Marrow Stroma Research Articles

Targeted Disruption of Bone Marrow Stromal Cell-Derived Gremlin1 Limits Multiple Myeloma Disease Progression In Vivo.

In most instances, multiple myeloma (MM) plasma cells (PCs) are reliant on factors made by cells of the bone marrow (BM) stroma for their survival and growth. To date, the nature and cellular composition of the BM tumor microenvironment and the critical factors which drive tumor progression remain imprecisely defined. Our studies show that Gremlin1 (Grem1), a highly conserved protein, which is abundantly secreted by a subset of BM mesenchymal stromal cells, plays a critical role in MM disease development. Analysis of human and mouse BM stromal samples by quantitative PCR showed that GREM1/Grem1 expression was significantly higher in the MM tumor-bearing cohorts compared to healthy controls (p < 0.05, Mann–Whitney test). Additionally, BM-stromal cells cultured with 5TGM1 MM PC line expressed significantly higher levels of Grem1, compared to stromal cells alone (p < 0.01, t-test), suggesting that MM PCs promote increased Grem1 expression in stromal cells. Furthermore, the proliferation of 5TGM1 MM PCs was found to be significantly increased when co-cultured with Grem1-overexpressing stromal cells (p < 0.01, t-test). To examine the role of Grem1 in MM disease in vivo, we utilized the 5TGM1/KaLwRij mouse model of MM. Our studies showed that, compared to immunoglobulin G (IgG) control antibody-treated mice, mice treated with an anti-Grem1 neutralizing antibody had a decrease in MM tumor burden of up to 81.2% (p < 0.05, two-way ANOVA). The studies presented here demonstrate, for the first time, a novel positive feedback loop between MM PCs and BM stroma, and that inhibiting this vicious cycle with a neutralizing antibody can dramatically reduce tumor burden in a preclinical mouse model of MM.

Microfracture Versus Drilling of Articular Cartilage Defects: A Systematic Review of the Basic Science Evidence.

Background:Microfracture (MFx) is one of the most common techniques used for the treatment of articular cartilage defects, although recently there has been a trend toward the use of drilling rather than MFx for the treatment of these defects.Purpose:To perform a systematic review of basic science studies to determine the effect of microfracture versus drilling for articular cartilage repair.Study Design:Systematic review.Methods:A systematic review was performed by searching PubMed, the Cochrane Library, and EMBASE to identify basic science studies comparing outcomes of MFx versus drilling. The search phrase used was microfracture AND (drilling OR microdrilling). Inclusion criteria were basic science studies that directly compared the effect of MFx versus drilling on subchondral bone, bone marrow stimulation, and cartilage regeneration.Results:A total of 7 studies met the inclusion criteria and were included in this systematic review. Of these, 4 studies were performed in rabbits, 1 study in sheep, and 2 studies in humans. All of the included studies investigated cartilage repair in the knee. In the animal studies, microfracture produced fractured and compacted bone and led to increased osteocyte necrosis compared with drilling. Deep drilling (6 mm) was superior to both shallow drilling (2 mm) and MFx in terms of increased subchondral hematoma with greater access to marrow stroma, improved cartilage repair, and increased mineralized bone. However, the overall quality of cartilage repair tissue was poor regardless of marrow stimulation technique. In 2 studies that investigated repair tissue after MFx and/or drilling in human patients with osteoarthritis and cartilage defects, the investigators found that cartilage repair tissue did not achieve the quality of normal hyaline articular cartilage.Conclusion:In the limited basic science studies that are available, deep drilling of cartilage defects in the knee resulted in improved biological features compared with MFx, including less damage to the subchondral bone and greater access to marrow stroma. Regardless of marrow stimulation technique, the overall quality of cartilage regeneration was poor and did not achieve the characteristics of native hyaline cartilage. Overall, there is a general lack of basic science literature comparing microfracture versus drilling for focal chondral defects.

Iron overload as a risk factor for poor graft function following allogeneic hematopoietic stem cell transplantation.

Hematological malignancies are increasingly treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, iron overload is a frequent adverse effect of allo-HSCT and is associated with poor prognosis. In the present study, we investigated hematopoiesis in iron-overloaded mice and elucidated the effects of iron overload on the bone marrow (BM) microenvironment. Iron-overloaded BALB/C mice were generated by injecting 20 mg/mL saccharated iron oxide intraperitoneally. Hematoxylin-eosin staining was performed to evaluate the effects of an iron overload in mice. BM cells obtained from C57BL/6 mice were transplanted into irradiated BALB/C mice (whole-body irradiation of 4 Gy, twice with a 4-hours interval) by tail vein injection. Two weeks after allo-HSCT, the hematopoietic reconstitution capacity was evaluated in recipients by colony-forming assays. Histopathological examinations showed brown-stained granular deposits, irregularly arranged lymphocytes in the liver tissues, and blue-stained blocks in the BM collected from mice received injections of high-dose saccharated iron oxide (20 mg/mL). Iron-overloaded mice showed more platelets, higher-hemoglobin (HGB) concentration, fewer granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte colony-forming units (CFU-E), and mixed granulocyte/erythrocyte/monocyte/megakaryocyte colony-forming units (CFU-mix) than healthy mice. Iron-overloaded recipients presented with reduced erythrocytes and HGB concentration in peripheral blood, along with decreased marrow stroma cells, CFU-GM, CFU-E, and CFU-mix relative to healthy recipients. Taken together, our findings demonstrate that iron overload might alter the number of red blood cells after transplantation in mice by destroying the BM microenvironment, thereby affecting the recovery of BM hematopoietic function.

Exploring the potential of human adipocytes in periodontal regeneration: A review

Stem cells, initially identified in embryonic tissues and later in numerous adult tissues, tend to possess the potentiality to differentiate into various cell types. Though most flexible of all stem cell lines, ethical issues restrict the use of embryonic cells. Furthermore, induced pluripotent stem cells (iPS) and adult stem cells (e.g: bone marrow stroma) can also be used. However, procurement of autologous bone marrow has its potential limitations. An alternate source of autologous adult stem cells which can be procured in large quantities, under local anesthesia, with minimal discomfort would be of keen interest. In the present context, human adult adipose tissue may be the best appropriate alternative source of mesenchymal stem cells. Studies have shown that adipose stem cells (ASCs) extracted from subcutaneous human adult adipose tissue tend to contain heterogeneous cell population called stromal vascular fraction (SVF). It may be used directly or cultured in for selection and expansion of an adherent population, and hence, they are called ASCs. The adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), are processed to obtain a fibroblast-like population of cells, also called processed lipoaspirate (PLA). PLA cells has the potentiality to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. This attributable feature of ASCs may be of significant importance in future clinical cell-based therapy for periodontal disease as well. This review describes current knowledge &amp; recent advances in ASCs &amp; their application. This review describes current knowledge and recent advances in ASCs and their application in periodontal regeneration.

SULF1 suppresses Wnt3A-driven growth of bone metastatic prostate cancer in perlecan-modified 3D cancer-stroma-macrophage triculture models.

Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in αSMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1's role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.

CXCR4 expression in the bone marrow microenvironment is required for hematopoietic stem and progenitor cell maintenance and early hematopoietic regeneration after myeloablation.

The bone marrow (BM) microenvironment/niche plays a key role in regulating hematopoietic stem and progenitor cell (HSPC) activities; however, mechanisms regulating niche cell function are not well understood. In this study, we show that niche intrinsic expression of the CXCR4 chemokine receptor critically regulates HSPC maintenance during steady state, and promotes early hematopoietic regeneration after myeloablative irradiation. At steady state, chimeric mice with wild-type (WT) HSPC and marrow stroma that lack CXCR4 show decreased HSPC quiescence, and their repopulation capacity was markedly reduced. Mesenchymal stromal cells (MSC) were significantly reduced in the BM of CXCR4 deficient mice, which was accompanied by decreased levels of the HSPC supporting factors stromal cell-derived factor-1 (SDF-1) and stem cell factor (SCF). CXCR4 also plays a crucial role in survival and restoration of BM stromal cells after myeloablative irradiation, where the loss of BM stromal cells was more severe in CXCR4-deficient mice compared to WT mice. In addition, transplantation of WT donor HSPC into CXCR4-deficient recipient mice demonstrated reduced HSPC homing and early hematopoietic reconstitution. We found that CXCR4 signaling attenuates irradiation-induced BM stromal cell loss by upregulating the expression of the antiapoptotic protein Survivin via the PI3K pathway. Our study suggests that SDF-1-CXCR4 signaling in the stromal microenvironment cells plays a crucial role in maintenance of HSPCs during homeostasis, and promotes niche regeneration and early hematopoietic reconstitution after transplantation. Modulation of CXCR4 signaling in the HSPC microenvironment could be a means to enhance hematopoietic recovery after clinical hematopoietic cell transplantation.

Superiority of synovial membrane mesenchymal stem cells in chondrogenesis, osteogenesis, myogenesis and tenogenesis in a rabbit model

Engineering complex tissues is perhaps the most ambitious goal of all tissue engineers. Despite significant advances in tissue engineering, which have resulted in successful engineering of simple tissues such as skin and cartilage, there are a number of challenges that remain in engineering of complex, hybrid tissue structures, such as osteochondral tissue. Mesenchymal stem cells (MSCs) have the capacity to highly proliferate in an undifferentiated state and the potential to differentiate into a variety of different lineages, providing a promising single cell source to produce multiple cell types. MSC obtained from adult human contribute to the regeneration of mesenchymal tissues such as bone, cartilage, fat, muscle, tendon and marrow stroma. In the present study, the regeneration capacity of multipotent MSCs derived from different tissues in the rabbit were compared. Specifically the aim of this study was to isolate and characterize rabbit adult stem cell populations from bone marrow, adipose, synovial membrane, rotator cuff, ligament and tendon and assess their cell morphology, growth rate, cell surface markers and differentiation capacity. MSCs derived from synovial membrane showed superiority in terms of chondrogenesis, osteogenesis, myogenesis and tenogenesis, suggesting that synovial membrane-derived MSCs would be a good candidate for efforts to regenerate musculoskeletal tissues.

Bone Marrow Stromal Cells Drive Key Hallmarks of B Cell Malignancies.

All B cell leukaemias and a substantial fraction of lymphomas display a natural niche residency in the bone marrow. While the bone marrow compartment may only be one of several sites of disease manifestations, the strong clinical significance of minimal residual disease (MRD) in the bone marrow strongly suggests that privileged niches exist in this anatomical site favouring central elements of malignant transformation. Here, the co-existence of two hierarchical systems, originating from haematopoietic and mesenchymal stem cells, has extensively been characterised with regard to regulation of the former (blood production) by the latter. How these two systems cooperate under pathological conditions is far less understood and is the focus of many current investigations. More recent single-cell sequencing techniques have now identified an unappreciated cellular heterogeneity of the bone marrow microenvironment. How each of these cell subtypes interact with each other and regulate normal and malignant haematopoiesis remains to be investigated. Here we review the evidences of how bone marrow stroma cells and malignant B cells reciprocally interact. Evidently from published data, these cell–cell interactions induce profound changes in signalling, gene expression and metabolic adaptations. While the past research has largely focussed on understanding changes imposed by stroma- on tumour cells, it is now clear that tumour-cell contact also has fundamental ramifications for the biology of stroma cells. Their careful characterisations are not only interesting from a scientific biological viewpoint but also relevant to clinical practice: Since tumour cells heavily depend on stroma cells for cell survival, proliferation and dissemination, interference with bone marrow stroma–tumour interactions bear therapeutic potential. The molecular characterisation of tumour–stroma interactions can identify new vulnerabilities, which could be therapeutically exploited.

Role of Vitamins A and D in BCR-ABL Arf\u2212/\u2212 Acute Lymphoblastic Leukemia

The effects of vitamin A and/or vitamin D deficiency were studied in an Arf−/− BCR-ABL acute lymphoblastic leukemia murine model. Vitamin D sufficient mice died earlier (p = 0.003) compared to vitamin D deficient (VDD) mice. Vitamin A deficient (VAD) mice fared worst with more rapid disease progression and decreased survival. Mice deficient for vitamins A and D (VADD) had disease progression similar to VAD mice. Regulatory T cells, previously shown to associate with poor BCR-ABL leukemia control, were present at higher frequencies among CD4+ splenocytes of vitamin A deficient vs. sufficient mice. In vitro studies demonstrated 1,25-dihydroxyvitamin D (1,25(OH)2VD3) increased the number of BCR-ABL ALL cells only when co-cultured with bone marrow stroma. 1,25(OH)2VD3 induced CXCL12 expression in vivo and in vitro in stromal cells and CXCL12 increased stromal migration and the number of BCR-ABL blasts. Vitamin D plus leukemia reprogrammed the marrow increasing production of collagens, potentially trapping ALL blasts. Vitamin A (all trans retinoic acid, ATRA) treated leukemic cells had increased apoptosis, decreased cells in S-phase, and increased cells in G0/G1. ATRA signaled through the retinoid X receptor to decrease BCR-ABL leukemic cell viability. In conclusion, vitamin A and D deficiencies have opposing effects on mouse survival from BCR-ABL ALL.

Mobilization-based transplantation of young-donor hematopoietic stem cells extends lifespan in mice.

Mammalian aging is associated with reduced tissue regeneration and loss of physiological integrity. With age, stem cells diminish in their ability to regenerate adult tissues, likely contributing to age‐related morbidity. Thus, we replaced aged hematopoietic stem cells (HSCs) with young‐donor HSCs using a novel mobilization‐enabled hematopoietic stem cell transplantation (HSCT) technology as an alternative to the highly toxic conditioning regimens used in conventional HSCT. Using this approach, we are the first to report an increase in median lifespan (12%) and a decrease in overall mortality hazard (HR: 0.42, CI: 0.273–0.638) in aged mice following transplantation of young‐donor HSCs. The increase in longevity was accompanied by reductions of frailty measures and increases in food intake and body weight of aged recipients. Young‐donor HSCs not only preserved youthful function within the aged bone marrow stroma, but also at least partially ameliorated dysfunctional hematopoietic phenotypes of aged recipients. This compelling evidence that mammalian health and lifespan can be extended through stem cell therapy adds a new category to the very limited list of successful anti‐aging/life‐extending interventions. Our findings have implications for further development of stem cell therapies for increasing health and lifespan.

Aldehyde Dehydrogenase as a Marker of Early Mesenchymal Progenitor Cells in Donor Bone Marrow Stroma

Aim. To analyze the growth rate of mesenchymal stromal cell (MSC) culture depending on the aldehyde dehydrogenase-positive (ALDH+) cell count. Materials &amp; Methods. The study involved bone marrow mesenchymal cell cultures of 10 donors (5 men and 5 women) with median age of 34.5 years (range 14-38 years). Nucleated cells were obtained by density gradient centrifugation. MSCs were cultivated according to the conventional protocol using platelet-rich donor plasma. Stromal cell identification and ALDH+ cell counting were performed by laser flow cytometry according to the criteria of the International Society for Cell Therapy. Results. The growth rate of MSC cultures and ALDH+ cell counts are maximum at primary and passage No. 1, becoming significantly lower by passage No. 3. The relationship between MSC culture growth rate and ALDH+ cell count was revealed. The older the donor, the lower MSC culture growth rate and ALDH+ cell count in bone marrow stroma. Conclusion. The data obtained indicate the relationship between bone marrow MSC culture growth rate, donor’s age, and ALDH+ cell count. ALDH-expressing cells proved to confer MSC population renewal. Based on the results acquired, we assume that the studied ALDH marker can serve as an objective criterion for placing mesenchymal cell elements into the category of early progenitor cells.

The Secretome of Hypoxia Conditioned hMSC Loaded in a Central Depot Induces Chemotaxis and Angiogenesis in a Biomimetic Mineralized Collagen Bone Replacement Material.

The development of biomaterials with intrinsic potential to stimulate endogenous tissue regeneration at the site of injury is a main demand on future implants in regenerative medicine. For critical-sized bone defects, an in situ tissue engineering concept is devised based on biomimetic mineralized collagen scaffolds. These scaffolds are functionalized with a central depot loaded with a signaling factor cocktail, obtained from secretome of hypoxia-conditioned human mesenchymal stem cells (MSC). Therefore, hypoxia-conditioned medium (HCM)-production is standardized and adapted to achieve high signaling factor-yields; a concentration protocol based on dialysis and freeze-drying is established to enable the integration of sufficient and defined amounts into the depot. In humid milieu-as after implantation-signaling factors are released by forming a chemotactic gradient, inducing a directed migration of human bone marrow stroma cells (hBMSC) into the scaffold. Angiogenic potential, determined by coculturing human umbilical vein endothelial cells (HUVEC) with osteogenically induced hBMSC shows prevascular structures, which sprout throughout the interconnected pores in a HCM-concentration-dependent manner. Retarded release by alginate-based (1 vol%) depots, significantly improves sprouting-depth and morphology of tubular structures. With the intrinsic potential to supply attracted cells with oxygen and nutrients, this bioactive material system has great potential for clinical translation.

Phase I Study of Cord Blood Transplantation with Intra-Bone Marrow Injection of Mesenchymal Stem Cells

Phase I Study of Cord Blood Transplantation with Intra-Bone Marrow Injection of Mesenchymal Stem Cells

Proteasome Inhibitors Impair the Innate Antiviral Immune Response and Potentiate Pelareorep-Based Viral Therapy in Multiple Myeloma

INTRODUCTION: Pelareorep is the infusible form of human reovirus (RV). Our single-agent phase 1 RV trial in relapsed multiple myeloma (MM) showed that pelareorep treatment selectively infected MM cells, as viral RNA was found in myeloma cells and not the bone marrow (BM) stroma. However, we did not observe apoptosis. Our ongoing phase 1 trial, which combines the proteasome inhibitor carfilzomib with RV, has demonstrated RV infection, apoptosis, and clinical responses. We investigated the molecular mechanisms behind the role of a PI (carfilzomib) in this setting. RESULTS: In all MM cell lines we tested (n=4), independently from their sensitivity to RV infection (Stiff et al., Mol Cancer Ther, 2016), viral replication and apoptosis was impaired when MM cells were directly exposed to PIs (carfilzomib and bortezomib). When this experiment was repeated in the setting of the bone marrow cellular fraction or peripheral blood mononuclear cells (PBMCs), it had the opposite effect, as the addition of PI to RV increased RV replication and apoptosis in MM cells. When we washed PBMCs after overnight exposure to RV+PI or to either single agent, then added MM cells, we observed higher infection and apoptosis in cancer cells co-cultured with RV+PI compared to levels from PBMCs treated with each of the single agents, suggesting that PIs increase the ability of PBMCs to serve as a reservoir for infectious reovirus. Monocytes (CD14+) can engage in phagocytosis of reovirus (Berkeley et al., Cancer Immunol Res, 2018), and accordingly we found that RV genome and capsid protein production were detected in CD14+ cells, but not in CD14-depleted PBMCs, and increased upon PI treatment compared to that in RV-treated CD14+ cells. Given that the NF-κB complex is a key proinflammatory signaling pathway associated with the early innate-antiviral immune response, and because PIs can block the degradation of the NF-κB inhibitor IκBα upon phosphorylation, we investigated the effect of the specific IκBα inhibitor Bay-11 in RV viral replication. Our data show that either PI or Bay-11 can inhibit RV-induced IκBα phosphorylation and its subsequent degradation upon RV infection in CD14+ cells, an effect associated with higher capsid formation in RV-treated CD14+ cells in combination with PI or Bay-11, compared to levels from RV alone. Cytokine profiling in PBMCs and CD14+ cells treated with RV in combination with either PI or Bay-11 showed a significant decrease in IFN-α and IFN-β (IFNs) levels and a concomitant increase in RV replication, in contrast to levels from RV alone (p&lt;0.001). We then decided to investigate whether CD14 depletion could affect RV delivery to the cancer cells invivo. Upon intra-femoral injection of 5TGM1 MM cells into syngeneic C57BL/KaLwRij mice, the mice in which the monocytes were depleted by clodronate-liposome treatment before intravenous RV injection showed lower capsid protein formation in the BM MM cells compared to that in mice where the monocytic population was intact. Because monocytes respond to infection by dividing into macrophages to eliminate pathogens, we wanted to investigate whether PI could impair this effect. Intriguingly, although higher levels of viral genome were detected in PI+RV-treated CD14+ cells compared to RV-treated cells (p&lt;0.01), PI abolished ex-vivo RV-induced monocyte differentiation (n=3, p&lt;0.001), without affecting the ability of RV-infected CD14+ cells to induce higher MM cell killing as compared to that from the CD14+ fraction treated with either single agent. Immune killing assays showed that RV-infected MM cells are more susceptible than non-infected cells to T cell effectors (p&lt;0.001), a mechanism that is further potentiated by the addition of PI. In fact, in contrast to the PI-induced blocking of monocyte expansion and activation in response to the virus (n=3, p&lt;0.001), our data suggest that the PI potentiate the expansion of CD8+ T cells, both ex vivo and in two RV+PI treated patients we analyzed so far. CONCLUSIONS: Here we report for the first time that PIs enhance pelareorep entry, infection, and killing of myeloma cells through its effect on the CD14+ fraction. Reovirus infection and replication within CD14+ cells are augmented by PI-induced NF-κB inhibition of the early innate pro-inflammatory immune response. We also report for the first time that carfilzomib induces direct T cell activation and potentiates T cell killing activity against RV-infected MM cells. Disclosures Krishnan: Takeda: Research Funding; Celgene, Z Predicta: Other: Stock Ownership; Amgen, Takeda: Speakers Bureau; Sutro BioPharma, zPredicta: Consultancy; Celgene, Janssen, Sanofi, BMS: Consultancy. Sborov:Celgene: Honoraria; Janssen: Consultancy. Hofmeister:Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees.

Mechanistic Characterization of Tafasitamab-Mediated Antibody-Dependent Cellular Phagocytosis Alone or in Combination with Lenalidomide

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. Approximately 60% of DLBCL patients can be cured using standard chemotherapy, including an anti-CD20 antibody (R-CHOP; rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). However, 40% of DLBCL patients have refractory disease or experience relapse. This highlights the need for more effective therapies for this subset of relapsing or refractory patients. Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal anti-CD19 antibody, currently in clinical development in patients with relapsed or refractory (r/r) DLBCL in combination with either the immunomodulatory drug lenalidomide (LEN) (L-MIND study) or the chemotherapeutic agent bendamustine (B-MIND study). The B-lymphocyte antigen CD19 is broadly and homogeneously expressed throughout B-cell development and across different B-cell malignancies, including DLBCL. CD19 has been reported to enhance B-cell receptor signaling, which is assumed important for B-cell survival. This makes CD19 a promising therapeutic target in B-cell malignancies. Macrophages are abundant in the bone marrow stroma and lymph nodes of lymphoma patients. These lymphoma-associated macrophages (LAMs) support proliferation, survival, and drug resistance of lymphoma cells; therefore, serving a tumor-promoting function (Farinha et al, Blood, 2005). However, macrophages can also act as immune effector cells for antitumor effects exerted by therapeutic monoclonal antibodies, like tafasitamab. Given their abundance in DLBCL, an attractive therapeutic approach would be to stimulate their tumoricidal activity in order to promote antitumor immunity. Consequently, the immunomodulatory agent LEN has the potential to synergize with tafasitamab by enhancing antibody dependent cellular phagocytosis (ADCP). Here, we demonstrate that monocyte-derived macrophages and isolated LAMs from DLBCL patients can kill lymphoma cell lines and autologous lymphoma cells by tafasitamab-mediated phagocytosis. Tafasitamab-mediated ADCP activity was correlated with FcγRIII/FcγRI levels and inversely correlated with SIRPα expression on macrophage/effector cells (Spearman r between 0.51 and 0.62). Analyzing immune checkpoint receptor expression on LAMs of DLBCL patients, we found increased expression of VISTA, SLAMF7 and SIRPα compared with normal macrophages from healthy volunteers (VISTA: 2.8-fold, p=0.0006; SLAMF7: 2.9-fold, p=0.03; SIRPα: 1.8-fold, p=0.04). In contrast, we found no significant difference in FcγR expression (CD16: 1.4-fold, p=0.12; CD32: 1.3-fold, p=0.32; CD64: 1.2-fold, p=0.12). Moreover, pretreatment with lenalidomide showed a 3 to 5-fold enhanced tafasitamab-mediated cytotoxicity by macrophages and isolated LAMs (Figure 1A). Of note, the increased cytotoxicity was tafasitamab-specific and neither attributable to direct cytotoxic effects of LEN, nor increased CD19 expression on target cells. In addition, LEN treatment decreased the expression of SIRPα on macrophages (control vs. LEN: 3,117±652 MFI [mean fluorescent intensity] vs 2,327±610 MFI) (Figure 1B). This suggests that the CD47-SIRPα pathway may be suppressed by downregulation of SIRPα by LEN, contributing to the observed ADCP enhancement. These results suggest that tafasitamab leverages the presence of tumor-promoting LAMs as effector cells. The inherent ADCP activity of tafasitamab can be further improved by the immunomodulatory agent LEN, potentially via reduction of SIRPα levels on macrophages. Finally, our results provide a rationale for targeting the CD47-SIRPα axis that should be further explored in preclinical and clinical studies. Figure 1 Disclosures Endell: MorphoSys AG: Employment, Patents &amp; Royalties. Boxhammer:MorphoSys AG: Employment, Patents &amp; Royalties. Bruns:Morphosys AG: Research Funding.

A Novel Combination Regimen of BET and FLT3 Inhibition for FLT3-ITD Acute Myeloid Leukemia

Background: Acute myeloid leukemia patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors such as quizartinib and gilteritinib improve overall survival in relapsed patients, but their efficacy is limited and most such patients die of the disease. This is because even with potent FLT3 inhibition, the disease persists within the bone marrow microenvironment, mainly due to bone marrow stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as c-Myc and Bcl2, but these drugs thus far have shown only limited single-agent clinical potential. PLX51107 is a novel BET inhibitor designed to inhibit BET activity in intermittent daily fashion to allow for greater tolerability. We investigated whether the addition of PLX51107 to potent FLT3 inhibition with quizartinib could overcome the protective effect of the bone marrow stroma through inhibition of c-Myc expression. Methods: We developed a plasma inhibitory activity assay for c-Myc (c-Myc PIA) to assess in vivo efficacy of the PLX51107 in patients and to identify c-Myc-inhibitory doses of the drug. We tested PLX51107 alone and in combination with quizartinib in murine models of AML and against primary FLT3-ITD AML cells co-cultured with human bone marrow stroma. In addition, we analyzed gene expression patterns in the treatment models to explore the basis of any observed synergistic cytotoxic effect. Results: In a murine xenograft model of AML using MV4-11 cells, PLX51107 alone induced suppression of tumor growth in association with a 90% decrease in c-Myc gene expression. The combination of PLX51107 and quizartinib induced complete tumor regression in 5 out of 7 animals after 14 days of treatment (Figure 1). Animals treated with a 5-day course of quizartinib alone displayed tumor regression persisting until day 26 of treatment, while the addition of PLX51107 resulted in tumor regression until day 39. In patients treated with a dose of 120 mg/day of PLX51107, the c-Myc PIA demonstrated a robust suppression of c-Myc expression for roughly 6 hours, returning to baseline between 7 and 9 hours post-treatment (Figure 2). The mean plasma concentration to achieve this inhibition was 3.3 uM, which, accounting for the difference in protein drug binding between plasma and culture medium with 10% FBS, corresponded to a concentration of 250 nM PLX51107 in culture medium. With this same concentration and schedule (i.e., at concentrations and exposure times equivalent to what human patients would experience taking 120 mg daily of PLX51107 and 60 mg daily of quizartinib), the combination induced synergistic cytotoxicity in a series of 10 different FLT3-ITD AML patient blast samples co-cultured with bone marrow stroma (Figure 3). C-Myc RNA and protein were directly suppressed in these primary samples, and ingenuity pathway analysis of RNA expression confirmed that c-Myc associated genes displayed the highest level of down-regulation. Conclusions: These studies suggest that combination therapy with approximately 120 mg/day PLX51107 and 60 mg/day quizartinib will be a more effective therapy for relapsed FLT3-ITD AML than 60 mg/day of quizartinib alone. The combination of FLT3 inhibition and BET inhibition may represent an attractive therapeutic option for FLT3-ITD AML. Disclosures Hizukuri: Daiichi Sankyo Co, Ltd: Employment. Severson:Plexxikon Inc.: Employment. Powell:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Ma:Plexxikon Inc: Employment. Narahara:Daiichi Sankyo Co, Ltd: Employment. Sumi:Daiichi Sankyo, Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levis:FUJIFILM: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Menarini: Consultancy, Honoraria.

Microenvironmental Triggers Induce a Chemoresistant, Differentiated Subset of S100A8/A9high AML Cells Via the Jak/STAT3 Signaling Axis

Introduction: The bone marrow stromal niche can serve as a protective environment in hematological malignancies such as AML. Intensive research of the bidirectional interactions between leukemic blasts and stromal cells already highlighted numerous mechanisms how malignant cells are capable of shaping their local milieu in an opportunistic fashion. However, the complex mechanisms remain to be fully elucidated. Previously, we found two small intracellular calcium-sensing molecules, S100A8 and S100A9, among the top upregulated genes in primary AML blasts upon stromal contact in an unbiased analysis. They are members of the S100 protein family that can modulate cellular responses such as proliferation, migration, inflammation, and differentiation. Dysregulation of S100 protein expression is described as a common feature in several human cancers. Particularly, expression of S100A8 in leukemic blasts predicts poor survival in de novo AML patients. Thus, we aimed to decipher the underlying pathways of the stroma-mediated S100A8/A9 upregulation as well as its functional consequences. Methods: We cultured AML cell lines in absence or presence of bone marrow stroma cell-conditioned medium. Subsequent, AML cells were analyzed for alterations in intracellular signaling pathways and metabolic characteristics by means of flow cytometry, fluorescence microscopy and quantitative real-time PCR. Additionally, AML cells were analyzed for their resistance towards chemotherapy under the very same culture conditions. Moreover, matched pairs of patient-derived AML samples from the peripheral blood and the bone marrow were compared for key findings from the in vitro analyses. Results: The previously seen upregulation of S100A8 and S100A9 in primary AML blasts upon stromal contact could be confirmed in AML cell lines both on the mRNA and the protein level and proved to be contact-independent and reversible. Interleukin 6-mediated triggering of the Jak/STAT3 signaling was shown to be one major pathway of this induction. The S100A8/A9high subpopulation is characterized by profound metabolic alterations, and by increased surface levels of maturation markers (i.e. CD11b and CD14) both conferring chemoresistance. Strikingly, both differentiation and chemoresistance could be reversed by inhibition of Jak/STAT signaling or metabolic interference. Ultimately, elevated frequencies of S100A8/A9high AML blasts with increased surface levels of maturation markers could be detected in bone marrow-derived specimen compared to matched samples from the peripheral blood in the same patient. Conclusion: We could demonstrate that bone marrow stroma-induced S100A8/A9high AML cells show increased maturation and chemoresistance which both can be targeted by metabolic or oncogenic signaling inhibition representing additional possible targets for further pre-clinical exploration. Disclosures Bruns: Morphosys AG: Research Funding.

Pre-Clinical Efficacy of the Novel Kinase Inhibitor Nintedanib on PAX5 Fusion Genes in Pediatric Ph-like B-Cell Precursor Acute Lymphoblastic Leukemia

Despite the current risk-based stratification protocol, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) still experience relapse. In the large subset of 'B-other' patients (negative for common fusion transcripts, non-high hyperdiploid and non-Down Syndrome), 'Ph-like' is a high risk subgroup with high incidence of relapses, which represents 30% of B-others or 10-15% of BCP-ALL patients. The PAX5 gene, encoding for a B-cell related transcription factor, is frequently involved in several translocations in Ph-like patients, determining the formation of fusion genes encoding for aberrant proteins. Our previous studies showed that PAX5 fusions sustain survival of leukemic cells by Lymphocyte kinase (LCK) hyperactivation, which can be targeted by the kinase inhibitor Nintedanib/BIBF1120. This study aims (i) to unravel the functional features of PAX5 fusion genes, elucidating the involved signaling pathways; (ii) to develop new pre-clinical strategies to target PAX5 fusion genes, testing the efficacy of the LCK inhibitor BIBF1120. We selected a cohort of 138 B-other cases among 400 childhood BCP-ALL cases enrolled in Italy in AIEOP-BFM ALL2000/R2006 protocols; by gene expression analysis (HG-U133Plus2.0 Affymetrix array), 59/138 presented as Ph-like, and by NGS, a driver fusion gene was identified in 34/59 (58%). Beside known genetic lesions, such as P2RY8/CLRF2 (N=9), EBF1 fusions (N=3), TCF3/HLF (N=1), IKZF1-fusion (N=1) and BCL9/MEF2D (N=1), and single cases with novel fusions, seven cases were carrying a PAX5 fusion gene, representing the most frequent rearrangement. Ex-vivo inhibition with BIBF1120 was setup on primary cells from 5 PAX5-fusion BCP-ALL samples, demonstrating significant efficacy in inducing leukemic cell apoptosis, both as single agent and in combination with standard chemotherapeutic agents (Annexin V viability assay of leukemic cells in co-culture on human bone marrow stroma). Strikingly, in 3/5 cases dexamethasone and BIBF1120 had a synergistic effect and were further tested in in vivo assays. A daily treatment was performed at bulk disease detection (BM aspiration, mean engraftment 20% CD10/CD19+) in patient-derived xenotransplanted NSG mice from 2 different PAX5 fusion cases. At the endpoint (after two weeks), in the PAX5/AUTS2 PDX mice we detected a mild effect in the BM by BIBF1120 alone (disease reduction 24%, p=0.057), further enhanced in combination with dexamethasone (-49%, p=0.005, with a mean engraftment in vehicle mice of 82.6%). In the spleen, the efficacy was highly significant both for BIBF1120 (-52%, p=0.025) and the combination (-91%, p=0.015, mean engraftment vehicle 69.5%). A similar statistical significant effect was observed also in peripheral blood, whilst, BIBF1120 alone showed a specific significant efficacy in CNS meninges. Analogous results have been observed in PDX from the PAX5/DACH2 PDX mice; BM leukemia decreased 47% using BIBF1120 alone (p=0.004), further diminished by the combination (-65%, p=0.0004, with a mean engraftment in vehicle mice of 65%). In the spleen, the efficacy was highly significant both with BIBF1120 (-45.6%, p=0.04) and for the combination (-96.3%, p=0.0008, mean engraftment vehicle 72.4%). Strikingly, BIBF1120 treatment alone showed analogous efficacy in PB and CNS, with leukemia decrease as low as -45% (p=0.04) and -76% (p=0.007), respectively, and the combination nearly achieved remission in PB (-94%, p=0.0001) and it was significant in CNS (-81.2%, p=0.03). Dexamethasone alone was not effective in the BM and spleen, whereas it decreased the leukemia bulk both in PB (-65%, p=0.0004) and CNS (-52.8%, p=0.03). Overall, BIBF1120 treatment was more effective than dexamethasone. Interestingly, phosphoflow analysis showed a marked inhibition by BIBF1120 of pAkt-Thr308 and its downstream effectors, such as pS6 and 4pEBP1, in ex vivo BM and spleen cells. In conclusions, PAX5 fusion genes are highly recurrent among Ph-like patients and can potentially be targeted by Nintendanib/BIBF1120, that showed a significant effect ex vivo and in vivo, even as a single agent. Disclosures No relevant conflicts of interest to declare.

Targeting cyclooxygenase by indomethacin decelerates progression of acute lymphoblastic leukemia in a xenograft model

AbstractAcute lymphoblastic leukemia (ALL) develops in the bone marrow in the vicinity of stromal cells known to promote tumor development and treatment resistance. We previously showed that the cyclooxygenase (COX) inhibitor indomethacin prevents the ability of stromal cells to diminish p53-mediated killing of cocultured ALL cells in vitro, possibly by blocking the production of prostaglandin E2 (PGE2). Here, we propose that PGE2 released by bone marrow stromal cells might be a target for improved treatment of pediatric ALL. We used a xenograft model of human primary ALL cells in nonobese diabetic-scid IL2rγnull mice to show that indomethacin delivered in the drinking water delayed the progression of ALL in vivo. The progression was monitored by noninvasive in vivo imaging of the engrafted leukemic cells, as well as by analyses of CD19+CD10+ leukemic blasts present in spleen or bone marrow at the termination of the experiments. The indomethacin treatment increased the level of p53 in the leukemic cells, implying that COX inhibition might reduce progression of ALL by attenuating protective paracrine PGE2 signaling from bone marrow stroma to leukemic cells.

Extracellular Ca2+ in Bone Marrow.

Our blood serum Ca2+ levels are maintained within a narrow range (Ca2+ homeostasis) through a complex feedback system. However, local bone marrow Ca2+ levels can reach high concentrations, at least transiently, due to bone resorption, which is one of the notable features of the bone marrow stroma. Bone homeostasis is maintained by both the balance between osteoblastic bone formation and osteoclastic bone resorption and the balance of mesenchymal stem cell differentiation into osteoblasts and adipocytes. It has been reported that under culture conditions of infrequent adipocyte differentiation (no treatment with insulin or dexamethasone), high extracellular Ca2+ enhances osteoblast but not adipocyte accumulation in bone marrow stromal cells. In contrast, under culture conditions of predominant adipocyte differentiation (treatment with insulin and dexamethasone), high extracellular Ca2+ enhances adipocyte but not osteoblast accumulation in bone marrow stromal cells. Thus, the increased extracellular Ca2+ caused by bone resorption might enhance osteoblast development to reform missing bone under conditions of infrequent adipocyte differentiation (such as the normal physiological state) and might accelerate adipocyte accumulation instead of osteoblastic bone formation under conditions of predominant adipocyte differentiation (such as aging, obesity, use of glucocorticoids, and postmenopause). Moreover, increased adipocyte accumulation in bone marrow suppresses lymphohematopoiesis and contributes to a dysfunction of osteogenesis.