeutic strategy for chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is a disease that affects the elderly, characterized by the accumulation of mature B cells in the bloodstream. With an estimation incidence of 21,250 new cases diagnosed and 4,320 deaths for 2021, CLL is still considered a non-curable disease despite the wide therapeutic alternatives. CLL, like other malignant diseases, involves the activity of different cells in the microenvironment that support the leukemic cells by providing survival signals, even in presence of therapeutic agents. Among these cells, nurse-like cells (NLC) are of great importance. NLCs are M2-like macrophages known to be developed from blood monocytes, which need close contact with CLL cells for both their differentiation and their protective function. They provide multiple survival signals to CLL cells, and protect them against agents like the BTK inhibitor ibrutinib. Despite their relevance, little research has been done to elucidate the program that drives NLC differentiation. Here, we addressed this question by looking at the DNA methylation signatures before and after NLC differentiation. We found that NLC differentiation produced a marked DNA hypomethylation when compared to freshly isolated monocytes, characterized by the enrichment in AP-1 transcription binding sites. AP-1 is known to be regulated by MAP kinases, so we decided to chemically inhibit these pathways and found that specific MEK inhibition lead to reduced numbers of NLCs in vitro. Further studies showed that indeed NLCs have an activated MEK signaling as seen by the basal phosphorylation of ERK in vitro by confocal microscopy. We then used the adoptive transfer Eµ-TCL1 mice model to seek the effect of MEK inhibition in vivo. By treating the mice with trametinib, an FDA-approved MEK inhibitor, we found an increase in mouse survival when compared with the vehicle control. In addition, we observed a reduced number of monocyte/macrophage populations, especially in those expressing EGR2, a known M2 marker, suggesting that MEK inhibition causes a disruption in CLL-supportive macrophages both in vitro and in vivo. In conclusion, we observed that NLC differentiation strongly depends on the MEK signaling pathway, and that its inhibition leads to reduced myeloid supportive cells for CLL cells, followed by a reduced leukemic progression and increased CLL survival in vivo. Thus, we propose that MEK inhibition could be a potential therapeutic alternative for CLL. Further research is ongoing to determine how MEK signaling is activated during NLC differentiation. Citation Format: Giovanna Merchand Reyes, Ramasamy Santhanam, Frank H. Robledo Avila, Christoph Weigel, Juan D. Ruiz Rosado, Xiaokui Mo, Santiago Partida Sanchez, Jennifer A. Woyach, Christopher C. Oakes, Susheela Tridandapani, Jonathan P. Butchar. Targeting cells in the microenvironment as a novel therapeutic strategy for chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB050.

Nurselike cells sequester B cells in disorganized lymph nodes in chronic lymphocytic leukemia via alternative production of CCL21

AbstractTumor microenvironment exerts a critical role in sustaining homing, retention, and survival of chronic lymphocytic leukemia (CLL) cells in secondary lymphoid organs. Such conditions foster immune surveillance escape and resistance to therapies. The physiological microenvironment is rendered tumor permissive by an interplay of chemokines, chemokine receptors, and adhesion molecules as well as by direct interactions between malignant lymphocytes and stromal cells, T cells, and specialized macrophages referred to as nurselike cells (NLCs). To characterize this complex interplay, we investigated the altered architecture on CLL lymph nodes biopsies and observed a dramatic loss of tissue subcompartments and stromal cell networks as compared with nonmalignant lymph nodes. A supplemental high density of CD68+ cells expressing the homeostatic chemokine CCL21 was randomly distributed. Using an imaging flow cytometry approach, CCL21 mRNA and the corresponding protein were observed in single CD68+ NLCs differentiated in vitro from CLL peripheral blood mononuclear cells. The chemokine was sequestered at the NLC membrane, helping capture of CCR7-high-expressing CLL B cells. Inhibiting the CCL21/CCR7 interaction by blocking antibodies or using therapeutic ibrutinib altered the adhesion of leukemic cells. Our results indicate NLCs as providers of an alternative source of CCL21, taking over the physiological task of follicular reticular cells, whose network is deeply altered in CLL lymph nodes. By retaining malignant B cells, CCL21 provides a protective environment for their niching and survival, thus allowing tumor evasion and resistance to treatment. These findings argue for a specific targeting or reeducation of NLCs as a new immunotherapy strategy for this disease.

BTK Inhibitors Impair Platelet-Mediated Antifungal Activity.

In recent years, the introduction of new drugs targeting Bruton’s tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.

Aberrantly expressed Wnt5a in nurse-like cells drives resistance to Venetoclax in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of neoplastic B lymphocytes with high levels of Wnt5a in the plasma. Currently, the cell source of Wnt5a remains controversial. The receptor of Wnt5a is ROR1, whose expression is associated with disease progression and resistance to venetoclax, a BCL-2 inhibitor approved for the treatment of CLL. In this study, we found that the levels of Wnt5a in the plasma of CLL patients were positively correlated with absolute monocyte counts, but not lymphocyte counts. We cultured monocyte-derived nurse-like cells (NLCs) from patients with CLL, and detected Wnt5a expressed in NLCs. Flow cytometry and transwell assays showed that the antibody neutralizing Wnt5a inhibited the enhanced survival and migration in CLL cells co-cultured with NLCs. Furthermore, we performed a drug screening with CLL cells cultured with or without NLCs with a library containing 133 FDA-approved oncology drugs by using high-throughput flow cytometry. We observed a significant resistance to venetoclax in CLL cells co-cultured with NLCs. Immunoblot revealed the activation of NF-κB with enhanced expression of MCL-1 and BCL-XL in CLL cells co-cultured with NLCs. Neutralizing Wnt5a or blocking NF-κB pathway significantly decreased the expression of MCL-1 and BCL-XL, which leads to enhanced sensitivity to venetoclax in CLL cells co-cultured with NLCs. In conclusion, our data showed that NLCs could be one of the sources of Wnt5a detected in patients with CLL, and Wnt5a-induced NF-κB activation in the CLL microenvironment results in resistance to venetoclax in CLL cells.

Activation and expansion of T-follicular helper cells in chronic lymphocytic leukemia nurselike cell co-cultures.

Interactions between chronic lymphocytic leukemia (CLL) cells and T-cell subsets in the lymph node microenvironment are thought to play a central role in disease biology. To study these interactions in a model of the CLL lymph node microenvironment, we characterized T-cell subsets in CLL nurselike cell (NLC) co-cultures. We focused on T-follicular helper (Tfh) cells, which are characterized by CXCR5 expression and localization to B-cell follicles. In co-cultures from 28 different CLL patients, we detected an expansion of Tfh cells based on PD-1, BCL6, and ICOS expression, with increased IL-21 and downmodulated CD40L surface expression. Regulatory T cells (Treg), which promote immune tolerance, also expanded in NLC co-cultures. T-cell receptor (TR) gene repertoire analyses confirmed the clonal expansion of CD4+ T cells, with an enrichment of TR clonotypes commonly expanded also in primary CLL samples. Multicolor confocal microscopy revealed that Tfh, but not Treg co-localize with proliferating CLL cells in CLL lymph node sections. Collectively, these data provide new insight into the cellular and molecular cross-talk between CLL and T-cell subsets, resulting in clonal expansion of T-helper cells and interaction of Tfh cells with proliferating CLL cells which may open new avenues for therapeutic targeting.

IL-10 Rescues CLL Survival through Repolarization of Inflammatory Nurse-like Cells.

Simple SummaryIn in vitro co-cultures of CLL cells and nurse-like cells (NLC), protection against apoptosis is only provided by M2-like NLC, and not M1-like NLC. In this study, we propose that fine-tuning of NLC polarization (and therefore survival of leukemic cells) is dictated by a balance between IL-10 and TNF.Tumor-associated macrophages (TAMs) in chronic lymphocytic leukemia (CLL) are also called nurse-like cells (NLC), and confer survival signals through the release of soluble factors and cellular contacts. While in most patient samples the presence of NLC in co-cultures guarantees high viability of leukemic cells in vitro, in some cases this protective effect is absent. These macrophages are characterized by an “M1-like phenotype”. We show here that their reprogramming towards an M2-like phenotype (tumor-supportive) with IL-10 leads to an increase in leukemic cell survival. Inflammatory cytokines, such as TNF, are also able to depolarize M2-type protective NLC (decreasing CLL cell viability), an effect which is countered by IL-10 or blocking antibodies. Interestingly, both IL-10 and TNF are implied in the pathophysiology of CLL and their elevated level is associated with bad prognosis. We propose that the molecular balance between these two cytokines in CLL niches plays an important role in the maintenance of the protective phenotype of NLCs, and therefore in the survival of CLL cells.

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Nurse-like Cells Express High-Levels of Wnt5a, Which Induces ROR1-Dependent Signaling That Promotes Migration and Survival of Neoplastic Cells in Chronic Lymphocytic Leukemia

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen expressed on chronic lymphocytic leukemia (CLL) cells, but not on normal postpartum tissues. The ligand that binds to ROR1 is Wnt5a, which we find present at high levels in the plasma of patients with CLL relative to that of age-matched healthy adults. Wnt5a can induce CLL-cell activation of Rho-GTPases in a ROR1-dependent manner, thereby enhancing leukemia-cell proliferation, migration, and survival. Currently, the cell-source(s) of Wnt5a in CLL is not known. Good candidates include Nurse-Like cells (NLCs), which are monocyte-derived accessory cells that develop in the context of CLL cells and that are found within the leukemia-cell microenvironment in lymphoid tissues. Already NLCs have been shown to elaborate factors that can promote leukemia-cell activation/survival, including chemokines (e.g. CXCL12, CXCL13) and members of the tumor-necrosis-factor family (e.g. BAFF and APRIL). Because activated monocytes can produce Wnt5a, we hypothesize that NLC also may produce this non-canonical Wnt factor. For this study, we generated NLCs from blood mononuclear cells of CLL patients through co-culture with leukemia B cells using established methods. NLCs, which stained positive for CD163 and CD68, were isolated free of leukemia cells via adherence. We isolated RNA from purified CLL cells or purified NLC of the same patient for real-time PCR to measure the levels of WNT5A transcripts. WNT5A transcripts were abundant in the RNA isolated from NLC, but were negligible in RNA isolated from CLL cells each patient tested (N = 5). We evaluated for Wnt5a via ELISA in the culture-medium of isolated NLCs of five different patients. The cultured medium of NLCs from each patient had detectable Wnt5a that increased in concentration over time. Furthermore, ultra-high resolution confocal fluorescent microscopy of NLC-CLL co-cultures showed Wnt5a was expressed at high-levels in NLCs, but was undetectable in CLL cells of each patient tested (N = 3). We performed co-culture studies and trans-well assays to assess the survival and migration of CLL cells when cultured with or without NLCs, and with or without a neutralizing mAb specific for Wnt5a or cirmtuzumab, which is a humanized anti-ROR1 mAb that blocks Wnt5a-induced, ROR1-dependent signaling. We found that anti-Wnt5a or cirmtuzumab, but not a mAb of irrelevant specificity, each could comparably and significantly inhibit the protective effects of NLCs for CLL cells in vitro (p= 0.008, N = 3 and p= 0.0032, N = 3, respectively by paired student's t-test). Moreover, anti-Wnt5a or cirmtuzumab each could comparably and significantly inhibit the migration of CLL cells enhanced by co-culture with NLCs in vitro (p < 0.0001, N = 3, by paired student's t-test). We also examined for Rho-GTPase activation in CLL cells co-cultured with NLCs. CLL cells co-cultured with NLCs, but not CLL cells cultured alone, had high-level activation of Rac1 and RhoA. The activation of RhoA and Rac1 in co-cultured CLL cells could be blocked by neutralizing anti-Wnt5a or cirmtuzumab, but not by control mAb of irrelevant specificity. We conclude that NLCs express Wnt5a, which can enhance CLL-cell migration and survival via a ROR1-dependent pathway. We speculate that the high-level Wnt5a noted in the plasma of patients with CLL is produced primarily by NLCs, which reside in lymphoid tissues. If so, then the high-level Wnt5a present in the plasma of CLL patients may extend the protective effects of NLCs beyond the CLL microenvironment in which NLCs reside. Treatment with cirmtuzumab to block ROR1-dependent Wnt5a-signaling may mitigate such effects, potentially enhancing the clearance of leukemia cells when used alone or in combination with drugs that target other survival-signaling pathways in CLL. DisclosuresKipps:AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Oncternal: Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

Increased Levels of Soluble RAGE in CLL Patient Plasma Has a Protective Effect on CLL Disease Progression

Background: Receptor for advanced glycation end-products (RAGE) is the primary receptor for the inflammatory molecule HMGB1. We previously demonstrated that HMGB1 differentiates nurse like cells (NLC), and that increased levels HMGB1 in the plasma of CLL patients and overexpression of RAGE in CLL lymph nodes is associated with a poorer clinical outcome. Moreover, we have shown that HMGB1 induced activation of NF-κB in primary CLL cells in vitro . Soluble RAGE (sRAGE) is a reported antagonistic decoy receptor of plasma HMGB1, and limits chronic inflammation. The pool of plasma sRAGE originates from 2 main sources - alternative splicing to produce endogenous secreted RAGE (esRAGE), or from surface RAGE ectodomain shedding catalyzed by ADAM10. In a healthy setting, HMGB1 and sRAGE plasma levels are inversely correlated, with increased levels of sRAGE limiting inflammatory responses. RAGE shedding in response to saturating levels of ligand (i.e. HMGB1) establishes a negative feedback loop, dampening inflammatory signaling by sequestering RAGE ligands. In chronic disease, the HMGB1-sRAGE inverse relationship is frequently reversed. The functional impact of sRAGE is poorly documented in cancer, and its role in CLL is currently unknown.We hypothesize that sRAGE could have a protective effect in CLL and could dampen HMGB1-mediated disease progression. The aim of this study is to characterise the clinical significance of sRAGE in CLL and investigate whether RAGE undergoes membrane shedding to confer a protective effect in CLL.Methods: sRAGE plasma levels were measured by ELISA in 54 CLL patients and 15 healthy controls. sRAGE protein mass was assessed by Western blotting and compared to matched patient ADAM10 whole cell expression. ADAM10 inhibitor studies and HMGB1-induced RAGE shedding was performed in RAGE-transfected HEK293 cell line and primary CLL cells; sRAGE production was analyzed by ELISA and surface RAGE expression determined by flow cytometry. sRAGE containing media was produced by HEK293 cell line transfected with esRAGE isoform gene sequence; sRAGE containing media (2ng/ml) was used to assess the effect on primary CLL NLC differentiation.Results: sRAGE plasma levels were significantly higher in CLL patients compared to healthy controls (median 60.0pg/ml ± 79.5 interquartile range and 31.0pg/ml ±46.9, respectively; P <0.01), but fell below levels capable of full HMGB1 inhibition for the same patient cohort (72.2ng/ml ±111.4). Absence of a negative correlation between HMGB1 and sRAGE suggested a breakdown in the negative feedback loop in CLL, compared to the classical inverse trend observed for the healthy controls. Patients with high levels of plasma sRAGE had an increased time to first treatment (P=0.0134; n=26) and sRAGE levels were increased in patients who had reached remission following treatment compared to pre-treatment samples, suggesting that sRAGE provides a protective effect on CLL disease progression.CLL plasma sRAGE is of shorter length than cellular full-length RAGE and esRAGE from primary CLL cells, suggesting sRAGE originates from ectodomain shedding. ADAM10 expression, the main RAGE sheddase, is independent of sRAGE plasma levels assessed by Western blotting. ADAM10 stimulation with ionomycin induced RAGE ectodomain shedding into cell culture medium in RAGE-transfected HEK293 cell line (2270.0ng/ml) compared to untreated cells, without altering surface RAGE or ADAM10 expression. Ionomycin-induced RAGE shedding was abrogated by pre-treatment with ADAM10 inhibitor, GI254023X (45.5ng/ml). Ionomycin induced apoptosis in primary CLL cells, so could not be investigated for RAGE shedding, but our data demonstrates that saturating HMGB1 levels induces RAGE shedding in CLL. Finally, sRAGE-containing media did not reduce primary CLL NLC differentiation in vitro after 1-2 weeks culture.Conclusions: Our data demonstrates for the first time that plasma sRAGE limits CLL disease progression by prolonging time to first treatment and sustaining stable disease following treatment. sRAGE originates from ectodomain shedding, is dependent on ADAM10 and inducible by HMGB1. Although sRAGE failed to block NLC differentiation in vitro, it contributes to the extracellular mileu ‘cocktail’ shaping the CLL microenvironment. We therefore propose that HMGB1 and sRAGE levels could be useful plasma markers for inflammatory microenvironment and therapy monitoring. DisclosuresGribben:Acerta: Honoraria; Pharmacyclics: Honoraria; Celgene: Honoraria; TG Therapeutics: Honoraria; Karyopharm: Honoraria; Janssen: Honoraria; Kite: Honoraria; Abbvie: Honoraria; Genentech/Roche: Honoraria.

Phase II/1b Trial of Ibrutinib Plus Prednisone and Rituximab Induction Followed By Venetoclax Plus Ibrutinib Maintenance in Newly Diagnosed High-Risk Chronic Lymphocytic Leukemia

Introduction: Therapy options for chronic lymphocytic leukemia (CLL) are rapidly changing with the approval of ibrutinib, an irreversible Bruton's tyrosine kinase inhibitor, and venetoclax, a Bcl2 inhibitor. In the RESONATE studies, ibrutinib showed significant improvements in overall response rates (ORR), progression-free-survival (PFS), and overall survival (OS). Nonetheless, complete response (CR) rates were only about 4%.During the first few weeks of treatment, ibrutinib will mobilize CLL cells from tissue sites into peripheral blood. Rituximab, a chimeric monoclonal CD20 antibody, when used with ibrutinib, has shown to immediately clear CLL from the blood with durable responses but with CR's of only 8%.Venetoclax is approved for relapsed or refractory CLL with 17p deletion. In a phase 2 study, at a median follow-up of 12.1 months, the ORR was ~79%, but with only 16% CR's. Amongst the 45 patients of whom minimal residual disease (MRD) was assessed, 24 were negative for MRD.In vitro studies have shown that nurse-like cells (NLCs) will protect CLL cells against spontaneous apoptosis by producing chemokines and interleukins such as SDF1, suggestive of a mechanism of ibrutinib resistance. SDF1 was shown to bind to its receptor CXCR4 located on CLL cells and activate a signal transduction pathway that would reduce spontaneous apoptosis. Dexamethasone was found to decrease NLC viability. This suggests that a combined therapy of ibrutinib plus steroids along with rituximab and venetoclax may be necessary to achieve deeper CR's in high-risk CLL.In addition, CLL with overexpression of exosome miR-155 is associated with a poor response to chemoimmunotherapy. In prior preclinical studies, exosome miR-150 and miR-155 have been shown to increase with BCR activation via α-IgM stimulation. We hope that via next generation microRNA sequencing from our patients' CLL plasma cells we will observe a decrease in the levels of exosome miR-150 and miR-155 in response to inhibition of BCR-pathway by ibrutinib as seen in preclinical studies.Methods: Patients with newly diagnosed high-risk CLL per FISH studies will be enrolled. Ibrutinib will be administered orally 420 mg daily, rituximab will be dosed at 375 mg/m2 intravenously on days 1, 8, 15, 22 of cycle 1 and on day 1 for cycle 2-6. Prednisone will be administered as 10 mg one week on, one week off per cycle. Each cycle will last 28 days. After 6 cycles, if patients have MRD, venetoclax will be added in on a weekly schedule with weekly ramp up from 20mg to 50mg, 100mg, and 200mg for a total duration of 6 months plus ibrutinib at 420mg as maintenance until disease progression or drug intolerance. There is the option to reintroduce venetoclax, prednisone, or rituximab if patients progress during maintenance.Results: The primary objectives of this study are to evaluate safety and MRD negative CR rates based on IWG-CLL criteria. For the evaluation of safety, the proportions of the 29 subjects with any adverse events, each specific adverse event, and study-related adverse events will be reported along with the 95% confidence intervals. The proportion of the 29 subjects who demonstrate a MRD negative CR will be reported along with the 95% confidence interval. A sample size of 29 will produce a two-sided 95% confidence interval with a width≤ 0.380.In addition, Kaplan-Meier method will be performed to evaluate time to first MRD negativity (TTMN), time to first relapse (TTFR), PFS and OS, respectively. Based on the Kaplan-Meier curve, the median time to TTMN, TTFR, PFS and OS, respectively, will be reported along with the 95% confidence interval if it exists. Correlation coefficients will be derived to describe the relationship between continuous endpoints and Kendall's tau will be derived to describe the relationship between categorical endpoints.There is no planned interim analysis for the CR rate. However, at any time point if an excessive number of adverse events are observed, the study will be terminated.Conclusion: In this Phase IB/II clinical trial, we seek to observe prolonged response rates of ibrutinib via inhibiting NLC's by adding prednisone. Also, we anticipate that rituximab will further amplify the CR's. For maintenance therapy, we hope that by combining venetoclax with ibrutinib in high-risk CLL patients who were unable to achieve negative MRD's, we will be able to induce deeper remissions. [Display omitted] DisclosuresPersky:Genentech: Consultancy; MorphoSys: Other: Independent Data Monitoring Committee member ; Verastem: Consultancy; Spectrum Pharmaceuticals: Research Funding.