Ex vivo characterization of acute myeloid leukemia patients undergoing hypomethylating agents and venetoclax regimen reveals a venetoclax-specific effect on non-suppressive regulatory T cells and bona fide PD-1+TIM3+ exhausted CD8+ T cells.

Acute myeloid leukemia (AML) is an aggressive heterogeneous disease characterized by several alterations of the immune system prompting disease progression and treatment response. The therapies available for AML can affect lymphocyte function, limiting the efficacy of immunotherapy while hindering leukemia-specific immune reactions. Recently, the treatment based on Venetoclax (VEN), a specific B-cell lymphoma 2 (BCL-2) inhibitor, in combination with hypomethylating agents (HMAs) or low-dose cytarabine, has emerged as a promising clinical strategy in AML. To better understand the immunological effect of VEN treatment, we characterized the phenotype and immune checkpoint (IC) receptors' expression on CD4+ and CD8+ T cells from AML patients after the first and second cycle of HMA in combination with VEN. HMA and VEN treatment significantly increased the percentage of naïve CD8+ T cells and TIM-3+ CD4+ and CD8+ T cells and reduced cytokine-secreting non-suppressive T regulatory cells (Tregs). Of note, a comparison between AML patients treated with HMA only and HMA in combination with VEN revealed the specific contribution of VEN in modulating the immune cell repertoire. Indeed, the reduction of cytokine-secreting non-suppressive Tregs, the increased TIM-3 expression on CD8+ T cells, and the reduced co-expression of PD-1 and TIM-3 on both CD4+ and CD8+ T cells are all VEN-specific. Collectively, our study shed light on immune modulation induced by VEN treatment, providing the rationale for a novel therapeutic combination of VEN and IC inhibitors in AML patients.

Case Report: Invasive fungal infection after anti-CD19 CAR-T cell therapy. Implication for antifungal prophylaxis.

CAR-T therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies. Patients who are receiving such therapy are susceptible to an increased incidence of infections due to post-treatment immunosuppression. The need for antifungal prophylaxis during the period of neutropenia remains to be determined. The clinical outcome of a 55-year-old patient with relapsed/refractory DLBCL who received axicabtagene ciloleucel is described here. The patient developed CRS grade II and ICANS grade IV requiring tocilizumab, prolonged use of steroids and anakinra. An invasive pulmonary aspergillosis arose after 1 month from CAR-T reinfusion, resolved with tracheal sleeve pneumonectomy. The patient is now in Complete Remission. This case suggests that antifungal prophylaxis should be considered. We have now included micafungin as a standard prophylaxis in our institution.

Blinatumomab differentially modulates peripheral blood and bone marrow immune cell repertoire: A Campus ALL study.

Blinatumomab is the first bi-specific T-cell engager approved for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia (B-ALL). Despite remarkable clinical results, the effects of blinatumomab on the host immune cell repertoire are not fully elucidated. In the present study, we characterized the peripheral blood (PB) and, for the first time, the bone marrow (BM) immune cell repertoire upon blinatumomab treatment. Twenty-nine patients with B-ALL received blinatumomab according to clinical practice. Deep multiparametric flow cytometry was used to characterize lymphoid subsets during the first treatment cycle. Blinatumomab induced a transient redistribution of PB effector T-cell subsets and Treg cells with a persistent increase in cytotoxic NK cells, which was associated with a transient upregulation of immune checkpoint receptors on PB CD4 and CD8 T-cell subpopulations and of CD39 expression on suppressive Treg cells. Of note, BM immune T-cell subsets showed a broader post-treatment subversion, including the modulation of markers associated with a T-cell-exhausted phenotype. In conclusion, our study indicates that blinatumomab differentially modulates the PB and BM immune cell repertoire, which may have relevant clinical implications in the therapeutic setting.

Release of IFNγ by Acute Myeloid Leukemia Cells Remodels Bone Marrow Immune Microenvironment by Inducing Regulatory T Cells.

PURPOSE: The stromal and immune bone marrow (BM) landscape is emerging as a crucial determinant for acute myeloid leukemia (AML). Regulatory T cells (Treg) are enriched in the AML microenvironment, but the underlying mechanisms are poorly elucidated. Here, we addressed the effect of IFNγ released by AML cells in BM Treg induction and its impact on AML prognosis. EXPERIMENTAL DESIGN: BM aspirates from patients with AML were subdivided according to IFNG expression. Gene expression profiles in INFγhigh and IFNγlow samples were compared by microarray and NanoString analysis and used to compute a prognostic index. The IFNγ release effect on the BM microenvironment was investigated in mesenchymal stromal cell (MSC)/AML cell cocultures. In mice, AML cells silenced for ifng expression were injected intrabone. RESULTS: IFNγhigh AML samples showed an upregulation of inflammatory genes, usually correlated with a good prognosis in cancer. In contrast, in patients with AML, high IFNG expression was associated with poor overall survival. Notably, IFNγ release by AML cells positively correlated with a higher BM suppressive Treg frequency. In coculture experiments, IFNγhigh AML cells modified MSC transcriptome by upregulating IFNγ-dependent genes related to Treg induction, including indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 inhibitor abrogated the effect of IFNγ release by AML cells on MSC-derived Treg induction. In vivo, the genetic ablation of IFNγ production by AML cells reduced MSC IDO1 expression and Treg infiltration, hindering AML engraftment. CONCLUSIONS: IFNγ release by AML cells induces an immune-regulatory program in MSCs and remodels BM immunologic landscape toward Treg induction, contributing to an immunotolerant microenvironment. See related commentary by Ferrell and Kordasti, p. 2986.

An IDO1-related immune gene signature predicts overall survival in acute myeloid leukemia.

The contribution of the bone marrow (BM) immune microenvironment to acute myeloid leukemia (AML) development is well-known, but its prognostic significance is still elusive. Indoleamine 2,3-dioxygenase 1 (IDO1), which is negatively regulated by the BIN1 proto-oncogene, is an interferon-γ-inducible mediator of immune tolerance. With the aim to develop a prognostic IDO1-based immune gene signature, biological and clinical data of 982 patients with newly diagnosed, nonpromyelocytic AML were retrieved from public datasets and analyzed using established computational pipelines. Targeted transcriptomic profiles of 24 diagnostic BM samples were analyzed using the NanoString's nCounter platform. BIN1 and IDO1 were inversely correlated and individually predicted overall survival. PLXNC1, a semaphorin receptor involved in inflammation and immune response, was the IDO1-interacting gene retaining the strongest prognostic value. The incorporation of PLXNC1 into the 2-gene IDO1-BIN1 score gave rise to a powerful immune gene signature predicting survival, especially in patients receiving chemotherapy. The top differentially expressed genes between IDO1lowand IDO-1high and between PLXNC1lowand PLXNC1high cases further improved the prognostic value of IDO1 providing a 7- and 10-gene immune signature, highly predictive of survival and correlating with AML mutational status at diagnosis. Taken together, our data indicate that IDO1 is pivotal for the construction of an immune gene signature predictive of survival in AML patients. Given the emerging role of immunotherapies for AML, our findings support the incorporation of immune biomarkers into current AML classification and prognostication algorithms.

An Abnormal Host/Microbiomes Signature of Plasma-Derived Extracellular Vesicles Is Associated to Polycythemia Vera.

Polycythemia Vera (PV) is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. Chronic inflammation is commonly observed in myeloproliferative neoplasms including PV. The inflammatory network includes the extracellular vesicles (EVs), which play a role in cell-cell communication. Recent evidence points to circulating microbial components/microbes as potential players in hemopoiesis regulation. To address the role of EVs in PV, here we investigated phenotype and microbial DNA cargo of circulating EVs through multidimensional analysis. Peripheral blood and feces were collected from PV patients (n=38) and healthy donors (n=30). Circulating megakaryocyte (MK)- and platelet (PLT)-derived EVs were analyzed by flow cytometry. After microbial DNA extraction from feces and isolated EVs, the 16S rDNA V3-V4 region was sequenced. We found that the proportion of circulating MK-derived EVs was significantly decreased in PV patients as compared with the healthy donors. By contrast, the proportion of the PLT-derived EVs was increased. Interestingly, PV was also associated with a microbial DNA signature of the isolated EVs with higher diversity and distinct microbial composition than the healthy counterparts. Of note, increased proportion of isolated lipopolysaccharide-associated EVs has been demonstrated in PV patients. Conversely, the gut microbiome profile failed to identify a distinct layout between PV patients and healthy donors. In conclusion, PV is associated with circulating EVs harbouring abnormal phenotype and dysbiosis signature with a potential role in the (inflammatory) pathogenesis of the disease.

Emerging Bone Marrow Microenvironment-Driven Mechanisms of Drug Resistance in Acute Myeloid Leukemia: Tangle or Chance?

Acute myeloid leukemia (AML) has been considered for a long time exclusively driven by critical mutations in hematopoietic stem cells. Recently, the contribution of further players, such as stromal and immune bone marrow (BM) microenvironment components, to AML onset and progression has been pointed out. In particular, mesenchymal stromal cells (MSCs) steadily remodel the leukemic niche, not only favoring leukemic cell growth and development but also tuning their responsiveness to treatments. The list of mechanisms driven by MSCs to promote a leukemia drug-resistant phenotype has progressively expanded. Moreover, the relative proportion and the activation status of immune cells in the BM leukemic microenvironment may vary by influencing their reactivity against leukemic cells. In that, the capacity of the stroma to re-program immune cells, thus promoting and/or hampering therapeutic efficacy, is emerging as a crucial aspect in AML biology, adding an extra layer of complexity. Current treatments for AML have mainly focused on eradicating leukemia cells, with little consideration for the leukemia-damaged BM niche. Increasing evidence on the contribution of stromal and immune cells in response to therapy underscores the need to hold the mutual interplay, which takes place in the BM. A careful dissection of these interactions will help provide novel applications for drugs already under experimentation and open a wide array of opportunities for new drug discovery.

Disease-Specific Derangement of Circulating Endocannabinoids and N-Acylethanolamines in Myeloproliferative Neoplasms.

Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family-palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF; n = 41), polycythemia vera (PV; n = 9), and essential thrombocythemia (ET; n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC-MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

The Yin and Yang of the Bone Marrow Microenvironment: Pros and Cons of Mesenchymal Stromal Cells in Acute Myeloid Leukemia.

Mesenchymal stromal cells (MSCs) have, for a long time, been recognized as pivotal contributors in the set up and maintenance of the hematopoietic stem cell (HSC) niche, as well as in the development and differentiation of the lympho-hematopoietic system. MSCs also have a unique immunomodulatory capacity, which makes them able to affect, both in vitro and in vivo, the function of immune cells. These features, namely the facilitation of stem cell engraftment and the inhibition of lymphocyte responses, have both proven essential for successful allogeneic stem cell transplantation (allo-SCT), which remains the only curative option for several hematologic malignancies. For example, in steroid-refractory acute graft-vs. host disease developing after allo-SCT, MSCs have produced significant results and are now considered a treatment option. However, more recently, the other side of the MSC coin has been unveiled, because of their emerging role in creating a protective and immune-tolerant microenvironment able to support the survival of leukemic cells and affect the response to therapies. In this light, it has been proposed that the failure of current treatments to efficiently override the stroma-mediated protection of leukemic cells accounts for the high rate of relapse in acute myeloid leukemia, at least in part. In this review, we will focus on emerging microenvironment-driven mechanisms conferring a survival advantage to leukemic cells overt physiological HSCs. This body of evidence increasingly highlights the opportunity to consider tumor-microenvironment interactions when designing new therapeutic strategies.

Mesenchymal stromal cells from myelodysplastic and acute myeloid leukemia patients display in vitro reduced proliferative potential and similar capacity to support leukemia cell survival.

BACKGROUND: Mesenchymal stromal cells (MSCs) are an essential element of the bone marrow (BM) microenvironment, playing a crucial function in regulating hematopoietic stem cell proliferation and differentiation. Recent findings have outlined a putative role for MSCs in hematological malignancy development. So far, conflicting results have been collected concerning MSC abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In particular, a considerable amount of evidence has been accumulated strongly supporting a permissive role of MSCs in malignancy evolution to MDS, while a potentially causative or promoting function performed by MSCs in AML has not yet been fully clarified. Here, we compared MSCs isolated from healthy, MDS, and AML subjects to investigate MSC alterations and to emphasize putative common and/or diverse features. METHODS: We isolated and expanded MSCs from AML patients (AML-MSCs) and MDS patients (MDS-MSCs), and we analyzed and compared their phenotypic and functional properties with respect to each other and versus healthy donor-derived MSCs (HD-MSCs). RESULTS: We found that stable MSC cultures could be easily established from HD and MDS mononuclear BM-derived cells, while a substantial fraction (25%) of AML patients failed to yield MSCs. Nevertheless, isolated MDS-MSCs and AML-MSCs, as well as HD-MSCs, contained the basic features of MSCs. Indeed, they displayed similar surface marker expression and efficient capacity to differentiate versus osteogenic and adipogenic lineage in vitro. We also proved that MDS-MSCs and AML-MSCs, analyzed by fluorescence in-situ hybridization, did not harbor leukemic cell cytogenetic abnormalities. Moreover, MDS-MSCs and AML-MSCs were similar in terms of ability to sustain AML cell viability and immune-regulatory capacity. However, we were also able to detect some differences between AML-MSCs and MDS-MSCs. Indeed, we found that the frequency of rescued MSCs was lower in the AML group than in the HD and MDS groups, suggesting that a reduced number of MSC precursors could inhabit AML BM. Instead, MDS-MSCs showed the lowest proliferative capacity, reflecting some intrinsic and particular defect. CONCLUSIONS: Overall, our results elucidated that MDS-MSCs and AML-MSCs did not show macroscopic and/or tumor-related defects, but both displayed functional features potentially contributing to favor a leukemia-protective milieu.

Immunosenescence and Immunotherapy in Elderly Acute Myeloid Leukemia Patients: Time for a Biology-Driven Approach.

Acute myeloid leukemia (AML) is a disease, which mainly affects the elderly population. Unfortunately, the prognosis of patients aged >65 years is dismal, with 1-year overall survival approaching 10% with conventional therapies. The hypothesis of harnessing the immune system against cancer, including leukemia, has been postulated for a long time, and several clinical attempts have been made in this field. In the last years, we increased our knowledge about the interplay between AML and immune cells, but no major improvement has been translated, up to now, from bench to bedside. However, the outstanding results coming from the modern immuno-oncology trials with new drugs have granted a new interest for immunotherapy in AML. Accordingly, the elderly population represents an ideal target, given the low percentage of patients eligible for allogeneic stem cell transplant. With that in mind, in the era of immunotherapy, we consider immunosenescence as the optimal background to start investigating a biology-driven approach to AML therapy in the elderly. By taking into account the physiological age-related changes of immune response, more personalized and tailored use of the new drugs and strategies harnessing the immune system against AML, has the potential to increase their efficacy and impact on clinical outcomes.

The More, The Better: "Do the Right Thing" For Natural Killer Immunotherapy in Acute Myeloid Leukemia.

Natural killer (NK) cells are circulating CD3- lymphocytes, which express CD56 or CD16 and an array of inhibitory receptors, called killer-immunoglobulin-like receptors (KIRs). Alloreactive KIR-ligand mismatched NK cells crucially mediate the innate immune response and have a well-recognized antitumor activity. Adoptive immunotherapy with alloreactive NK cells determined promising clinical results in terms of response in acute myeloid leukemia (AML) patients and several data demonstrated that response can be influenced by the composition of NK graft. Several data show that there is a correlation between NK alloreactivity and clinical outcome: in a cohort of AML patients who received NK infusion with active disease, more alloreactive NK cell clones were found in the donor repertoire of responders than in non-responders. These findings demonstrate that the frequency of alloreactive NK cell clones influence clinical response in AML patients undergoing NK cell immunotherapy. In this work, we will review the most recent preclinical and clinical data about the impact of alloreactive NK cells features other than frequency of alloreactive clones and cytokine network status on their anti-leukemic activity. A better knowledge of these aspects is critical to maximize the effects of this therapy in AML patients.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) promotes survival and migration of acute myeloid leukemia cells through CD63/PI3K/Akt/p21 signaling.

We and others have shown that the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), a member of the inflammatory network exerting pleiotropic effects in the bone marrow (BM) microenvironment, regulates the survival and proliferation of different cell types, including normal hematopoietic progenitor cells. Moreover, TIMP-1 has been shown to be involved in cancer progression. However, its role in leukemic microenvironment has not been addressed. Here, we investigated the activity of TIMP-1 on Acute Myelogenous Leukemia (AML) cell functions. First, we found that TIMP-1 levels were increased in the BM plasma of AML patients at diagnosis. In vitro, recombinant human (rh)TIMP-1 promoted the survival and cell cycle S-phase entry of AML cells. These kinetic effects were related to the downregulation of cyclin-dependent kinase inhibitor p21. rhTIMP-1 increases CXCL12-driven migration of leukemic cells through PI3K signaling. Interestingly, activation of CD63 receptor was required for TIMP-1's cytokine/chemokine activity. Of note, rhTIMP-1 stimulation modulated mRNA expression of Hypoxia Inducible Factor (HIF)-1α, downstream of PI3K/Akt activation. We then co-cultured AML cells with normal or leukemic mesenchymal stromal cells (MSCs) to investigate the interaction of TIMP-1 with cellular component(s) of BM microenvironment. Our results showed that the proliferation and migration of leukemic cells were greatly enhanced by rhTIMP-1 in presence of AML-MSCs as compared to normal MSCs. Thus, we demonstrated that TIMP-1 modulates leukemic blasts survival, migration and function via CD63/PI3K/Akt/p21 signaling. As a "bad actor" in a "bad soil", we propose TIMP-1 as a potential novel therapeutic target in leukemic BM microenvironment.