Leukemic Stem Cell Niche Research Articles

Cordycepin (3'dA) Induces Cell Death of AC133+ Leukemia Cells via Re-Expression of WIF1 and Down-Modulation of MYC.

Wnt/β-catenin signaling is critically required for the development and maintenance of leukemia stem cells (LSCs) in acute myeloid leukemia (AML) by constitutive activation of myeloid regeneration-related pathways. Cell-intrinsic activation of canonical Wnt signaling propagates in the nucleus by β-catenin translocation, where it induces expression of target oncogenes such as JUN, MYC and CCND1. As the Wnt/β-catenin pathway is now well established to be a key oncogenic signaling pathway promoting leukemic myelopoiesis, targeting it would be an effective strategy to impair LSC functionality. Although the effects of the adenosine analogue cordycepin in repressing β-catenins and destabilizing the LSC niche have been highlighted, the cellular and molecular effects on AML-LSC have not been fully clarified. In the present study, we evaluated the potency and efficacy of cordycepin, a selective repressor of Wnt/β-catenin signaling with anti-leukemia properties, on the AC133+ LSC fraction. Cordycepin effectively reduces cell viability of the AC133+ LSCs in the MUTZ-2 cell model and patient-derived cells through the induction of apoptosis. By Wnt-targeted RNA sequencing panel, we highlighted the re-expression of WIF1 and DKK1 among others, and the consequent downregulation of MYC and PROM1 (CD133) following MUTZ-2 cell exposure to increasing doses of cordycepin. Our results provide new insights into the molecular circuits involved in pharmacological inhibition mediated by cordycepin reinforcing the potential of targeting the Wnt/β-catenin and co-regulatory complexes in AML.

Epigenetic Modification of Mesenchymal Stromal Cells Derived from Bone Marrow and Embryonal Tumors to Facilitate Immunotherapeutic Approaches in Pediatric Malignancies.

Mesenchymal stromal cells (MSC) are part of the bone marrow architecture and contribute to the homeostasis of hematopoietic stem cells. Moreover, they are known to regulate immune effector cells. These properties of MSC are pivotal under physiologic conditions, and they may aberrantly also protect malignant cells. MSCs are also found in the leukemic stem cell niche of the bone marrow and as part of the tumor microenvironment. Here, they protect malignant cells from chemotherapeutic drugs and from immune effector cells in immunotherapeutic approaches. Modulation of these mechanisms may improve the efficacy of therapeutic regimens. We investigated the effect of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA, Vorinostat™) on the immunomodulatory effect and cytokine profile of MSC derived from bone marrow and pediatric tumors. The immune phenotype of MSC was not markedly affected. SAHA-treated MSC showed reduced immunomodulatory effects on T cell proliferation and NK cell cytotoxicity. This effect was accompanied by an altered cytokine profile of MSC. While untreated MSC inhibited the production of certain pro-inflammatory cytokines, SAHA treatment led to a partial increase in IFNγ and TNFα secretion. These alterations of the immunosuppressive milieu might be beneficial for immunotherapeutic approaches.

Abstract 5608: Deciphering role of NLRP3 inflammasome in acute myeloid leukemia microenvironment

Abstract Acute Myeloid Leukemia (AML), the most common form of leukemia in adults. Despite intensive chemotherapy regimen, approximately 60% of patients subsequently relapse. Inflammatory cells and mesenchymal stem cells (MSC) which are part of bone marrow (BM) microenvironment, play a critical role in cancer progression. Current strategies are focused on understanding cell-cell contact dependent/independent communication within the leukemic stem cell niche. NLRP3 is a part of innate immune mechanisms, resulting in the release of IL-1β and IL-18 immunomodulatory cytokines. Current study was aimed at analyzing NLRP3 pathway regulation at phenotypic and genotypic level in AML BM-MSC compared to healthy BM-MSC and their cross-talk with immune function in AML stem cell niche. MSC were isolated and cultured from AML BM that exhibited heterogenous fibroblastoid shape. Multi-color flow cytometry of AML BM-MSC revealed characteristic high expression of CD90 and minimal expression of CD45. They also expressed immune checkpoint markers TIM3, PDL-1, adenosine receptors A1R/A2BR, vimentin and NLRP3. Cultured AML BM MSC and fresh leukemic blasts demonstrated tunneling nanotubules, increased vesicles and increase in mitochondria which were of irregular shapes and sizes as seen by transmission electron microscopy. Microarray data revealed genes of NLRP3 damage-associated molecular patterns (DAMP) pathway viz. Nlrp3, NME8, Pycard and IL-18 were distinctly upregulated in AML BM-MSC compared to uninvolved BM-MSC. AML peripheral blood (PB) plasma showed immunosuppressive effect on healthy peripheral blood mononuclear cells. NK cell cytotoxicity was found to be significantly reduced in presence of culture supernatant of AML cell line and further reduced with supernatant of cells activated for NLRP3 pathway. Immunofluorescence demonstrated NLRP3 expression in AML BM-MSC and also in vesicles released from MSC. NLRP3 pathway activation was observed in AML cell line which was significantly abrogated upon treatment with NLRP3 inhibitor. NLRP3 pathway is stimulated using basic DAMP agent LPS and secondary activators ATP or Nigericin. Activation was visualized by colocalization of NLRP3 and caspase-1, in the presence/absence of NLRP3 inhibitor. Expression of IL-1β was observed using immunofluorescence and its release in culture supernatants was measured by ELISA. Inhibition of NLRP3 corroborated with decreased secretion of IL-1β in AML cell line. Thus, a positive correlation of IL-1β release was observed with expression of NLRP3. Paraffin sections of AML BM are being evaluated for NLRP3 pathway markers by immunohistochemistry. Understanding remodelling of BM-MSC, leukemia target and immune cell dynamics through NLRP3 pathway regulation in stroma microenvironment may provide crucial leads in therapeutic modulation of the immune response within the stem cell niche. Citation Format: Monalisa Sahoo, Manasi Nagare, Naythan Dcunha, Meena Patkar, Manju Sengar, Navin Khattry, Anant Gokarn, Sachin Punatar, Sridhar Epari, Tanuja Shet, Jyoti Kode. Deciphering role of NLRP3 inflammasome in acute myeloid leukemia microenvironment [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 5608.

Regulation of Malignant Myeloid Leukemia by Mesenchymal Stem Cells.

Bone marrow microenvironment (BMM) has been proven to have benefits for both normal hematopoietic stem cell niche and pathological leukemic stem cell niche. In fact, the pathological leukemia microenvironment reprograms bone marrow niche cells, especially mesenchymal stem cells for leukemia progression, chemoresistance and relapse. The growth and differentiation of MSCs are modulated by leukemia stem cells. Moreover, chromatin abnormality of mesenchymal stem cells is sufficient for leukemia initiation. Here, we summarize the detailed relationship between MSC and leukemia. MSCs can actively and passively regulate the progression of myelogenous leukemia through cell-to-cell contact, cytokine-receptor interaction, and exosome communication. These behaviors benefit LSCs proliferation and survival and inhibit physiological hematopoiesis. Finally, we describe the recent advances in therapy targeting MSC hoping to provide new perspectives and therapeutic strategies for leukemia.

CNST is Characteristic of Leukemia Stem Cells and is Associated With Poor Prognosis in AML.

Consortin (CNST) is a protein located on the trans-Golgi network that can target transmembrane proteins to the plasma membrane. Although CNST was discovered more than 10 years ago, there are still not enough studies on its function. During our search for possible new acute myeloid leukemia (AML) markers, we found that CNST was overexpressed in almost all patients with AML. By analyzing profiling data from public databases, we found that CNST expression inversely correlated with overall survival among AML patients. There was a great variation in CNST expression among different subtypes of AML, and the expression was the highest in the t(8,21) subtype, which was probably due to the direct regulation of CNST transcription by RUNX1-RUNX1T1. In addition, we analyzed the expression of CNST in different cells of the hematopoietic system. We found that CNST was associated with the low differentiation degrees of hematopoietic cells and had the highest expression level in leukemia stem cells (LSCs). Finally, we analyzed the CNST-related gene network and found that the genes negatively correlated with CNST are involved in various immune-related pathways, which indicates that CNST is likely related to immune evasion, LSC niche retention, and assembly of stress granules. In conclusion, our study suggests that CNST has the potential to be a diagnostic and prognostic biomarker for AML.

Human acute myeloid leukemia stem cells: evolution of concept.

The history of human acute myeloid leukemia stem cells (AMLSCs) began in a seminal study performed by Lapidot and Dick, proving that only CD34+CD38- human primary acute myeloid leukemia (AML) cells can repopulate in severe combined immunodeficient mice. The concept of leukemic stem cells (LSCs) has impeded a huge change in the treatment strategy against AML from killing proliferating leukemic cells to eradicating quiescent/dormant LSCs. As next-generation sequencing technologies have developed, multiple and recurrent genetic mutations have been discovered in large cohorts of patients with AML, and the updated understanding of leukemogenesis has improved the old concept of LSC to a revised version of a serial developmental model of LSC; that is, pre-LSCs are generated as seeds by the first hit on epigenetic regulators, and then, leukemia-initiating LSCs emerge from seeds by the second hits on genes involved in transcription and signaling. Dreams for universal and targetable AMLSC biomarker sparing healthy hematopoietic stem cells have weakened after the confrontation of significant heterogeneity of AMLSCs from genomic and immunophenotypic viewpoints. However, there is still hope for effective targets for AMLSCs since there is evidence that grouped gene signatures, such as 17-gene LSC score, and common epigenetic signatures, such as HOXA clusters, independent of various gene mutations, exist. Recently, the LSC niche in the bone marrow has been actively investigated and has expanded our knowledge of the physiology and vulnerability of AMLSCs. Presently, an applicable treatment that always works in AMLSCs is lacking. However, we will find a way, we always have.

Leukemic Stem Cells: From Leukemic Niche Biology to Treatment Opportunities.

Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults. While complete remission can be obtained with intensive chemotherapy in young and fit patients, relapse is frequent and prognosis remains poor. Leukemic cells are thought to arise from a pool of leukemic stem cells (LSCs) which sit at the top of the hierarchy. Since their discovery, more than 30 years ago, LSCs have been a topic of intense research and their identification paved the way for cancer stem cell research. LSCs are defined by their ability to self-renew, to engraft into recipient mice and to give rise to leukemia. Compared to healthy hematopoietic stem cells (HSCs), LSCs display specific mutations, epigenetic modifications, and a specific metabolic profile. LSCs are usually considered resistant to chemotherapy and are therefore the drivers of relapse. Similar to their HSC counterpart, LSCs reside in a highly specialized microenvironment referred to as the “niche”. Bidirectional interactions between leukemic cells and the microenvironment favor leukemic progression at the expense of healthy hematopoiesis. Within the niche, LSCs are thought to be protected from genotoxic insults. Improvement in our understanding of LSC gene expression profile and phenotype has led to the development of prognosis signatures and the identification of potential therapeutic targets. In this review, we will discuss LSC biology in the context of their specific microenvironment and how a better understanding of LSC niche biology could pave the way for new therapies that target AML.

Splenic Red Pulp Macrophages Provide a Secondary Stem Cell Niche for Chronic Myeloid Leukemia Stem Cells

Leukemic stem cells (LSCs) are responsible for initiation, progression and relapse of the disease. Chronic myeloid leukemia (CML) is a classical stem-cell driven malignancy and represents an attractive model to study LSCs. Resistance of LSCs to treatment is mediated by cell-intrinsic characteristics but also by the interactions of LSCs with their microenvironment. LSCs depend on signals from their surrounding niche in the bone marrow (BM) and spleen in order to maintain stem cell characteristics such as quiescence and self-renewal. The BM as a niche for LSCs is well-investigated and several therapeutic targets are under consideration.Splenomegaly is a hallmark of CML. Nevertheless, the role of the spleen on leukemia and LSCs in unknown. Therefore, this project aims to investigate the potential role of the spleen as an independent secondary niche for CML stem cells and its contribution to disease development and progression.In order to study the role of the spleen in CML development and progression, we retrovirally transduced FACS-purified Lineage- Sca-1+ c-Kit+ BM cells (LSKs) with pMSCV-p210BCR/ABL-IRES-GFP and injected transduced LSKs into splenectomized or sham operated mice. Splenectomy prior to CML induction significantly improved survival compared to sham operated controls (median survival 31 vs. 22 days; p=0.0006) with 20 % of splenectomized animals surviving longer than 90 days (endpoint). In addition, splenectomized animals showed a 3.7-fold reduction (p=0.002) in LSC numbers in the BM when analyzed 18 days after CML induction, suggesting that the spleen influences the disease burden in the BM.We next analyzed the leukemic compartment in the BM and spleen and found a significant enrichment of leukemic stem and progenitor cells in the spleen (20 % spleen vs. 10 % BM; % leukemic stem and progenitor cells of total leukemic cells; p=0.01; 19-fold increase in total amount of LSCs in the spleen; p =0.007). To functionally confirm the phenotypic increase in splenic LSCs, we performed secondary transplantations of total leukemic cells from the BM or spleen at limiting dilution into lethally irradiated secondary recipients. The analysis revealed a significantly higher frequency of LSCs in the spleen compared to the BM (1/41'703 vs. 1/432'594; p=0.02).In addition, we determined the localization of LSCs in the spleen by confocal microscopy on optically cleared spleens. Splenic LSCs were exclusively localized within the red pulp and in close proximity to macrophages. It was previously shown that hematopoietic stem cells reside in direct contact with macrophages in the red pulp of the spleen during extramedullary hematopoiesis (Dutta et al., JEM, 2015). Therefore, we depleted macrophages in CML mice using clodronate liposomes. Spleen weight (867mg vs. 249mg; p<0.0001) and numbers of splenic LSCs (23-fold; p=0.001) were significantly reduced in clodronate treated animals. Importantly, clodronate treatment did not affect LSC viability, as indicated by comparable amounts of AnnexinV+ cells in both groups.In summary, we show that the interaction of splenic niche cells (red pulp macrophages) with LSCs results in the generation of a big reservoir of LSCs which contribute to CML progression. Therefore, our study strongly suggest the presence of an independent secondary splenic LSC niche in addition to the well characterized BM LSC niche in CML. DisclosuresNo relevant conflicts of interest to declare.

CXCL2 benefits acute myeloid leukemia cells in hypoxia.

Drug resistance and relapse of acute myeloid leukemia (AML) is still an important problem in the treatment of leukemia. Leukemia outbreak causes severe hypoxia in bone marrow (BM), remolding BM microenvironment (niche), and transforming hematopoietic stem cell (HSC) niche into leukemia stem cell (LSC) niche. AML cells and the microenvironment usually conduct "cross-talk" through cytokines to anchor resistant AML cells into LSC niche, thus supporting their survival. Therefore, this study was aimed to investigate the role of CXCL2 in the hypoxic AML niche. AML hypoxic niche was simulated by hypoxic culture of THP-1 and HL-60 cells in vitro, thus to study the effects of CXCL2 on the proliferation and migration of AML cells. The expression of hypoxia-inducible factor-1α (HIF-1α) and the activation of survival-related kinases such as PIM2 and mTOR under CoCl2 -simulated hypoxic conditions were detected. The correlation between CXCL2 and the prognosis of AML with big data was verified. (a) CXCL2 promoted the proliferation and migration of AML cells. (b) CXCL2 up-regulated the expression of PIM2 by enhancing the transcriptional activity of HIF-1α. (c) CXCL2 activated mTOR in AML cells. (d) CXCL2 was associated with poor prognosis in AML. CXCL2 promotes survival, migration, and drug resistance pathway of AML cells in hypoxia and is associated with poor prognosis in AML. Therefore, CXCL2 can be considered as an important factor in promoting the development of AML cells in hypoxia.

Clinical Implications of Inflammation in Acute Myeloid Leukemia.

Recent advances in the description of the tumor microenvironment of acute myeloid leukemia, including the comprehensive analysis of the leukemic stem cell niche and clonal evolution, indicate that inflammation may play a major role in many aspects of acute myeloid leukemia (AML) such as disease progression, chemoresistance, and myelosuppression. Studies on the mechanisms of resistance to chemotherapy or tyrosine kinase inhibitors along with high-throughput drug screening have underpinned the potential role of glucocorticoids in this disease classically described as steroid-resistant in contrast to acute lymphoblastic leukemia. Moreover, some mutated oncogenes such as RUNX1, NPM1, or SRSF2 transcriptionally modulate cell state in a manner that primes leukemic cells for glucocorticoid sensitivity. In clinical practice, inflammatory markers such as serum ferritin or IL-6 have a strong prognostic impact and may directly affect disease progression, whereas interesting preliminary data suggested that dexamethasone may improve the outcome for AML patients with a high white blood cell count, which paves the way to develop prospective clinical trials that evaluate the role of glucocorticoids in AML.

Splenic CD24low Red Pulp Macrophages Provide an Alternate Niche for Chronic Myeloid Leukemia Stem Cells

Chronic myeloid leukemia (CML) is a typical stem-cell driven malignancy, driven by leukemia stem cells (LSCs). LSCs are resistant to conventional therapies. This resistance is mediated by cell-intrinsic mechanisms and interactions with the microenvironment. LSCs depend on signals from a specialized microenvironment, a so called niche, to maintain their stem cell characteristics. In CML the bone marrow (BM) as a niche is well-investigated and several therapeutic targets, which aim at LSCs by interrupting their interaction with the BM-niche are under investigation. However, even though splenomegaly is a hallmark of CML the contribution of the splenic microenvironment to CML development has not been studied so far. This project aims to investigate the role of the splenic microenvironment as an independent secondary LSC niche and its contribution to disease development. To induce a CML-like disease in mice we retrovirally transduced FACS-sorted Lineage- Sca-1+ cKit+ BM cells with pMSCV-p210BCR/ABL-IRES-GFP and injected the transduced cells into non-irradiated mice. To find out if the spleen contributes to disease development we induced CML in splenectomized and sham operated mice. Splenectomized mice survived significantly longer compared to sham operated controls (median survival 31 vs. 22 days; p=0.0006) with 20% of the splenectomized mice surviving longer than 90 days. Moreover, the number of LSCs in the BM of splenectomized mice was reduced 3.7-fold (p=0.002). Flowcytometric analysis of the spleen and BM compartments of CML bearing mice revealed that the majority of the leukemic stem and progenitor cells (LSPCs) were located in the spleen (19-fold more LSCs in the spleen; p =0.007). Moreover we found the leukemic compartment in the spleen to be enriched for LSPCs compared to the BM (20 % spleen vs. 10 % BM; % LSPCs of total leukemic cells; p=0.01). To confirm this phenotypic observation functionally we performed a limiting dilution transplantation of leukemic cells from spleen and BM. In line with the phenotypic observation we found a higher frequency of LSCs in the spleen compared to the BM (1/41'703 vs. 1/432'594; p=0.02). We next analyzed the gene expression of LSPCs from spleen and BM. We found that the gene expression profile of splenic LSPCs showed higher expression of stemness-related genes and reduced expression of myeloid differentiation genes compared to BM LSPCs, indicating that the spleen is more supportive of primitive LSPCs. Knowing that the spleen contributes to disease development by providing an alternate niche for LSCs we next analyzed the spleens using confocal microscopy. We found that the LSCs resided exclusively in the red pulp. Previous studies have shown that HSCs reside in direct contact with red pulp macrophages (RPMs) during extramedullary hematopoiesis (Dutta et al., JEM, 2015). In addition we found that in spleens from human CML patients CD34+ leukemia cells localized together with macrophages (p=0.001). Furthermore we could show that RPMs are capable of producing both SCF and G-CSF. To test the role of RPMs as a potential niche component in vitro we co-incubated LSCs and RPMs overnight before plating the LSCs in a colony formation assay. We found that the co-incubation with RPMs improved the colony formation capacity of LSCs (CFUs 166 vs. 138; p=0.0356). To test the role of RPMs in vivo we depleted macrophages in CML mice using clodronate liposomes. This resulted in significantly reduced splenomegaly (867mg vs. 249mg; p&lt;0.0001) and reduced numbers of splenic LSCs (23-fold; p=0.001). This was further confirmed in a genetically engineered mouse model lacking RPMs (Spic-/-) (735mg vs. 450mg; p=0.0112 and 22-fold fewer LSCs; p&lt;0.0001). Finally, while in naïve mice RPMs can be differentiated into a CD24low and CD24high population, the CD24high population is lost in CML bearing mice. In summary, we found that the spleen provides an alternate niche for LSCs, thereby contributing to CML development. Compared to the BM niche the splenic niche is more supportive of primitive LSPCs, as shown by the higher frequency of LSCSs found in the spleen and the higher expression of stemness related genes in splenic LSCs compared to BM LSCs. Moreover we identified CD24low RPMs as a unique and central component of the splenic LSC niche. Even though we could show that RPMs are capable of producing SCF and G-CSF the exact mechanisms by which RPMs support LSCs remains to be investigated. Disclosures No relevant conflicts of interest to declare.

Identifying the Leukemia Stem Cell Niche Mediating Drug Resistance in Flt3-ITD Acute Myeloid Leukemia

The Flt3-ITD is one of the most common mutations in AML, and defines a distinct subtype of disease with unique features and biology, and with poor prognosis related to high rates of recurrence. Although several FLT3 TKIs have been developed for clinical use, responses are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC drug resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. Administration of pIpC leads to deletion of TET2 and development of AML-like disease characterized by leukocytosis, accumulation of blasts, anemia and thrombocytopenia. Transplantation of selected STHSC, MPP and GMP populations revealed that LIC were almost exclusively found within the phenotypic ST-HSC population (calculated stem cell frequencies: &amp;lt;1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD AML patients LIC capacity was similarly restricted to more primitive HSPC populations (Lin-CD34+CD38-) and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We performed flow cytometry on collagenase-digested bone fragments from AML mice to characterize bone marrow stromal cells in Flt3-ITD AML mice. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found that several stromal populations are expanded in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+), a 3.9-fold increase in endothelial cells (CD45-Ter119-CD31+), and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). The expression of CXCL12, a key factor that mediates niche localization of HSC and LSC, was greater than 2-fold higher in osteoprogenitors, but not significantly different in endothelial cells, and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC. We transplanted murine AML cells into CXCL12flox/flox UBC-cre or CXCL12flox/flox Osx-cre mice to assess the effect of global or osteoprogenitor-specific CXCL12-KO, respectively, on AML progression and TKI response. We found that this model of AML was resistant to single agent Flt3 TKI (AC220) treatment. Global CXCL12-KO using CXCL12flox/flox UBC-cre mice modestly improved response to TKI. We show that a combination regimen including standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and a Flt3 TKI (AC220) results in more effective and selective targeting of leukemia cells in this model. We are currently treating osteoprogenitor-specific CXCL12-KO AML mice with the combination chemotherapy + TKI regimen to investigate the contribution of osteoprogenitors to disease progression and drug resistance in Flt3-ITD AML LSC. In conclusion, our results suggest that LSC in Flt3-ITD AML are present within a primitive phenotypic ST-HSC population more so than in MPP and GMP populations as often seen in some other types of AML. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

Targeting Leukemia Stem Cell-Niche Dynamics: A New Challenge in AML Treatment.

One of the most urgent needs in AML is to improve the disease cure rate as relapse still occurs in 60–80% of patients. Recent evidence suggests that dismal clinical outcomes may be improved by a better definition of the tight interaction between the AML cell population and the bone marrow (BM) microenvironment (“the niche”); the latter has been progressively highlighted to have an active role in the disease process. It has now been well established that the leukemic population may misinterpret niche-derived signals and remodel the niche, providing a shelter to AML cells and protecting them from the cytotoxic effects of chemoradiotherapy. Novel imaging technological advances and preclinical disease models have revealed that, due to the finite number of BM niches, leukemic stem cells (LSCs) and normal hematopoietic stem cells (HSCs) compete for the same functional areas. Thus, the removal of LSCs from the BM niche and the promotion of normal HSC engraftment should be the primary goals in antileukemic research. In addition, it is now becoming increasingly clear that AML-niche dynamics are disease stage specific. In AML, the niche has been linked to disease pathogenesis in the preleukemic stage, the niche becomes permissive once leukemic cells are established, and the niche is transformed into a self-reinforcing structure at a later disease stage. These concepts have been fostered by the demonstration that, in unrelated AML types, endosteal vessel loss occurs as a primary AML-induced niche alteration, and additional AML-induced alterations of the niche and normal hematopoiesis evolve focally and in parallel. Obviously, this endosteal vessel loss plays a fundamental role in AML pathogenesis by causing excessive vascular permeability, hypoxia, altered perfusion, and reduced drug delivery. Each of these alterations may be effectively targeted by various therapeutic procedures, but preservation of endosteal vessel integrity might be the best option for any future antileukemic treatment.

Normal and leukemic stem cell niche interactions

Normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) interact with the stem cell niche bone marrow in different ways. Understanding the potentially unique microenvironmental regulation of LSCs is key to understanding in-vivo leukemogenic mechanisms and developing novel antileukemic therapies. When leukemic cells are engrafted in the stem cell niche, the cellular nature of the niche - including mesenchymal stromal cells - is reprogramed. Altered mesenchymal cells selectively support leukemic cells and reinforce the pro-leukemic environment. As the niche plays an active role in leukemogenesis, its remodeling may significantly influence the leukemogenic pattern, and cause differences in clinical prognosis. Notably, niche cells could be stimulated to revert to a pronormal/antileukemic state, creating potential for niche-based antileukemic therapy. Bone marrow microenvironments are under dynamic regulation for normal and leukemic cells, and there is bi-directional control of leukemic cells in the niche. Leukemic cells are both protected by stroma and able to reprogram stromal cells to transform the niche to a state, which reinforces leukemogenesis. Because of its dynamic nature, the niche could be converted to an environment with antileukemic properties, making it an attractive target for therapy.

Ludwig Boltzmann Cluster Oncology (LBC ONC): first 10years and future perspectives.

SummaryIn 2008 the Ludwig Boltzmann Cluster Oncology (LBC ONC) was established on the basis of two previous Ludwig Boltzmann Institutes working in the field of hematology and cancer research. The general aim of the LBC ONC is to improve treatment of hematopoietic neoplasms by eradicating cancer-initiating and disease-propagating cells, also known as leukemic stem cells (LSC) in the context of leukemia. In a first phase, the LBC ONC characterized the phenotype and molecular aberration profiles of LSC in various malignancies. The LSC phenotypes were established in acute and chronic myeloid leukemia, in acute lymphoblastic leukemia and in chronic lymphocytic leukemia. In addition, the concept of preleukemic (premalignant) neoplastic stem cells (pre-L-NSC) was coined by the LBC ONC and was tested in myelodysplastic syndromes and myeloproliferative neoplasms. Phenotypic characterization of LSC provided a solid basis for their purification and for the characterization of specific target expression profiles. In a second phase, molecular markers and targets were validated. This second phase is ongoing and should result in the development of new diagnostics parameters and novel, more effective, LSC-eradicating, treatment strategies; however, many issues still remain to be solved, such as sub-clonal evolution, LSC niche interactions, immunologic control of LSC, and LSC resistance. In the forthcoming years, the LBC ONC will concentrate on developing LSC-eradicating strategies, with special focus on LSC resistance, precision medicine and translation of LSC-eradicating concepts into clinical application.

Regulation of Malignant Hematopoiesis by Bone Marrow Microenvironment.

Hematopoietic stem cells (HSCs) that give rise to all kinds of hematopoietic lineage cells on various demands throughout life are maintained in a specialized microenvironment called “niche” in the bone marrow (BM). Defining niche cells and unveiling its function have been the subject of intense study, and it is becoming increasingly clear how niche cells regulate HSCs in normal hematopoiesis. Leukemia stem cells (LSCs), which are able to produce leukemic cells and maintain leukemic clones, are assumed to share common features with healthy HSCs. Accumulating evidence suggests that LSCs reside in a specialized BM microenvironment; moreover, LSCs could control and rebuild the microenvironment to enhance their progression and survival. This article discusses the recent advances in our knowledge of the microenvironment supporting malignant hematopoiesis, including LSC niche.

De novo AML exhibits greater microenvironment dysregulation compared to AML with myelodysplasia-related changes

The interaction between the bone marrow microenvironment and malignant hematopoietic cells can result in the protection of leukemia cells from chemotherapy in both myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We, herein, characterized the changes in cytokine expression and the function of mesenchymal stromal cells (MSC) in patients with MDS, AML with myelodysplasia-related changes (MRC), a well-recognized clinical subtype of secondary AML, and de novo AML. We observed a significant inhibitory effect of MDS-MSC on T lymphocyte proliferation and no significant differences in any of the cytokines tested. AML-MSC inhibited T-cell proliferation only at a very low MSC/T cell ratio. When compared to the control, AML-MRCderived MSC presented a significant increase in IL6 expression, whereas de novo AML MSC presented a significant increase in the expression levels of VEGFA, CXCL12, RPGE2, IDO, IL1β, IL6 and IL32, followed by a decrease in IL10 expression. Furthermore, data indicate that IL-32 regulates stromal cell proliferation, has a chemotactic potential and participates in stromal cell crosstalk with leukemia cells, which could result in chemoresistance. Our results suggest that the differences between AML-MRC and de novo AML also extend into the leukemic stem cell niche and that IL-32 can participate in the regulation of the bone marrow cytokine milieu.

CD44: More than a mere stem cell marker

CD44 is a cell adhesion molecule that plays an important role in tumor progression and metastasis. The role of CD44 in tumorigenesis is due to its binding to extracellular matrix components, including hyaluronan (HA) and osteopontin (OPN), and to messenger molecules, such as growth factors present in the tumor microenvironment. HA and OPN are highly abundant in the leukemic stem cell niche and in solid tumors of various cancer types, where they contribute to the maintenance of the stemness of malignant cells. CD44 has consequently been recognized as a cancer stem cell marker in several types of cancers, which has been a topic of much recent research. In this review we have addressed the question of how CD44 might promote cancer cell stemness by interacting with extracellular matrix components, growth factors and cytokines.

Hypoxia regulates proliferation of acute myeloid leukemia and sensitivity against chemotherapy

Reduced oxygen partial pressure (pO2, hypoxia) is an important component of the bone marrow microenvironment and the hematopoietic stem cell niche. It is unclear whether this applies to the leukemic stem cell as well and if differences in pO2 between the normal hematopoetic and the leukemic stem cell niche exits. Here, we demonstrate that while there is no detectable difference in the hypoxic level of bone marrow infiltrated by acute myeloid leukemia (AML) and healthy bone marrow, physiological hypoxia of 1% O2 itself leads to cell cycle arrest of AML blasts (both cell lines and primary AML samples) in the G0/G1 phase with upregulation of p27 and consecutive decrease of cells in the S phase. Hence, susceptibility of AML blasts toward cytarabine as S phase dependent drug is significantly decreased as shown by decreased cytotoxicity in vitro. In addition, cells exposed to hypoxia activate PI3K/Akt and increase expression of anti-apoptotic XIAP. Inhibition of PI3K can restore cytarabine sensitivity of AML blasts at hypoxic conditions. In conclusion, hypoxia mediated effects encountered in the bone marrow might contribute to chemoresistance of AML blasts.

EphA3 Is Expressed on Leukemia Stem Cells, and Eph/Ephrin Signalling Features in the Remodelling of the Leukemia Stem Cell Niche

EphA3 is a member of the Eph receptor tyrosine kinase family, the largest such family in the human genome. The bi-directional signalling of Eph receptors and their ligands (the ephrins) is responsible for correct migration and positioning of cells during development, as well as adult processes such as angiogenesis and neurogenesis. EphA3 was first discovered as a receptor expressed on the surface of LK63, a B-cell leukemia cell line. It is not expressed on normal human hematopoietic cells, but expression has been noted on the cell surface of leukemic blasts from a high percentage of patients with hematopoietic neoplasia of all lineages. It is particularly commonly expressed in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), where it is present in approximately 50% of patients. A therapeutic antibody against EphA3 has been developed and is in Phase II clinical trials for MDS and AML.Consistent with data from human MDS patients, the NUP98-HOXD13 (NHD13) mouse model of MDS spontaneously expresses EphA3 on a percentage of cells from all populations throughout the hematopoietic compartment. The proportion of positive cells varies from mouse to mouse, but the proportion positively correlates with disease severity, indicating a possible causative relationship. Studies crossing the NHD13 transgene onto an EphA3 knockout background are ongoing and will definitively answer this question.Recent studies have demonstrated the alteration (or “remodelling”) of the hematopoietic stem cell (HSC) micro-environment (or “niche”) by malignant hematopoietic cells, and the role that this plays in maintaining the leukemia stem cell (LSC) population. This self-reinforcing niche remodelling is critical to the maintenance of hematopoietic disease, particularly during a course of chemotherapy. These niche changes have also been shown to be sufficient to drive malignancy, and are therefore an attractive target for the design of disruptive therapeutics. EphA3 and other Eph/ephrin family members are expressed on normal mesenchymal stem/stromal cells (MSCs) and other stromal component cells of the HSC niche. EfnA5 (EphrinA5) is the high-affinity ligand for EphA3, and we observed an increased expression of EfnA5 on stromal cells in the NHD13 mice, indicating that the EphA3-EfnA5 interaction in these mice may facilitate LSC maintenance in the LSC niche. More broadly, it appears that Eph/Ephrin signalling may be a component of the remodelling of the niche to favour LSCs over normal HSCs. Possible indirect means by which this remodelling may occur include production of the cytokines CCL3 (decreased in NHD13 mice) or CXCL12 (variably decreased in NHD13 mice, and its receptor CXCR4 is increased on the cell surface of NHD13 HSCs). Imaging studies confirm preferential homing of EphA3-positive LSCs to niche locations, including co-localisation with CXCL12. It is therefore likely that the remodelled stem cell niche maintains stemness and leukemogenicity of the LSCs via interaction of the Eph/ephrin family members abnormally expressed on the LSCs and stromal cells, respectively. DisclosuresNo relevant conflicts of interest to declare.