Abstract: Multipotent mesenchymal stromal/stem cells (MSC) have shown potential clinical utility. However, previous assessments of MSC behavior in recipients have relied on visual detection in host tissue following sacrifice, failing to monitor in vivo MSC dispersion in a single animal and limiting the number of variables that can be observed concurrently. In this study, we used noninvasive, in vivo bioluminescent imaging to determine conditions under which MSC selectively engraft in sites of inflammation. MSC modified to express firefly luciferase (ffLuc-MSC) were injected into healthy mice or mice bearing inflammatory insults, and MSC localization was followed with bioluminescent imaging. The inflammatory insults investigated included cutaneous needle-stick and surgical incision wounds, as well as xenogeneic and syngeneic tumors. We also compared tumor models in which MSC were i.v. or i.p. delivered. Our results demonstrate that ffLuc-expressing human MSC (hMSC) systemically delivered to nontumor-bearing animals initially reside in the lungs, then egress to the liver and spleen, and decrease in signal over time. However, hMSC in wounded mice engraft and remain detectable only at injured sites. Similarly, in syngeneic and xenogeneic breast carcinoma-bearing mice, bioluminescent detection of systemically delivered MSC revealed persistent, specific colocalization with sites of tumor development. This pattern of tropism was also observed in an ovarian tumor model in which MSC were i.p. injected. In this study, we identified conditions under which MSC tropism and selective engraftment in sites of inflammation can be monitored by bioluminescent imaging over time. Importantly, these consistent findings were independent of tumor type, immunocompetence, and route of MSC delivery.

Abstract: Acute myeloid leukemia (AML) is initiated and maintained by a relatively rare leukemia stem cells (LSCs) capable of self-renewal and proliferation. Recent data showed that LSCs (Lagadinou et al. Cell Stem Cell 2013) and residual cytarabine (Ara-C)-resistant AML cells (representing minimal residual disease, MRD) (Farge et al. Cancer Discovery 2017) are highly dependent on mitochondrial function for survival. This unique metabolic biology makes chemoresistant LSCs and AML cells vulnerable to pharmacological blockade of the oxidative phosphorylation (OXPHOS). We have reported that a novel OXPHOS inhibitor IACS-010759 potently inhibits mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells in vitro and in vivo (Molina et al. Nat Med 2018). In this study, we aimed to determine the effects of OXPHOS inhibition with IACS-010759 on residual AML cells surviving standard chemotherapy (Doxorubicin/Ara-C, DA) in cell line and patient-derived xenograft (PDX) AML models. Consistent with our hypothesis, OCI-AML3 cells treated with DA in vitro induced elevated levels of reactive oxygen species, higher mitochondrial mass and membrane potential (Fig. 1A), indicating reliance on the mitochondrial metabolism. Further, Ara-C treatment resulted in significantly increased basal and maximal oxygen consumption rates (OCR) (36%±8%, p=0.03; 36%±3%, p=0.003, respectively) compared to control. In turn, targeting OXPHOS with IACS-010759 at 30 nM fully inhibited basal and Ara-C-induced OCR. These findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor. To test the efficacy of combining IACS-010759 (5 mg/kg) and standard chemotherapy (Doxorubicin: 1.5 mg/kg; Ara-C: 50 mg/kg) in vivo, we injected NRG mice with genetically engineered OCI-AML3/Luc/GFP cells. Bioluminescent imaging demonstrated significantly reduced leukemia burden in DA/IACS-010759 combination group compared to vehicle on days 15 and 42 (p 〈 0.01) (Fig. 1B). DA/IACS-010759 combination significantly extended survival, compared to the vehicle or single-agent treatment arms (Fig. 1C). Mouse body weight monitoring indicated that therapy was well tolerated We next examined the efficacy of IACS-010759 on leukemia cells surviving chemotherapy in a chemosensitive PDX AML model of minimal residual disease (Fig. 1D). Treatment of mice inoculated with a human AML PDX harboring FLT3-ITD mutation with DA reduced circulating leukemia burden (0.8 ± 0.6% vs 45.8 ± 8.2% blasts in vehicle-treated mice, p=0.001). The residual AML cells in DA-treated mice expanded and caused rapidly progressive leukemia (78.2 ± 6.2% vs 95.3 ± 1.0% in vehicle-treated mice, p=0.047) on week 6 post DA. Daily oral treatment of mice with IACS-010759 (7.5 mg/kg) as a single agent reduced leukemia burden, and delayed leukemia recurrence when administered post completion of DA (Fig. 1E). A SPADE tree was built based on 13 surface markers and colored by expression intensity of CD34 using CyTOF mass cytometry data (Fig. 1F). The data demonstrated reduced frequency of CD34+CD38lowCD123+ AML LSCs and increase in CD11c+ differentiated cells in both IACS and IACS/DA groups (Fig. 1G & H). In contrast, chemotherapy alone failed to significantly reduce fractions of LSCs or induce differentiation. Proliferation measured by Ki67 was greatly reduced by IACS/DA combination in all populations including LSCs (1.4 ± 0.3% vs 5.5 ± 0.4% in vehicle group, p 〈 0.01). The expression of Hypoxia-Inducible Factor 1α (HIF-1α) was downregulated, consistent with the decreased oxygen consumption induced by IACS-010759 (not shown). In conclusion, minimal residual AML cells surviving chemotherapy depend on OXPHOS for survival. OXPHOS inhibition with complex I inhibitor IACS-010759 is effective in reducing LSCs and MRD, alone and in combination with chemotherapy in vivo. Our data advocate for combining IACS-010759 with chemotherapy for improved control of MRD upon identification of a recommended Phase II dose in a clinical trial of IACS-010759 in AML (NCT02882321). Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Kantarjian:Pfizer: Honoraria, Research Funding; Cyclacel: Research Funding; AbbVie: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Ariad: Research Funding; Novartis: Research Funding; Agios: Honoraria, Research Funding; BMS: Research Funding; Astex: Research Funding; Amgen: Honoraria, Research Funding; Jazz Pharma: Research Funding. Daver:Jazz: Consultancy; Hanmi Pharm Co., Ltd.: Research Funding; Agios: Consultancy; Immunogen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; Karyopharm: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy; Servier: Research Funding; NOHLA: Research Funding; Glycomimetics: Research Funding; Otsuka: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy. Konopleva:Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

Abstract: Abstract 2391 Connective tissue growth factor (CTGF) regulates extracellular matrix production, chemotaxis, cell proliferation and integrin expression. Recent reports suggest that recombinant CTGF transforms mesenchymal stromal cells (MSCs) into fibroblast like cells and inhibits their differentiation potential. We have recently shown that stable knockdown of CTGF expression in bone marrow derived MSCs rendered them quiescent and increased their adipocyte differentiation potential (Battula et al., ASH 2010 abstract 3845, Blood, Vol. 116). Based on these findings, we hypothesized that inhibition of CTGF expression modifies stem cell properties of MSCs and may affect leukemic cell homing. To test this hypothesis, bone marrow derived MSCs were transduced with lentivirus expressing either CTGFshRNA or control vector. Down-regulation of CTGF mRNA by ∼75% was observed in CTGF-KD-MSCs compared to empty vector control-MSCs. To examine the stem cell potential of CTGF-KD-MSCs, embryonic stem cell markers including oct-4, nanog, sox2 were analyzed. Real-time RT-PCR analysis revealed 2–3 fold up regulation of these genes in CTGF-KD-MSCs compared to controls indicating increased stemness. To test if leukemic cells home differentially to CTGF-KD-MSCs, we utilized our recently developed extra-medullary human bone marrow model by implanting a carrier impregnated with BM-MSC mixed with endothelial progenitor cells (EPCs) subcutaneously into NOG mice. These implants generate extra-medullary bone marrow (EXM-BM) in 6–8 weeks and resemble the normal bone marrow microenvironment. In-vivo imaging confirmed that both cell types formed EXM-BM with positive Osteosense binding which reflects the presence of hydroxyapatite. H & E staining of bone sections revealed that CTGF-KD-MSCs derived EXM-BM displayed more spongy bone compared to control-MSCs. To examine the extent of adipocytic differentiation in CTGF-KD-MSCs derived bone pellets, expression of adipocyte-specific markers PPAR-γ and C/EBPα was analyzed. Cells residing in the spongy EXM-BM region expressed high levels of nuclear PPAR-γ and C/EBPα, confirming differentiation into mature adipocytes. These markers were negative in the stromal/endothelial compartment of CTGF-KD-MSCs derived EXM-BM or control EXM-BM. When Nalm-6 (B-ALL cell line) cells expressing firefly luciferase and YFP were transplanted into these mice, leukemic cells migrated 10 fold more avidly towards CTGF-KD-MSCs derived EXM-BM compared to control EXM-BM in the same mouse (n=5). Immunohisto-chemical analysis demonstrated preferential homing of leukemic cells inside spongy regions of the EXM-BM derived from CTGF-KD-MSCs and control-MSCs. To investigate the mechanism behind the leukemic cell homing into CTGF-KD-MSCs derived EXM-BM, expression of stromal cell-derived factor-1α (SDF1α), a major factor involved in leukemic cell homing to bone marrow, was analyzed in both cell types by qRT-PCR. CTGF-KD-MSCs expressed 3-fold higher levels of SDF-1α compared to control cells indicating that SDF1α secretion by CTGF-KD-MSCs derived bone marrow might enhance the leukemia cell homing. In summary, these findings suggest that CTGF plays a crucial role in the regulation of stemness and differentiation of MSCs and affects the homing of leukemic cells to the bone marrow microenvironment. Targeting adipocyte progenitors may reduce the lodging of leukemic cells in the bone marrow niche and enhance chemosensitivity of leukemic cells within the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

Abstract: The bone marrow microenvironment (BME) critically supports hematopoietic stem cells and protects leukemia cells from chemotherapy, immune surveillance, and related stresses. A critical component of the BME is the mesenchymal stem cell (MSC). Dan Link’s group demonstrated that MSC are essential for human hematopoiesis, particularly as a source of SDF-1, which regulates homing, proliferation, and differentiation of HSC. Moreover, studies from our group and others have demonstrated that MSC protect leukemia cells from chemotherapy. At present, very little is known about MSC derived from AML patients, and an understanding of the proteomic makeup of these cells in the leukemia microenvironment could help to elucidate mechanisms involved in supporting their pro-tumor function. We used reverse phase protein array analysis (RPPA) to compare the expression of 151 proteins in MSC derived from AML BMs (N = 106) with those from healthy donors (N = 71). The expression of 45 of these proteins was deemed significantly different (p 〈 0.01) between the two sets. AML MSC expressed higher levels of p53 and p21 (CDKN1A), and the expression of the latter was correlated with other proteins within each MSC set. Using beta-galactosidase staining, AML MSC were found to undergo senescence more frequently than normal MSC. Elevated p21 in AML MSC is consistent with this finding. While 15 proteins were positively, and 20 proteins negatively, correlated with p21 expression in normal MSC, there were only three proteins positively, and nine negatively, correlated in AML-derived MSC. In normal MSC, SMAD1 (a key component in MSC growth and differentiation involving multiple receptors like TGF beta and BMP) expression and AKT signaling were low when p21 is expressed. However, in AML MSC this association was not seen, albeit a negative correlation with ITGAL was observed. SMAD1 expression was higher in normal MSC. In normal MSC, the expression of SMAD1 was negatively correlated with PPARG and NPM1, and was positively correlated with the expression of phosphorylated ELK. The opposite relationship was seen in AML MSC (i.e., PPARG and NPM1 exhibited positive correlation with SMAD1 and phosphorylated ELK was negatively correlated with the protein). While the significance of these relationships remains to be determined it is interesting to note that PPARG is a key regulator of adipocyte differentiation in MSC, so perhaps this alteration of SMAD/PPARG in AML MSC could impede their differentiation potential. In an accompanying abstract from our group, we report that AML MSC are primed toward osteoblastic differentiation and do not differentiate into adipocytes (Battula VL et al, ASH 2014). The RPPA data on PPARG is consistent with this finding. SMAD1 also positively regulates miR-21. Since p21 is a miR-21 target, it seems possible that the differences in expression could be attributed to SMAD1 and miR-21 signaling. We analyzed miR-21 expression in normal and AML-derived MSC (N = 10, each) using qRT-PCR and found a statistically significant (p =0.014) increase in its expression in normal MSC relative to their disease counterparts. When anti-miR-21 was transduced into healthy donor MSC, which caused a 3-fold increase in p21 (but no difference in cyclin D1 expression, another miR-21 target whose expression was also increased in AML MSC). AML MSC also exhibited higher protein expression of the B55 alpha subunit (PPP2R2A) of protein phosphatase 2A (PP2A). This expression contrasted interestingly with that of leukemia cells, since we have previously reported low PPP2R2A levels in AML blasts associated with shorter remission durations (Ruvolo et al Leukemia 2011). Furthermore, AKT phosphorylation was negatively correlated with PPP2R2A expression in AML blasts, and normal MSC, but there was no correlation between PPP2R2A and phosphorylated AKT in AML MSC. Also, expression of PPP2R2A was positively correlated with the expression of the survival protein NOL3 (ARC) which may provide new clues to possible survival mechanisms in AML MSC. In summary, these findings represent insights into the proteomic profiling of normal and AML MSC. Results suggest that senescence (via p21), differentiation potential (involving SMAD/PPARG pathway), and survival signaling (including PP2A/AKT) are altered in AML MSC. Studies are underway to determine how these variations in MSC properties impact the AML microenvironment. Disclosures Carter: Tetralogic Pharmaceuticals: Research Funding.

Abstract: Abstract:  The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)‐derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271 + population. The recognized CD271 + populations were fractionated by fluorescence‐activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU‐F. The results showed that only the CD271 bright but not the CD271 dim population contained CFU‐F. Two‐color flow cytometry analysis revealed that only the CD271 bright population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271 bright population but no other BM cells. The new MSC‐specific molecules included the platelet‐derived growth factor receptor‐β (CD140b), HER‐2/erbB2 (CD340), frizzled‐9 (CD349), the recently described W8B2 antigen, as well as cell‐surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9‐3C2F1, and HEK‐3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM‐MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.

Abstract: Abstract 2593 Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of proteins involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in 75% of acute lymphoblastic leukemia (Br J Haematol, 2007; 138(6):740–8), and that increased expression of CTGF is associated with inferior outcome in B-ALL (Blood, 2007; 109(7):3080–3). In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knock-down CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA (479.3±37.2 and 57.3±5.9 copies per 100 copies of ABL1, respectively). Silencing of CTGF (CTGF-knockdown, CTGF-kd) resulted in significant suppression of leukemia cell growth (57% in RS4;11 and by 70% in REH) compared to control vector. CTGF knockdown moderately reduced adhesion of RS4;11 to fibronectin (27%±0.1%). In the in vitro culture system, CTGF knockdown significantly enhanced growth inhibition and apoptosis induction after 48 hour exposure to chemotherapy agents (annexinV(+): Vincristine 25.8±3.5%, Vincristine/CTGF-kd 42.6±2.8%; Dexamethasone 66.3±1.8%, Dexamethasone/CTGF-kd 99.3±0.6%; Methotrexate, 17.4±0.6%, Methotrexate/CTGF-kd 39.5±3.9). Analysis of signaling pathways showed that CTGF down-regulation inhibits Src phosphorylation at Tyr416. Remarkably, phosphorylation of AKT at Ser473, and of mTOR downstream targets S6 Ribosomal Protein and 4E-BP1 were significantly inhibited in CTGF-knockdown RS4;11 cells, concomitantly with upregulation of expression of AKT targets Bim and p27. No changes in the levels of apoptotic regulators cIAP1 and Bcl-xL were found. This data suggest that CTGF regulates growth and chemosensitivity of ALL cells through Src and AKT/mTOR signaling. We previously reported that an anti-CTGF monoclonal antibody significantly extended median survival of mice implanted with xenografts derived from a primary CTGF expressing ALL sample in NOD/SCID mice. We are now investigating the effects of combining anti-CTGF treatment with cytotoxic chemotherapy in this model. Blocking CTGF signaling may represent a useful adjunct to cytotoxic therapies in acute lymphoblastic leukemia. Disclosures: Spong: Fibrogen: Employment, Equity Ownership.

Abstract: The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows stem cell traits to breast epithelial cells. Mesenchymal stem cells (MSC) have the capacity to self-renew and differentiate into multiple tissue lineages. We hypothesized that the activation of EMT by ectopic expression of Twist, Snail or TGF-β in immortalized human mammary epithelial cells (HMEC) will result in the generation of cells with a phenotype and functionality similar to MSC. We found that the EMT-derived cells not only showed similar morphology but also displayed the typical MSC phenotype i.e. CD44+, CD24− and CD45−. Alternatively, MSC expressed EMT inducing genes such as Twist, Snail and FOXC2. Interestingly, CD140b (PDGFR-β), a marker for naive MSC, was exclusively expressed in EMT-derived cells compared to their epithelial counterparts. Moreover, functional analysis revealed that EMT-derived but not the control cells differentiate into Alizarin Red S-positive mature osteoblasts, Oil Red O-positive adipocytes and Alcian Blue-positive chondrocytes similar to MSC. We also observed that EMT-derived but not control cells invade and migrate towards MDA-MB-231 tumor cells in-vitro similar to MSC, displaying the characteristic tropism of MSC for tumor cells as previously reported by us. In-vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSC. In conclusion, we demonstrated that the EMT-derived cells are similar to MSC in gene-expression, multi-lineage differentiation, migration towards tumor cells and their ability to home to wounds. These results also suggest that EMT-derived MSC are active participants in cancer growth and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2314.