NFĸB Targeting in Bone Marrow Mesenchymal Stem Cell-Mediated Support of Age-Linked Hematological Malignancies

Mesenchymal stem cells (MSCs) can become dysfunctional in patients with hematological disorders. An unanswered question is whether age-linked disruption of the bone marrow (BM) microenvironment is secondary to hematological dysfunction or vice versa. We therefore studied MSC function in patients with different hematological disorders and found decreased MHC-II except from one sample with acute myeloid leukemia (AML). The patients’ MSCs were able to exert veto properties except for AML MSCs. While the expression of MHC-II appeared to be irrelevant to the immune licensing of MSCs, AML MSCs lost their ability to differentiate upon contact and rather, continued to proliferate, forming foci-like structures. We performed a retrospective study that indicated a significant increase in MSCs, based on phenotype, for patients with BM fibrosis. This suggests a role for MSCs in patients transitioning to leukemia. NFĸB was important to MSC function and was shown to be a potential target to sensitize leukemic CD34+/CD38− cells to azacitidine. This correlated with their lack of allogeneic stimulation. This study identified NFĸB as a potential target for combination therapy to treat leukemia stem cells and showed that understanding MSC biology and immune response could be key in determining how the aging BM might support leukemia. More importantly, we show how MSCs might be involved in transitioning the high risk patient with hematological disorder to AML.


Introduction
Mesenchymal Stem Cells (MSCs), also referred as bone marrow (BM) stromal cells, are found in adult and fetal tissues [1][2][3]. In adults, MSCs are predominantly found in adipose tissue and BM [1,2]. MSCs are distinct from BM stromal cells, which are mostly comprised of hematopoietic supporting fibroblasts, differentiated from MSCs [3][4][5]. In BM, MSCs can be located at different anatomical sites-central sinus, trabeculae, endosteal region and compact bone [5,6]. These locations are also sites of hematopoietic activity in which the function of hematopoietic stem cells (HSCs) are supported with MSCs and their differentiated cells [7]. The latter include fibroblasts, adipocytes and osteoblasts [8]. Thus, the functional relationship between MSCs and hematopoietic activity are part of the process to maintain BM homeostasis.
MSCs are source of multiple cytokines and then can also respond to cytokines through specific receptors [9][10][11][12]. As third party cells within an inflammatory milieu such as graft versus host disease (GvHD), MSCs can be licensed as immune suppressor cells to modulate the inflammation [10]. At the perivascular region, MSCs can be considered as gate-keeper cells since they can exert plasticity to function as immune suppressor or enhancer cells. In this regard, MSCs can elicit immune stimulation against invading agents. Such response can increase inflammation, which will This work is in partial fulfillment for LSS doctoral thesis negatively regulate MSCs as immune suppressor to prevent exacerbated inflammation to avert inflammatorymediated hematopoietic suppression and premature BM aging [10,13,14].
BM MSCs are linked to the pathogenesis of hematological malignancies [15]. In light of the immune properties of MSCs and their key role in the hematopoietic niche, it is expected that MSCs might not be able to regulate autoimmune-mediated hematological disorders [15]. Indeed, MSCs from patients with idiopathic thrombocytopenia (ITP) exhibit reduced capacity to elicit immune suppression such as enhanced suppressor T-cells to control the autoimmune process of ITP [16]. However, the dysfunction in ITP MSCs could be reversed as reported for thalidomide [17]. MSCs from aplastic anemia patients, another autoimmune-mediated hematological disorder, also show reduced efficiency to support hematopoiesis [18].
The clinical and experimental evidence indicated that MSCs support the survival of leukemia cells, including drug resistance. Asparaginase, which is used to treat patients with acute lymphocytic leukemia (ALL) to deplete arginine from the leukemic cells, can survive by arginine released from the patients' MSCs [19]. Acute myeloid leukemia cells (AML) can survive chemotherapy through the transfer of MSC-derived mitochondria [20]. Chronic lymphocytic leukemia (CLL) cells mediate their own survival by releasing microvesicles that differentiate MSCs into cancer associated fibroblasts [21]. In addition, PDGF from CLL cells can switch MSCs towards angiogenesis [22]. In some cases of known mutation such as myeloproliferative disorder (MPD), the associated MSCs do not seem to harbor the same mutation [23]. In contrast, AML and myelodysplastic syndrome (MDS) have chromosomal abnormalities similar to the tumor cells [24,25]. However, others reports show a lack of clonal-associated markers in MDS MSCs with functional competence with respect to hematopoietic support [26]. This function is independent of their ability to be immune suppressor veto cells, underscoring their immune dysfunction in hematological disorders [27]. MSCs from AML show impaired proliferation, differentiation and hematopoietic support [28]. In light the discussed reports on MSCs on hematological disorder, this study focused on the functional immune properties of MSCs to determine how these cells contribute to hematological malignancies. We noted enhanced ability of patients' MSCs to be licensed as immune suppressor cells with an inflammatory milieu and showed that these cells are increased in MF MSCs. Functionally, MSCs appear to require CD74-NFĸB axis.

Reagents
Dulbecco's modified Eagle's medium (DMEM) with high glucose, RPMI 1640, Phytohemagglutinin (PHA, diluted in tissue culture grade phosphate buffered saline/PBS) and L-glutamine were purchased from Life Technologies Gibco (Grand Island, NY, USA). Fetal calf serum (FCS), paraformaldehyde, tissue culture grade PBS, pH 7.4, bovine serum albumin (BSA) and Ficoll-Hypaque were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bortezomib and azacytidine were obtained from University Hospital Pharmacy, Newark, NJ. RelA Ser529/536 and control peptides were purchased from Novus Biologicals (Centennial, CO). The peptides were dissolved in PBS and used in the assay at 30 µM.

Ethical Statement
The use of human BM aspirates and peripheral blood (PB) from healthy subjects and patients were approved by the Institutional Review Board of Rutgers University, Newark Campus and VA New Jersey Health Care System, East Orange, New Jersey. All subjects signed the consent forms. Patients' BM aspirates remaining after clinical analyses were used for the research studies.

BM Biopsy Slides
Slides (Table 2) from different BM biopsies at VA New Jersey Health Care system for immunohistochemistry. The slides were provided to the study team who were blinded 2179 with respect to patients' diagnoses. After labeling and summary of the staining, Dr. Chang provided the codes which were matched to diagnosis and age.

Culture of Mesenchymal Stem Cells (MSCs)
MSCs were cultured from BM aspirates of healthy donors and patients with hematological disorders, as described [29]. Briefly, unfractionated aspirates were diluted in DMEM with 10% FCS (D10 media) and then added to vacuum gas plasma-treated plates (BD Falcon; Franklin Lakes, NJ). After 3 days, red blood cells and granulocytes were removed by Ficoll-Hypaque density gradient centrifugation and the mononuclear fractions replaced in the plates with fresh D10 media. At weekly intervals, fresh media replaced 50% of the culture media. The adherent cells were serially passage at 80% confluence. At passage 4, the cells were negative for CD34, CD45 and CD14; positive for CD29, CD73, CD90, CD105 and CD44, and differentiated into adipogenic and osteogenic cells [10].

Flow Cytometry
At 80% confluence, MSCs were de-adhered with dissociation solution, washed with 1 × PBS (pH 7.4), and then incubated for 30 min at room temperature with primary antibodies at the dilutions shown above or isotype control. Cells were fixed with 0.2% paraformaldehyde and then analyzed on the FACScan (FACS Caliber; Becton Dickinson, Franklin Lakes, NJ).

Mixed Lymphocyte Response (MLR)
The MLR assay was previously described [10]. Briefly, peripheral blood mononuclear cells (PBMCs) (responders) were incubated with MSC stimulator cells in 96-well flat bottom tissue culture plates. The stimulator cells were γ-irradiated at 20 Gy with a Cesium source. After 72 h incubation, each well was pulsed with 1 μCi of [methyl-3H] TdR. After 16 h, the cells were harvested on glass fiber filters, which were analyzed on a scintillation counter for TdR incorporation. Counts per minute (CPM) were converted to disintegration per minute (DPM), which was used to calculate simulation indices (SI) = Experimental DPM/PBMC background DPM.

Blast Cell Isolation
The selection of CD34+/CD38− followed a 2-step isolation protocol. First, CD34+ cells were isolated from the peripheral blood (PB) of patients with leukemia using Dynabeads M-450 (ThermoFisher) as described [30]. Briefly, PB mononuclear cells (PBMCs) were isolated by Ficoll Hypaque density gradient centrifugation and 10 7 PBMCs/mL were resuspended in cold Ca 2+ Mg 2+ -free PBS containing 2% BSA (isolation buffer). The cell suspension was incubated in the cold for 30 min with 10 8 Dynabeads M-450 CD34. During incubation, the tubes were gentle agitated at 5-min intervals. Cells that bound to the Dynabeads were magnetically selected with Dynal MPC and then washed with isolation buffer. The cells were uncoupled from the beads by incubating at 37 °C for 15 min with DETACHaBEAD CD34 liquid. We verified the isolation efficiency to be ~90%, based on flow cytometry with PE-anti-CD34 and Tricolor-anti-CD45. Non-specific labeling was assessed with non-immune PE-IgG1. Second, CD34+ cells were if they express CD38 by negative selection. This was accomplished by incubating with murine anti-CD38 followed by depletion with Dynabead-linked anti-mouse IgG (ThermoFisher).

Drug Treatment
Leukemia blasts (10 3 ) were added to round bottom 96-well tissue culture plates in 150 µL RPMI 1640 containing 10% FCS. The cells were treated with bortezomib and/or azacytidine, in triplicate, with bortezomib studied at 100 ng/ mL and azacytidine at 1 μg/mL. At days 3 and 5, the total number of viable cells were determined by cell count and trypan blue exclusion.

Mitogen Assay
PBMCs from heathy donors were stimulated with 1% PHA in RPMI 1640 containing 10% FCS, as described [10]. Healthy or patients' MSCs (10 3 /0.2 mL) were added to each well. Control wells contained media instead of MSCs. The cultures were pulsed with 1 μCi/well of [methyl-3H]TdR. Cells were harvested with a PhD cell harvester onto glassfiber filters.
[3H]TdR incorporation was measured in a scintillation counter.

Immunohistochemistry
Antigens from coded paraffin-embedded human BM biopsies were retrieved by overnight incubation at 56 °C. This was followed by dewaxing with xylene, ethanol dehydrationed and rehydration with deionized water. The slides were incubated overnight at 37 °C in the dark within a humidified chamber in which the slides were covered with the primary fluorochrome-tagged antibodies (CD31 or CD105). The antibody concentrations are stated above. Unbound antibodies were washed with 1X PBS and then immediately analyzed on the EVOS FL Auto 2 Imaging System.

Western Blot
Whole cell extracts were obtained from uninduced MSC and induced MSCs. The MSCs were stimulated with IL-1α and TGFβ1 or unstimulated. Cells were trypsinized and washed in PBS (pH 7.4). Following this, 30 µl of 1 × Lysis Buffer (Promega, Madison, WI) was added and the cells were subjected to freeze/thaw cycles in a dry ice/ethanol bath. Cell-free whole cell lysates were obtained by centrifugation at 4,000 g for 5 min at 4 °C. Total protein was determined with a Bio-Rad DC protein assay kit (Hercules, CA). Extracts (200 µg) were analyzed by western blots using 4-20% SDS-PAGE pre-cast gels (Bio-Rad) and the proteins were transferred onto polyvinylidene difluoride membranes (Perkin Elmer Life Sciences, Boston, MA). Membranes were incubated overnight at 4 °C with primary antibodies and then detected the following day by 2-h incubation with HRPconjugated IgG. All primary and secondary antibodies were used at final dilutions of 1/1000 and 1/2000, respectively. HRP was developed with chemiluminescence detection reagent (Perkin Elmer Life Sciences). The membranes were stripped with Restore Stripping Buffer (Pierce, Rockford, IL) for reprobing with other antibodies.

Adhesion Assay
Adhesion assays were performed to assess contact-dependent interaction between MSCs and cancer cells, using breast cancer cells as a model. MSCs from healthy subjects were cultured in phenol red-free DMEM supplemented with 10% FBS. Next day, MSCs were transfected with scramble siRNA or Rel A siRNA (Dharmacon-Horizon Discovery (Lafayette, CO). The latter was transfected with Dharma-FECT (Thermo Scientific) according to the manufacturers recommendations. Parallel studies were incubated with 30 µM RelA Ser929/536 or control peptide. After 24 h, the cells were seeded at 2 × 10 4 cells per well in a vacuum gas plasma-treated 96-well plate and then allowed to adhere to the plates overnight. MDA-MB-231 were labeled with CFSE in the dark at room temperature, quenched with phenol-free DMEM and 10 4 cells added in each well containing monolayer MSCs. After 48 h, total fluorescence (preaspiration) was assessed on a microplate reader (SoftMax Pro5 SpectraMax 3) at 485 nm/535 nm. This was followed by washing 3 times with 1 × PBS followed by reading for fluorescence (post-aspiration). Percent adhesion was calculated based on the readout pre-aspiration/washing and post-aspiration/washing via the formula: [RFU post-aspiration -RFU µ,empty well ] × 100/[RFU pre-aspiration -RFU µ,empty well ] in which RFU = relative fluorescence unit.

Statistical Analyses
Statistical analyses were performed with analysis of variance and Tukey-Kramer multiple comparisons test. p < 0.05 was considered significant.

MSCs from Patients with Hematological Disorders
MSCs from the enrolled patients were expanded in culture to address the experimental questions posed in this manuscript. The morphology of Passage 3 MSCs from patients and healthy donors were similar (Fig. 1a-Representative images). Similarly, they exhibited comparable phenotypes for the following cell surface markers: CD105+, CD73+ and CD45− (Fig. 1b). The patients' MSCs were multipotent, based on differentiation into osteogenic cells and neurons, as described [31] (Fig. 1c).

Third Party Patients' MSCs in Immune Suppression
MSCs from AML patients show heterogeneity with respect to MHC-II expression whereas those from other patients analyzed in this study exhibit low MHC-II (Fig. 1). Unlike macrophages in which IFNγ is proportional to MHC-II expression, in MSCs, while low IFNγ induces MHC-II, high levels decrease MHC-II [31]. In cases of high IFNγ such as an inflammatory milieu caused by GvHD, MSCs can be licensed as veto cells [10,34]. We therefore asked if patients' MSCs can be licensed as immune suppressor cells, similar to healthy MSCs. This was addressed by adding patients' MSCs as third party cells in 2-way MLR. First, we verified allogeneic difference between the responder and stimulator PBMCs in MLR assay without MSCs (Fig. 2a, open bar). As third party cells in the 2-way MLR studies, healthy MSCs decreased the response by 4 folds, consistent with their reported veto property [10,34]. Similar studies with patients' MSCs varied depending on the source-except for AML MSCs, which enhanced the proliferative response whereas other patients' MSCs significantly (p < 0.05) resulted in decreased stimulation indices (SI) (Fig. 2a, b).

Patients' MSCs Within an Inflammatory Milieu Caused by Mitogen
We next asked if patients' MSCs also suppress mitogenic response. This was addressed in PHA-stimulated PBMCs containing MSCs. Control cultures containing PBMCs and healthy MSCs without PHA (media) resulted in >fivefold SI (Fig. 2c, far left bar). This was expected because MSCs were not added to an inflammatory milieu and the PBMCs responded to the allogeneic difference of healthy MSCs. Similar analyses with MSCs from MDS patient showed twofold SI which was significantly (p < 0.05) less than the decreased by healthy MSCs (Fig. 2c, left group). The reduced ability of MDS MSCs to elicit an allogeneic response was in line with undetectable MHC-II (Fig. 1).

MSC Distribution in BM Biopsies from Myelofibrosis (MF) Patients
We noted enhanced immune suppressive licensing of MSC from AML, MDS and MPD patients (Fig. 2). The question is whether MSC dysfunction is secondary to the respective hematological malignancies. Since patients with myeloproliferative neoplasms (MPNs) in particular MF, can transition to AML [35], we asked if MSCs are increased in MF BM. Particularly, increased MSCs in the BM of MF patients may contribute to enhanced angiogenesis and fibroblasts in MF BM and may contribute to the risk of these patients transitioning to leukemia [36]. Indeed, increased angiogenesis in MF patients is in line with the role of MSCs in angiogenic processes-production of angiogenic factors, differentiation into endothelial cells and supporting the architecture of blood vessels as pericytes [37,38].
We performed immunohistochemistry for CD31 (endothelial cells) and CD105 (MSCs or endothelial cells) with tissues from BM biopsies of patients with fibrosis (MF and other hematological disorder) or non-hematological disorders ( Fig. 3 and Table 2). There were few areas in which the red fluorescence (CD31) was in contact with green fluorescence (CD105), suggesting pericytes around blood vessels. In all cases including non-hematological disorder, there were single areas of green (CD105) with wide-spread CD31, indicative of increased angiogenesis in MF BM. A summary of the staining is summarized in the lower section (Fig. 3), showing increased MSCs in patients with MF and idiopathic myelofibrosis (IMF) and to a lesser extend for slides from non-hematological disorders.

Loss of Contact Inhibition by AML MSCs
Flow cytometry for MHC-II on MSCs from patients' BM indicated that AML MSCs, as compared to the other sources, showed varied MHC-II (Fig. 1d). Regardless of MHC-II, (change this to a comma and delete the period)  (Fig. 2a). Yet, they were able to inhibit the proliferation of PBMCs stimulated with the mitogen, PHA (Fig. 1d). We therefore focused on AML MSCs due to their ability to support chemoresistance [39].
We asked if the patients' MSCs at passage 5 showed evidence of transformation. All MSCs were seeded at the same time and then observed for time to achieve confluence. We noted rapid confluence by AML MSCs (Fig. 4-top right panel). Unlike AML MSCs, which showed evidence of transformation, MDS MSCs grew slowly with no evidence of transformation (Fig. 4 third  row). Others studies using a large cohort of AML MSCs observed no evidence of disease mutation but reported on increased clonogenic potential [40]. This report is in line with the loss of contact differentiation, expected for healthy MSCs.

Phenotype of AML MSCs
Due to the loss of contact by AML MSCs, we asked if these cells are still phenotypically MSCs. We addressed this question with a panel of markers that are known to be present or absent on MSCs (Fig. 5a). The latter included hematopoietic cells, myeloid and lymphoid markers and the former, CD90, CD44, CD73, CD105. In addition, we also evaluated for MHC-II expression. We evaluated AML MSCs for their ability to differentiate into osteogenic cells (Fig. 5b). The results indicated phenotypic and functional MSCs, despite the loss of contact inhibition.

CD74-NFĸB Axis in MSC Function
MSCs expanded from AML patients MSCs were less efficient as veto cells, implying their inability to suppress T-cell activation (Fig. 2). As compared to the other patients, MSCs from AML expressed MHC-II (Figs. 1,  5a). We therefore focused on the surface glycoprotein CD74 for the following reasons: it serves as the receptor for migration inhibitory factor (MIF) to mediate cancer progression [41]; CD74 serves as the invariant chain for MHC-II and could induce intracellular signal transduction via second messengers to promote cell proliferation, noted for AML MSCs (Fig. 5a). Thus, CD74 expression on AML MSCs may explain the increased proliferation as well as MHC-II expression. Indeed, similar to healthy subjects, flow cytometry indicated CD74 on AML MSCs (Fig. 6a).
CD74 can induce intracellular signaling via NFĸB, ERK, and PI(3)K pathways to promote cell proliferation [42]. Since NFĸB can also maintain MSC multipotency, we asked if inhibiting its activity could affect the function of MSCs [43]. We therefore used a model of breast cancer cells due to their ability to interact with MSCs [44]. Specifically, we asked if inhibiting NFĸB activity would interfere with their ability to interact with MSCs. We inhibited NFĸB activity in MSCs with a pharmacological Rel A inhibitor or with Rel A siRNA. Control MSCs were incubated with vehicle or scramble siRNA. After 24 h, MSCs were incubated with fluorescence labeled MDA-MB-231 breast cancer cells. After 48 h, we assessed cancer cell adhesion to MSCs by fluorescence scanning. We noted significant (p < 0.05) decreases when NFĸB was inactivated (Fig. 6b). In summary, the results support the involvement of CD74-NFĸB axis in MSC function.

Enhanced Sensitivity by Bortezomib and Azacytidine on AML Blasts
We isolated CD34+/CD38− cells from the blood of four patients with hematologic malignancies (CML, MDS transitioned to AML, AML, MF transitioned to AML). We cultured the cells in the presence of azacytidine and/ or bortezomib for 48 h and then accessed cell viability. Each of the four patient cells tested resulted in <5% viable cells as compared to a range of 56-93% after 3-and 5-day  Table 3). The resistance to single drug occurred despite enhanced cell proliferation. The results indicated that bortezomib seems to prepare the patient's CD34 + CD38-cells to azacytidine treatment.

Discussion
This study addressed an early time point of hematological malignancy that could be relevant to preventing the transition of age-linked hematological disorders to leukemia. The former includes patients with MF, PV, MDS and other MPD. The licensing ability of healthy MSCs as immune suppressor cells were maintained in MSCs cultured from the BM of patients with CML and MPDs (Fig. 2a). In contrast, MSCs from AML patient acted as immune stimulator cells (Fig. 2). Although MSCs from one AML patients expressed MHC-II, similar detection was not identified on MSCs from other AML patients (Fig. 1d). Thus, we could not explain the stimulatory response of AML MSCs when they were exposed to allogeneic MHC-II, suggesting that AML MSCs might be imparting immune cell activation in vivo when they are within an autologous system. This point is intriguing as it suggest that anti-inflammatory agents might pose benefit in reducing the proliferative effects of MSCs in AML patients. Since AML can expand in the gum, these findings suggest a close working relationship between the dentist and hematologist to manage patients with AML [45]. In contrast, healthy MSCs, which expressed MHC-II were effective veto cells [10]. The observed difference in AML MSCs was noted at cell confluence when they lost contact inhibition (Fig. 4). This contrasted healthy and other patients' MSCs that differentiated upon contact (not shown). Indeed, AML MSCs have been shown to be a support AML growth and also protect them via mitochondrial transfer [19,20,46]. Upon contact, AML MSCs began to form foci, suggesting cell transformation, leading to the following questions: Does transformation occur before the onset of AML? If so, do the transformed MSCs initiate the transformation of hematopoietic cells leading to leukemia? Alternatively, does the AML alter the microenvironment by inducing perhaps through direct contact and/or through their secretome to transformation MSCs that might support their growth? The latter could occur through interaction with genetic material transfer in secretome such as microvesicles as well as mitochondrial transfer and the former, through adhesion molecules and/ or gap junction. This study therefore initiated several questions for future studies, not only by our group, but others who may have access to patients and archived samples. It should be noted that despite the apparent transformation by the AML MSCs, these cells were similar to healthy stem cells with respect to phenotype and multipotency (Fig. 5). Similar to healthy MSCs, one of the methods used to assess multipotency is transdifferentiation into peptidergic neurons. Indeed AML MSCs were able to form neurons, although with reduced efficiency, was much less as compared to healthy MSCs. This supports some functional changes that might be important to assess their role in AML support.
Due to reduced ability of AML MSCs to elicit an allogeneic response compared to healthy MSCs and also MSCs from other patients, we studied the ability of AML MSCs to reduce an inflammatory response, induced by the mitogen, PHA. Interestingly, AML MSCs decreased the mitogen response significantly more than healthy MSCs (Fig. 2, right group). The question is why AML MSCs do not cause overt suppression as third party cells in MLR as compared to MSCs from CML and MPD (Fig. 1a). These are highly significant observations that would require in-depth studies since this would be relevant for current and future treatments. Also the findings might be relevant in predicting responses when hematopoietic transplantation is done for patients with hematological malignancies.
Since MPN, MDS and MF are significant age-related disorders, they also posed a high risk for transformation into acute leukemias. However, similar transition also occurs in young patients with similar diagnosis. The current treatment for high grade myelodysplastic syndromes are azacytidine and decitabine, with low response rates at approximately 10%. Similar treatment for MF patients with Ruxolitinib does not seem to increase patient survival. Patients with MF have been reported to have increased reticulin fibers, increased angiogenesis and increased fibroblasts. However, earlier reports showed colony forming units-fibroblasts from the BM of MF patients [47]. However, it is unlikely that fibroblasts will form colonies, which are established with MSCs. Interestingly, MSCs from patients with JAK2 + MPD supported the neoplastic cells [48]. Indeed, analyzing a large cohort of retrospective samples from patients with different hematological malignancies indicated the expected presence of phenotypic MSCs in all patients but more importantly urgently enhanced amounts in patients with MF and in other hematological disorders showing fibrosis (Fig. 3). Since these patients are at high risk to transition into leukemias, this finding was highly significant in the quest to prevent leukemia from patients with disorders such as MDS and MF.
Findings from the immunohistochemical analyses led us to seek potential mechanisms by which MSCs may be involved with leukemia. We could not find any difference in CD74, which could serve as an invariant chain of MHC-II [41] (Fig. 6a). Since CD74 could also activate intracellular signaling, partly through NFĸB activation [42], we asked if the latter could be involved in MSC function. Using a model of breast cancer-MSC interaction [44,49], we showed that inhibition of RelA in MSCs significantly decreased the interaction (Fig. 6b), supporting a role for NFĸB in MSC function. The effect of NFĸB in MSC function was in line with its reported role in maintaining cancer stem cells [50]. We identified a role for NFĸB with bortezomib treatment, which sensitized the otherwise resistant leukemia stem cells to azacytidine (Table 3). Since one of the patients was transitioning from MDS to AML with ~20% blast, the data suggested that this combination could be a potential treatment for patients at risk for transition into acute leukemia.
In summary, the combined treatment shown in Table 3 strongly support the need for a large clinical trial by combining an NFĸB inhibitor such as bortezomib or carfilzomib with an anti-proliferative drugs. In this study we used azacytidine suggesting that perhaps there is a change in the epigenetic program of the leukemia cells that is counter to cell survival. The studies also indicate that MSC function might hold the key to future therapies for hematological disorders and their function might be predictor of responses to varied treatments. Several hematological disorders are immune-mediated. Due to the dual properties of MSCs as immune enhancers and suppressors, it is important to examine these cells at the molecular level. A key finding is the MHC-II invariant chain CD74 that persisted on AML MSCs. This may prevent tumor antigen loading and presentation to T-cells, allowing for immune subversion. These findings will need to be expanded to MSCs derived from other patients because in addition to what is shown in Table 3 for combined treatment, the available anti-CD74, milatuzumab, merits further investigation for combined treatment. The findings in this study might Fig. 5 Characterization of AML MSCs. a Expanded MSCs from AML aspirates were analyzed by flow cytometry for myeloid (CD11b), lymphoid (CD19), hematopoietic cell (CD45), HLA-DR and know markers of MSCs. The results represent three different analyses. b MSCs at 80% confluence were studied for osteogenic differentiation ◂ begin to shed a different perspective on the reason for the concentration of leukemic cells in gum where MSCs can be present and with similar function as those in the BM [51,52]. The question is why the gum is an extramedullary site of leukemia and it is a distant site secondary to bone marrow? Insights were gained from hematopoietic activity reported in gum and the role of extracellular vesicles from dental stem cell to change the function of similar cells in Fig. 6 NFĸB signaling in MSCs. a Representative histogram for flow cytometry for CD74 on MSCs from healthy donors and AML. b MSCs were knockdown for RelA or pretreated with the inhibitor, RelA Ser529/530 and then evaluated for adhesion to MDA-MB-231 breast cancer cells. The results are the mean ± SD, n = 3. Each experiment was performed in triplicate and each with MSCs from a different donor bone marrow. Thus, we can surmise that leukemia in bone marrow and gum could undergo parallel development. A more important question, is whether the current treatment for leukemia is effective for the cells in the dental regions. An interesting question is why leukemia cannot develop in the gum then move to the bone marrow where it have a more complex niche to expand. If hematopoietic activity can occur with dental pulp stem cells as support, it will not be impossible for leukemia to be develop in the same area with age-linked changes. In this vein, the studies provide insights on how the aging BM microenvironment, particularly MSCs could be poised to support the growth of leukemia cells.