Bone Marrow Mononuclear Cells Of Patients Research Articles

Abstract 1237: IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. CD38 is an attractive therapeutic target for MM due to its high expression on plasma cells, but it is also expressed on multiple subsets of normal immune cells. Approved therapies including anti-CD38 monoclonal antibodies such as daratumumab are available, however resistance inevitably develops, and toxicities such as reduced immune cell counts put patients at increased risk for infections. To address this and other issues associated with current therapies, we are developing IGM-2644, an engineered high affinity, high avidity bispecific anti-CD38xCD3 IgM T cell engager (TCE) antibody for the treatment of plasma cell disorders. IGM-2644 kills CD38-expressing tumor cells through complement-dependent cytotoxicity (CDC) and T cell-dependent cellular cytotoxicity (TDCC). Live cell imaging of tumor cells in the presence of normal human serum as a source of complement has shown that IGM-2644, compared to daratumumab, has exhibited superior CDC with faster kinetics and higher maximal killing, as well as increased activity on tumor cell lines that express low levels of CD38. IGM-2644 also has displayed enhanced cellular dependent cytotoxicity over daratumumab across tumor cell lines with a range of CD38 expression levels in vitro. In ex vivo assays with MM patient bone marrow mononuclear cells (BMMC), IGM-2644 has shown superior tumor cell depletion compared to daratumumab even with low T cell to tumor cell ratios. Furthermore, IGM-2644 can deplete tumor cells in BMMC from daratumumab-refractory MM patients. Similarly, using in vivo humanized xenograft tumor mouse models with various levels of CD38 expression, greater anti-tumor activity with IGM-2644 over daratumumab was observed. When compared to daratumumab, IGM-2644 demonstrated lower immune cell fratricide in an in vivo xenograft tumor mouse model, suggesting a potentially differentiated safety profile. In summary, IGM-2644 is a bispecific anti-CD38xCD3 IgM TCE antibody that demonstrates superior tumor cell killing when compared to daratumumab in vitro and especially in xenograft tumor models in vivo with low CD38 expression while having low immune cell depletion, potentially reducing the risk of infections. The safety and efficacy of IGM-2644 has been initially studied in a phase 1 clinical trial for relapsed/refractory (r/r) MM. Applicability of IGM-2644 for the treatment of other plasma cell disorders is currently being explored, including autoimmune diseases. Citation Format: Kevin C. Hart, Daniel Santos, Keyu Li, Rui Yun, Min Chai, Andras Kabai, Gene Li, Poonam Yakkundi, Roel P. Funke, Yinghui Guan, Thomas Manley, Liqin Liu, Angus M. Sinclair, Albert F. Candia, Bruce Keyt, Maya F. Kotturi. IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1237.

Safety and efficacy of intra-arterial bone marrow mononuclear cell transplantation in patients with acute ischaemic stroke in Spain (IBIS trial): a phase 2, randomised, open-label, standard-of-care controlled, multicentre trial

Pilot clinical trials have shown the safety of intra-arterial bone marrow mononuclear cells (BMMNCs) in stroke. However, the efficacy of different doses of intra-arterial BMMNCs in patients with acute stroke has not been tested in a randomised clinical trial. We aimed to show safety and efficacy of two different doses of autologous intra-arterial BMMNC transplantation in patients with acute stroke. The IBIS trial was a multicentre phase 2, randomised, controlled, investigator-initiated, assessor-blinded, clinical trial, in four stroke centres in Spain. We included patients (aged 18-80 years) with a non-lacunar, middle cerebral artery ischaemic stroke within 1-7 days from stroke onset and with a National Institutes of Health Stroke Scale score of 6-20. We randomly assigned patients (2:1:1) with a computer-generated randomisation sequence to standard of care (control group) or intra-arterial injection of autologous BMMNCs at one of two different doses (2 × 106 BMMNCs/kg or 5 × 106 BMMNCs/kg). The primary efficacy outcome was the proportion of patients with modified Rankin Scale scores of 0-2 at 180 days in the intention-to-treat population, comparing each BMMNC dose group and the pooled BMMNC group versus the control group. The primary safety endpoint was the proportion of serious adverse events. This trial was registered at ClinicalTrials.gov, NCT02178657 and is completed. Between April 1, 2015, and May 20, 2021, we assessed 114 patients for eligibility. We randomly assigned 77 (68%) patients: 38 (49%) to the control group, 20 (26%) to the low-dose BMMNC group, and 19 (25%) the high-dose BMMNC group. The mean age of participants was 62·4 years (SD 12·7), 46 (60%) were men, 31 (40%) were women, all were White, and 63 (82%) received thrombectomy. The median NIHSS score before randomisation was 12 (IQR 9-15), with intra-arterial BMMNC injection done a median of 6 days (4-7) after stroke onset. The primary efficacy outcome occurred in 14 (39%) patients in the control group versus ten (50%) in the low-dose group (adjusted odds ratio 2·08 [95% CI 0·55-7·85]; p=0·28), eight (44%) in the high-dose group (1·89 [0·52-6·96]; p=0·33), and 18 (47%) in the pooled BMMNC group (2·22 [0·72-6·85]; p=0·16). We found no differences in the proportion of patients who had adverse events or dose-related events, but two patients had a groin haematoma after cell injection in the low-dose BMMNC group. Intra-arterial BMMNCs were safe in patients with acute ischaemic stroke, but we found no significant improvement at 180 days on the mRS. Further clinical trials are warranted to investigate whether improvements might be possible at different timepoints. Instituto de Salud Carlos III co-funded by the European Regional Development Fund/European Social Fund, Mutua Madrileña, and the Regional Ministry of Health of Andalusia.

Abstract 1959: CDK9 inhibition combined with hypomethylating agents target MCL-1 dependency in MDS and AML

Abstract Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic stem cell disorders. Approximately 70% of patients diagnosed with higher-risk (HR) MDS progress to acute myeloid leukemia (AML). Cyclin-dependent kinase 9 (CDK9) influences transcription through phosphorylation and activation of RNA polymerase II (RPB1), which increases the levels of key oncogenic survival genes like myeloid cell leukemia-1 (MCL-1). MCL-1 dependent malignancies can be identified through a functional assay by adding a NOXA mimetic MCL-1 antagonist peptide to patient samples and measuring the subsequent induction of apoptosis. The assay has been tested in bone marrow mononuclear cells (BMMCs) from AML and MDS patients to examine MCL-1 dependency prior to therapy with alvocidib. Approximately 25% of AML patient BMMCs and 60% of MDS patient BMMCs are MCL-1 dependent (data not shown). We hypothesized that the hypomethylating agents (HMAs) azacitidine and decitabine, which are currently approved for treatment of MDS, can increase MCL-1 dependency through re-expression of pro-apoptotic proteins like NOXA, normally repressed through DNA methylation in malignancies. In the MV-4-11 AML cell line and primary CD34+ MDS BMMCs, alvocidib treatment resulted in a dose dependent reduction in p-RPB1 and MCL-1. In cell viability assays using CellTiter-Glo, treatment with alvocidib and azacitidine resulted in IC50 values of ~100 nM and ~3000 nM, respectively in both cell types. Treatment with HMAs increased NOXA expression and alvocidib suppressed MCL-1 expression, whereas sequential treatment of azacitidine and alvocidib showed both NOXA induction and MCL-1 suppression. Sequential treatment of azacitidine and alvocidib showed synergistic apoptosis induction compared with either treatment alone. Azacitidine treatment sensitized MV-4-11 and MDS BMMCs to MCL-1 inhibition in an MCL-1 dependency assay. In an in vivo efficacy study using the MOLM13 AML xenograft model, the combination of alvocidib and azacitidine or decitabine inhibited tumor growth 71% and 84%, respectively. Alvocidib, azacitidine and decitabine alone showed 51%, 5.7% and 19% tumor growth inhibition (TGI), respectively. Pharmacodynamic analysis in a Phase 1 trial with alvocidib (clinicaltrials.gov, NCT03593915) showed DNA methylation of NOXA gene locus was reduced and NOXA gene expression was upregulated, while MCL-1 gene expression was suppressed in peripheral blood mononuclear cells (PBMCs) after azacitidine and alvocidib treatment. These pre-clinical and clinical data suggest that an alvocidib/HMA combination may constitute a viable therapeutic regimen whose rationale focuses on hypertargeting of NOXA/MCL-1. Taken together, these studies indicate that the combination of alvocidib and HMAs drives AML/MDS cells toward MCL-1 dependent apoptosis. Citation Format: Yuta Matsumura, Ethika Tyagi, Satya Pathi, Dan D. Vo, Tianxiang Zhu, Suman Verma, Clifford J. Whatcott, Stephen P. Anthony, Adam Siddiqui, Jason M. Foulks, David J. Bearss, Steven L. Warner. CDK9 inhibition combined with hypomethylating agents target MCL-1 dependency in MDS and AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1959.

Upregulated expression of leukocyte immunoglobulin-like receptor A3 in patients with severe aplastic anemia.

Severe aplastic anemia (SAA) is a rare and potentially life-threatening disease characterized by pancytopenia and bone marrow (BM) hypoplasia. In a previous study by our group, increased expression of leukocyte immunoglobulin-like receptors A (LILRA), LILRA3 in myeloid dendritic cells (mDCs) and LILRA5 in CD34+ cells in SAA was detected using proteomics techniques, highlighting their potential role in disease pathogenesis. In the present study, the expression of LILRA1-6 mRNA was assessed in the BM mononuclear cells of patients with SAA using reverse transcription-quantitative (RT-q)PCR. The expression of homogenic LILRA3 and LILRA5 isoform on mDCs, as well as CD34+, CD3+CD8+, CD19+ and CD14+ cells, was detected using flow cytometry. mDCs were then induced, cultured and sorted. The expression of LILRA3 was confirmed using RT-qPCR and western blot analyses. The serum levels of soluble LILRA3 were measured using ELISA. Furthermore, the relationship between LILRA3 expression and disease severity was assessed. The results indicated increased LILRA3 mRNA expression in patients with SAA. The percentage of LILRA3+ in BM mDCs and CD34+ cells was increased. Compared with controls, the relative LILRA3 mRNA expression and the relative protein intensity were highly increased in SAA mDCs. The serum LILRA3 levels in patients with SAA were also increased. The proportion of LILRA3+CD11C+ human leukocyte antigen (HLA)-DR+/CD11C+HLA-DR+ cells was positively correlated with the ratio of LILRA3+CD34+/CD34+ cells and the expression of LILRA3 mRNA. Taken together, the expression of LILRA3 on mDCs of patients with SAA was increased, which may affect the function of mDCs. LILRA3 may have a significant role in the immune pathogenesis of SAA.

Profiling the Cell Surface Landscape of Proteasome Inhibitor-Treated and -Resistant Multiple Myeloma to Guide Immunotherapy Targeting, Diagnosis, and Biology

Introduction: Proteasome inhibitor (PI) resistance remains a major clinical challenge in multiple myeloma (MM). As the proteasome plays a central role in cellular protein homeostasis, we hypothesized both PI treatment and resistance might rewire protein transport and recycling pathways, thereby leading to broad changes in cell surface protein expression. Defining the cell surface proteome has become increasingly important in MM to quantify immunotherapy target expression, identify potential biomarkers of drug response or resistance, and reveal proteins mediating interaction with the tumor microenvironment. Unbiased mass spectrometry approaches allow for profiling of hundreds of surface proteins simultaneously, allowing for novel protein discovery and extending beyond the limits of flow or mass cytometry. Methods: PI-resistant cells derived from cell lines AMO-1, L363, and RPMI-8226, and ARH-77 were grown in 90nM Bortezomib or Carfilzomib as previously described (Soriano et al, Leukemia (2016)). N-glycoproteomics was performed in triplicate on PI-resistant and parental lines. Briefly, glycosylated cell surface proteins were biotinylated prior to enrichment with Neutravidin beads and downstream LC-MS analysis. For perturbation studies, AMO-1 and RPMI-8226 cells were treated for 48 hours with Bortezomib, Lenalidomide, or CB-5083 prior to N-glycoproteomics. Flow cytometry was performed to validate surface protein abundance changes in PI-resistant lines, as well as with MM cell lines and ex vivo MM patient bone marrow mononuclear cells (PBMCs) treated with bortezomib and lenalidomide. For ex vivo MM patient cell surface proteomics, plasma cells were isolated from PBMCs with anti-CD138 magnetic beads prior to N-glycoproteomic analysis. Mass spectrometry data was analyzed in Maxquant and statistical analysis was performed in Perseus and R. Results: Supporting our hypothesis, we found that the MM surfaceome is broadly re-wired in the PI-resistant state. Using N-glycoproteomics, we identified EVI2B, CD53, CD50, and ITGB7 as downregulated and CD151, CD10, and SERC3 as upregulated in PI-resistant myeloma cell lines (up and downregulated defined as at least a +/- log2 fold change of 1.5 and p < .05). Notably, ITGB7 is under development as an immunotherapy target for MM (Hosen et al, Nat Med (2017)). To understand how surface remodeling in the PI-resistant state compares with short-term exposure to PI, we treated MM cells with bortezomib and again found broad surfaceome remodeling, including loss of immunotherapy target BCMA. We identified GITR, CD53, and ITGB7, among other proteins, as downregulated in both bortezomib-exposed and PI-resistant MM, suggesting that expression of proteins involved in therapeutic resistance might be rapidly modulated in initial cycles of therapy. To compare surfaceome changes identified in PI-exposure and resistance and those seen under anti-MM therapeutics with alternative mechanisms of action, we performed N-glycoproteomics on MM cells treated with the clinical immunomodulating agent Lenalidomide and the cytotoxic p97/VCP inhibitor CB-5083. Interestingly, we found MUC1, a proposed myeloma oncoprotein, to be upregulated under lenalidomide in AMO-1 but downregulated in the PI-resistant state. We validated surface protein abundance changes in PI-resistant MM cell lines and drug-treated MM cell lines or ex-vivo MM patient samples by flow cytometry. For additional validation, we are currently optimizing methods for N-glycoproteomics on CD138+ cells isolated from primary MM patient bone marrow aspirates. Initial results suggest several markers identified in cell lines are also highly expressed in primary PI-resistant MM. Conclusions: Unbiased N-glycoproteomics reveals broad cell surface protein remodeling in PI-resistant MM. Short-term treatment with bortezomib or lenalidomide also leads to significant surface alterations, with differential expression of proteins including MUC1, GITR, and BCMA potentially playing a role in drug resistance and informing rational combination with immunotherapies. Disclosures Martin: Roche and Juno: Consultancy; Amgen, Sanofi, Seattle Genetics: Research Funding. Wong:Celgene: Research Funding; Janssen: Research Funding; Roche: Research Funding; Fortis: Research Funding. Wiita:Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; UCSF: Patents & Royalties.

HDAC and HMT Inhibitors in Combination with Conventional Therapy: A Novel Treatment Option for Acute Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) is characterized by PML-RARA translocation, which causes the blockage of promyelocyte differentiation. Conventional treatment with Retinoic acid and chemotherapeutics is quite satisfactory. However, there are still patients who relapse or develop resistance to conventional treatment. To propose new possibilities for acute leukemia treatment, we studied the potential of histone deacetylase (HDAC) inhibitor and histone methyl transferase (HMT) inhibitor to enhance conventional therapy in vitro and ex vivo. NB4 and HL60 cell lines were used as an in vitro model; APL patient bone marrow mononuclear cells were used as an ex vivo model. Cell samples were treated with Belinostat (HDAC inhibitor) and 3-Deazaneplanocin A (HMT inhibitor) in combination with conventional treatment (Retinoic acid and Idarubicin). We demonstrated that the combined treatment used in the study had slightly higher effect on cell proliferation inhibition than conventional treatment. Also, enhanced treatment showed stronger effect on induction of apoptosis and on suppression of metabolism. Moreover, the treatment accelerated granulocytic cell differentiation and caused chromatin remodelling (increased H3K14 and H4 acetylation levels). In vitro and ex vivo models showed similar response to the treatment with different combinations of 3-Deazaneplanocin A, Belinostat, Retinoic acid, and Idarubicin. In conclusion, we suggest that 3-Deazaneplanocin A and Belinostat enhanced conventional acute promyelocytic leukemia treatment and could be considered for further investigations for clinical use.

Cotargeting the JAK/STAT signaling pathway and histone deacetylase by ruxolitinib and vorinostat elicits synergistic effects against myeloproliferative neoplasms.

The majority of patients with Philadelphia-negative myeloproliferative neoplasms (MPNs) harbor a gain of function mutation V617F in Janus kinase (JAK) 2. Although JAK2 inhibitors such as ruxolitinib have been shown to be clinically efficacious, the hematological toxicity and eventual drug resistance limit its use as monotherapy. Other gene mutations or dysregulation correlated with the disease phenotype and prognosis have been found to contribute to the complexity and heterogeneity of MPNs, giving rise to an increasing demand for combination therapies. Here, we combine ruxolitinib and the histone deacetylase inhibitor vorinostat as a rational combination strategy for MPNs. We tested the combination of ruxolitinib and vorinostat in cells with the JAK2V617F mutation, such as HEL cells, c-Kit+ cells from JAK2V617F transgenic mice and bone marrow mononuclear cells (BMMNCs) from patients with MPN. Our results showed significant synergistic effects of this combination strategy. Cotreatment with ruxolitinib and vorinostat synergistically induced apoptosis, cell cycle arrest and inhibition of the colony-forming capacity of HEL cells by attenuating the JAK/signal transducer and activator of transcription (STAT) and protein kinase-B (AKT) signaling pathways. In particular, cotreatment with ruxolitinib and vorinostat prevented the formation of large colonies of colony-forming unit-granulocyte/erythroid/macrophage/megakaryocytes (CFU-GEMMs) and colony-forming unit-granulocyte/macrophages (CFU-GMs) derived from the BMMNCs of patients with MPN. Taken together, these data provided preclinical evidence that the combination of ruxolitinib and vorinostat is a potential dual-target therapy for patients with MPN.

Intraportal therapy of autologous bone marrow mononuclear cells in patients on the waiting list for liver transplantation

This study was carried out to explore the methodology of intraportal infusion of autologous bone marrow mononuclear cells in patients in the waiting list for liver transplantation. The study included eight patients. The Model for End-Stage Liver Disease (MELD) score as a predictor of hepatic failure exceeds 10 points, i.e., 12-22 points. According to Child-Turcotte-Pugh (CTP) classification, the severity of cirrhosis ranged from class B to C. The bone marrow volume of 224-320 ml was harvested by bilateral aspiration from the posterior iliac crest. The total volume of the mononuclear cells (MNC) was 47.1-58.3 ml (median, 47 ml). The absolute viable count of isolated MNC was 1.24 · 109-5.84 · 109 and CD34+ cells 2.54 · 106-48.20 · 106. After portal vein catheterization, direct transhepatic portography was performed, and portosystemic collaterals were found in all cases. Portal flowmetry was performed to determine the optimal selective perfusion rate of the segmental branches of the portal vein. Portal vein infusion was performed at 0.3-1.5 ml/sec. Serious side effects were not detected during bone marrow harvesting and MNC infusion. All patients had stable condition (no deterioration was initially noticed) after MNC infusion according to the MELD score and CTP criteria during a 3-month observation. Moreover, improvement in physical health status was observed and obtained using the SF-36 questionnaire. This study confirmed the safety of using intraportal therapy of autologous bone marrow MNC in patients on the waiting list for liver transplantation. Herein, we presented the technique of target delivery of autologous bone marrow MNC to the liver to minimize their loss during intraportal infusion. Prolonged follow-up time and improvement of the present technique are needed for further evaluation of the efficiency of autologous bone marrow MNC.

Evaluation of autologous bone marrow mononuclear cells treatment for heart failure due to non-ischemic cardiomyopathy using PET myocardial perfusion/metabolic imaging

Objective To evaluate the clinical improvements after autologous bone marrow mononuclear cells (BMMNCs) percutaneously injected into coronary artery in patients with heart failure due to non-ischemic cardiomyopathy using PET myocardial perfusion/metabolic imaging. Methods From February 2011 to October 2012, 40 patients with heart failure due to non-ischemic cardiomyopathy were selected. The test group including 15 patients (13 males, 2 females, average age (57.5±14.5) years) received the autologous BMMNCs intracoronary injection on the basis of drug treatment. The other 25 cases (21 males, 4 females, average age (58.0±12.0) years) were taken as the control group and only received the drug treatment. All patients were followed up for 24 months, and the myocardial perfusion/metabolism imaging, echocardiography, brain natriuretic peptide (BNP) test, 6-minute walking experiment were performed. The data were analyzed by two-sample t test. Results During the follow-up period, the test group had no ventricular arrhythmia and other serious complications, and the patients′ symptoms had been improved. There was no change in myocardial perfusion after treatment of autologous BMMNCs, but the myocardial metabolic defect by volume reduced from (43.79±17.99) cm3 to (28.19±9.27) cm3 (t=3.33, P 0.05). In the test group, the left ventricular end-diastolic diameter decreased from (64.0±8.0) mm to (59.0±7.0) mm 24 months after the treatment (t=2.04, P<0.05), and the left ventricular ejection fraction was significantly higher than that before treatment: (45.0±4.0)% vs (27.0±6.0)% (t=10.81, P<0.01). Conclusion PET myocardial perfusion/metabolic imaging can be used as tools in evaluating the therapeutic effect of autologous BMMNCs in patients with heart failure due to non-ischemic cardiomyopathy. Key words: Cardiomyopathies; Heart failure; Stem cell transplantation; Positron-emission tomography; Tomography, X-ray computed; Ammonia; Deoxyglucose

Ex Vivo Modeling of Multiple Myeloma Provides Basis for Studying Treatment Combinations and Immunotherapy

BackgroundMultiple myeloma (MM) remains incurable despite treatment advances. While passive immunotherapy such as anti-CD38 antibodies is highly effective, active immunotherapy may provide long-lasting remissions by virtue of triggering memory. A phase 1 nivolumab study, an antibody targeting programmed death-1 (PD1), was unable to yield any responses in multiply relapsed MM patients. Conversely, preliminary trial data of lenalidomide combined with pembrolizumab, a different anti-PD1 antibody, found significantly higher response rates. These two differing outcomes reflect our limited understanding of checkpoint inhibition and immunotherapy in MM. There is a paucity of preclinical models to guide therapeutic studies. Cell lines and xenografted murine models are incapable of exploring active immunotherapy due to a lack of microenvironment and endogenous immune cell signals. Furthermore, malignant cells responsive to drugs in 2-dimensional (2D) cultures are known to display a more resistance in 3D. We have previously demonstrated that B-cell malignancies can be accurately studied using a 3D culture system of patient bone marrow mononuclear cells (BMCs) and can better inform translational trials. Herein we describe an ex vivo, 3D tissue culture model of patient-derived MM samples to more accurately test therapeutics including checkpoint inhibition using ipilimumab, a monoclonal antibody targeting cytotoxic T-lymphocyte antigen 4 (CTLA) which is crucial in co-stimulatory signaling of effector T-cells.MethodsA 3D extracellular matrix was created using matrigel in 12-well plates. BMCs were isolated from marrow aspirates of 5 MM patients at time of diagnosis and individually cultured. Each patient sample was tested for sensitivity against increasing concentrations of ipilimumab (1X, 3X, and 10X clinical doses) added into supportive medium. Plates were monitored visually by microscopy followed by harvest on day 21 using enzymatic degradation. Unique clonotypic heavy chain immunoglobulin rearrangement (IgH VDJ) from each sample was sequenced, validated and used for semi-quantitative PCR. Semi-QT PCR with clone-specific primers estimated malignant cell survival after harvest. Flow cytometry was used to define cell populations present in culture and to correlate with clonotypic PCR data. T-cell mediated activity was examined by reverse transcription of trizol-extracted, T-cell RNA after harvest.ResultsAll samples were successfully cultured, followed for 21 days and harvested. Flow cytometry confirmed presence of T-cell subsets, B-cells, NK cells and dendritic cells before and after culture in 3D. Minimal depletion of clonotypic cells was observed at 3x clinical levels of drug. At 10x simulated clinical therapeutic levels, 3 MM samples demonstrated >90% death of clonotypic MM cells while the other 2 demonstrated 62% and 72% death, respectively, compared to untreated control cultures. The extent to which the drug diffuses into the matrigel is as yet unknown. Flow cytometry of harvested cells suggest that T-cells demonstrate a modest shift toward CD4 and CD8 effector cells. Preliminary mechanistic data from one MM sample using trizol-extracted RNA and reverse transcriptase PCR harvested at 21 days from 3D culture suggests that anti-malignant, cytotoxic T-cell effect may be driven by granzyme B expression. Expanded data from the remaining samples will be presented.ConclusionsWe demonstrate that an ex vivo 3D tissue culture model of MM is both feasible and informative in studying immunotherapy. By culturing unselected BMCs which include stromal cells, immune cells and malignant populations, the 3D culture more closely mimics the tumor microenvironment with both the patient's immune system present as well as stromal supportive signals. In this study, we show that in the presence of active immune effector cells, ipilimumab has activity against patient-derived MM cells. The data suggests the importance of targeted cytotoxic T-cell activation as a primary mechanism of action. We have previously studied standard MM therapeutics such as cytotoxic chemotherapy, immunomodulatory drugs, and proteasome inhibitors in the same way. Consequently, this model is well positioned to study other immunotherapies such as other checkpoint inhibitors, cellular therapy, and combinations. Further testing with therapeutics targeting PD1/PDL1, and adenosine receptors are underway. DisclosuresVenner:Takeda: Honoraria; Celgene: Honoraria, Research Funding; J+J: Research Funding; Janssen: Honoraria; Amgen: Honoraria. Belch:Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Takeda: Honoraria.

Abstract 675: Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia

Abstract GSK3 alpha has been shown to be a new target in the treatment of acute myeloid leukemia (AML); however current GSK3 inhibitors are unselective and target both GSK3 alpha and GSK3 beta. Interestingly, a known pan-GSK3 inhibitor LiCl has been previously investigated for the treatment of AML yet has met limited clinical success. This could be partly due to the fact that pan-GSK3 inhibition results in beta-catenin stabilization, which has been shown to mediate hematopoietic self-renewal and leukemogenesis. As beta-catenin stabilization requires inhibition of both kinases and most GSK3 inhibitors target GSK3 alpha and GSK3 beta with equal potency, these compounds may possess some significant limitations. We have previously shown GSK3 alpha to be a prominent target of the intended MET inhibitor tivantinib (ARQ197) and that tivantinib shows some specificity for GSK3 alpha over GSK3 beta. We thus hypothesized that tivantinib would be an effective therapy for the treatment of AML. Consistently, tivantinib potently inhibited cellular viability across several AML cell lines. Using an unbiased, mass-spectrometry based chemical proteomics approach; we confirmed GSK3 alpha (and to a slightly lesser extent GSK3 beta) to be targeted by tivantinib in AML. Tivantinib strongly induced apoptosis as compared to the pan-GSK3 inhibitor LiCl in these cells while LiCl showed larger effects on cell differentiation. Interestingly tivantinib caused less stabilization of beta-catenin as compared to LiCl. Subsequent drug combination studies identified the Bcl-2 inhibitor ABT-199 to synergize with tivantinib and to amplify apoptosis as seen by PARP1 cleavage. Furthermore, the combination of tivantinib with ABT-199 completely abrogated beta-catenin stabilization. Tivantinib was able to significantly inhibit colony formation of primary AML patient bone marrow mononuclear cells (BMNCs) and ABT-199 combination showed significant benefit over tivantinib or ABT-199 alone. In summary, tivantinib has potent anticancer activity in AML based on targeting GSK3 alpha; and tivantinib single agent or combination with ABT-199 may represent a novel and exciting opportunity for the treatment of AML. Citation Format: Brent M. Kuenzi, Lily L. Remsing Rix, Sateesh S. Kunigal, Fumi Kinose, Claire E. Knezevic, Gabriela Wright, Jodi L. Kroeger, Jeffrey E. Lancet, Eric Padron, Uwe Rix. Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2015-675

Silencing HO-1 sensitizes SKM-1 cells to apoptosis induced by low concentration 5-azacytidine through enhancing p16 demethylation.

Heme oxygenase-1 was reported previously as a resistance target on acute myelocytic leukemia (AML). We found that HO-1 was resistant to 5-azacytidine (AZA) treatment of myelodysplastic syndrome (MDS), and explored further the relative mechanisms. Patient bone marrow mononuclear cells (n=48) diagnosed as different levels of MDS were collected. Cell growth was evaluated by MTT assay; cell cycle and apoptosis were detected by flow cytometry; mRNA expression was assessed by real-time PCR, protein expression was analyzed through western blotting. Methylation was assessed by MSP. The survival time, and weight of mice were recorded. HO-1 overexpression was observed in SKM-1 cells after AZA treatment comparing to other cell lines. The HO-1 expression in MDS patients with high-risk was higher than in low-risk patients. After HO-1 was silenced by lentivirus-mediated siRNA, the proliferation of SKM-1 cells was effectively inhibited by low concentration AZA, and the cell cycle was arrested in the G0/G1 phase. Upregulation of p16 and changing of p16-relative cell cycle protein was observed after silencing HO-1 in AZA treated SKM-1 cells. In addition, DNMT1 was downregulated following the decrease of HO-1 expression. In vivo, silencing HO-1 inhibited SKM-1 cell growth induced by AZA in a NOD/SCID mouse model. Silencing HO-1 sensitized SKM-1 cells toward AZA, which may be attributed to the influence of HO-1 on AZA-induced p16 demethylation. HO-1 may be one of the targets that enhance the therapeutic effects of AZA on MDS malignant transformation inspiring new treatment methods for high-risk and very high-risk MDS patients in clinical practice.

Abstract 1868: Clinical significance of cathepsin L and B expression in pediatric acute myeloid leukemia

Abstract Introduction: Cathepsin L and B are commonly expressed cysteine proteinases that play a major role in lysosomal bulk proteolysis, protein processing, matrix degradation, and tissue remodelling. In tumors there is deregulation of these proteases that interferes with these normal biological processes. In a pilot study done in our lab expression of cathepsin L and B were found to be significantly elevated in peripheral blood mononuclear cells (PBMCs) of pediatric AML patients. However the expression of these proteases in bone marrow aspirate, which represents almost true population of blasts, has never been studied before. Methods: Enzymatic activities of cathepsin L and B were assayed in PBMCs and bone marrow mononuclear cells (BMMCs) of AML patients and healthy controls. Further expression of these proteases and its inhibitor were confirmed by western blotting and real time assay. Cell cycle analysis in PBMCs and BMMCs of these patients was performed by flow cytometry using propidium iodide. Results: In the PBMCs of pediatric AML patients activity of cathepsin L and B was found to be significantly elevated as compared to healthy controls. Similarly, increased activity of cathepsin L and B was also observed in the BMMCs when compared with PBMCs of these patients. Furthermore, protein and mRNA expression also confirmed the increased level of these proteases in pediatric AML. The protein and mRNA expression of their endogenous inhibitor cystatin-C was found to be decreased in PBMCs and BMMCs of AML patients. Moreover, expression of these proteases correlated positively with the percentage of blasts and percentage of cells in S-phase. Conclusion: This study for the first time demonstrates the increased expression of cathepsin L and B in the BMMCs and PBMCs of pediatric AML patients, along with a positive correlation with percentage of blasts, thereby suggesting a potential role of these proteases in the severity of this disease. Citation Format: Garima Pandey, Sameer Bakhshi, Ratnakar Singh, Shyam S. Chauhan. Clinical significance of cathepsin L and B expression in pediatric acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1868. doi:10.1158/1538-7445.AM2014-1868

CCN1 enhances angiogenic potency of bone marrow transplantation in a rat model of hindlimb ischemia.

Implantation of autologous bone marrow mononuclear cells (BM-MNCs) has been performed in ischemic tissues, for stimulation of angiogenesis, but the limited number of BM-MNCs in patients with hindlimb ischemia disease may offset their overall therapeutic efficacy. CCN1 is a novel and essential regulator during angiogenesis. We evaluated whether CCN1 and BM-MNC are capable of promoting angiogenesis in hindlimb ischemia. In this study, we created the rat model of hindlimb ischemia, and then the rats were randomly divided into four groups: CCN1 infusion plus BM-MNC transplantation (CCN1 + BM-MNCs group), CCN1 infusion plus PBS injection (CCN1 group), vehicle infusion plus BM-MNC transplantation (BM-MNCs group) and vehicle infusion plus PBS injection (control group). The combination of CCN1 and BM-MNC therapy could increase blood perfusion, capillary/muscle fiber ratio and tissue oxygenation in ischemic hindlimb. Moreover, CCN1 could not only inhibit the apoptosis of BM-MNCs, but also enhance the adhesiveness of BM-MNCs to HUVEC. Taken together, CCN1 enhanced angiogenesis of BM-MNC transplantation, and combining CCN1 with BM-MNC transplantation is a useful alternative for ischemic limbs.

Humanized Anti-CD26 Monoclonal Antibody Inhibits Osteoclast Bone Resorption Activity Via Blockade Of P38MAPK Signaling In Multiple Myeloma

BackgroundBone disease is a hallmark of multiple myeloma (MM) due to increased osteoclast (OC)-mediated bone resorption and decreased osteoblast (OB)-mediated bone formation. CD26 is a 110-kDa cell surface glycoprotein with dipeptidyl peptidase IV (DPPIV) activity and has a well-defined role in T cell activation and several tumor developments including malignant lymphoma, whereas its expression in OCs further suggests a role in osteoclastgenesis (OCG). Interestingly, CD26 is associated with increased phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) which also occurs downstream of RANK signaling in OCG and plays an important role in OC function. Our previous studies showed that CD26 is preferentially expressed in normal human OCs, and is also intensely expressed in activated OCs in MM, but not in MM cells themselves. In this study, we investigated the efficacy of humanized anti-CD26 monoclonal antibody (YS110) on OCs derived from MM patient bone marrow (BM) and MM growth within the BM microenvironment. MethodsOCs derived from MM patient BM mononuclear cells were assayed with macrophage-colony stimulating factor (M-CSF) (25ng/ml) plus receptor activator of NF- κB ligand (RANKL) (50ng/ml) with or without YS110 for OC formation and maturation for TRAP/CD26 staining and functional assay with bone resorptive pit formation assay. To assess the mechanisms of action of YS110 against OC differentiation in MM, RANK signaling proteins were examined by immunoblotting. ResultsSignificantly reduced numbers of multinucleated TRAP/CD26 positive mature OCs (>3 nuclei per OC) were observed in YS110-treated versus non-treated MM OC culture. YS110 blocked RANKL/M-CSF induced phosphorylation of p38MAPK and its downstream activation of mi/Mitf in MM-derived OC precursor cells, which otherwise increased OC differentiation. In contrast, YS110 did not show significant inhibition against other MAPKs involved in RANK signaling pathway in OC precursor cells. Importantly, YS110 remarkably diminished bone erosion area formed by mature OCs on dentine slices in pit formation assay. YS110 also consistently induced changes of cell supernatants in a dose-dependent manner in MM OC culture by ELISA. In fact, OC- or MM- related growth factors such as TRAP5b, collagen type 1, APRIL and BAFF which are secreted by OCs were also down-regulated. DPPIV inhibitor had no significant inhibitory effects on OCG in MM. These results suggest that the mechanism of action by YS110 on OCG in MM result from the blockade of RANKL induced p38MAPK-mi/Mitf phosphorylation in MM-derived OC precursor cells. Next, although YS110 did not demonstrate direct inhibition of proliferation of MM cells, to further investigate the role of CD26 in MM cells in the BM microenvironment, co-cultures of MM cell lines (KMS11, KMS12, KMS18, KMS20, KMS21, KMS26, KMS27, KMS28, KMS34, KMM1, U266) with MM-derived CD26-stained OCs were performed. We examined the expression of CD26 in co-cultured MM cells and co-cultured OCs by flow cytometry and immunoblotting. On day 1 of the co-culture, the expression of CD26 was barely detected in MM cells. However, on day 5, the expression level of CD26 was shown to be further increased in OCs. Moreover, interestingly, the expression of CD26 was also significantly up-regulated in MM cells. By immunohistochemistry, co-cultured MM cells were intensely stained positive for CD26 as well as co-cultured OCs. P38MAPK was activated in co-cultured CD26-stained MM cells; it regulates OC differentiation and bone resorption activity in MM. MM cells in the BM microenvironment may induce functional changes which promote tumor development and progression, as well as microenvironmental alternations. Now on going studies are confirming the anti-myeloma efficacy of YS110 on co-cultures of MM cells with OCs. ConclusionsOur data strongly demonstrate that targeting CD26 with YS110 inhibits MM-derived OC differentiation and function through blocking p38MAPK-mi/Mitf cascade of RANK signaling in MM-derived OC precursor cells. These results suggests that CD26 in OCs may be a novel therapeutic target for the treatment of myeloma-assoiated bone disease. It was further confirmed that the direct interaction between MM cells and OCs results in the up-regulation of CD26 expression in MM cells and that targeting CD26-p38MAPK signaling cascade may have a novel therapeutic potential not only in MM-derived OCs but in MM cells. Disclosures:No relevant conflicts of interest to declare.

Intracavernous administration of bone marrow mononuclear cells: a new method of treating erectile dysfunction?

While PDE5 inhibitors have revolutionized treatment of ED, approximately 30% of patients are non-responsive. A significant cause of this is vascular and smooth muscle dysfunction, as well as nerve atrophy. Autologous administration of bone marrow mononuclear cells (BMMC) has been performed in over 2000 cardiac patients without adverse effects, for stimulation of angiogenesis/regeneration. Despite its ease of access, and dependence on effective vasculature for function, comparatively little has been perform in terms of BMMC therapy for ED. Here we outline the rationale for use of autologous BMMC in patients with ED, as well as provide early safety data on the first use of this procedure clinically.

Association of Th1 and Th2 cytokines with transient inflammatory reaction during lenalidomide plus dexamethasone therapy in multiple myeloma

Transient inflammatory reactions have been reported in a subpopulation of patients with multiple myeloma (MM) during lenalidomide (Len) plus dexamethasone (DEX) therapy. Here, we examined serum levels of Th1 (IL-2 and IFN-γ) and Th2 cytokines (IL-6 and TNF-α) in nine refractory or relapsed MM patients treated with Len plus low-dose DEX. Six patients showed elevation of C-reactive protein (CRP) after the initiation of therapy. In these patients, IFN-γ and IL-6 were also elevated in two and three patients, respectively. The remaining three patients showed no appreciable changes in CRP or these cytokines. Furthermore, Len enhanced the production of both Th1 and Th2 cytokines in normal peripheral blood mononuclear cells and in patient bone marrow mononuclear cells containing primary myeloma cells and lymphocytes. These results suggest that the modulation of the Th1 and Th2 cytokine production by Len may contribute to transient inflammatory reaction in MM patients.

Intra-Arterial Infusion of Autologous Bone-Marrow Mononuclear Cells in Patients with Moderate to Severe Middle-Cerebral-Artery Acute Ischemic Stroke

Intra-Arterial Infusion of Autologous Bone-Marrow Mononuclear Cells in Patients with Moderate to Severe Middle-Cerebral-Artery Acute Ischemic Stroke

Absence of accelerated atherosclerotic disease progression after intracoronary infusion of bone marrow derived mononuclear cells in patients with acute myocardial infarction—Angiographic and intravascular ultrasound—Results from the TErapia Celular Aplicada al Miocardio Pilot study

We tried to evaluate a putative negative effect on coronary atherosclerosis in patients receiving intracoronary infusion of unfractionated bone marrow mononuclear cells (BMMC) following an acute ST-elevation myocardial infarction. Peripheral blood mononuclear cells or enriched CD133(+) BMMC have been associated with accelerated atherosclerosis of the distal segment of the infarct related artery (IRA). Thirty-seven patients with ST-elevation myocardial infarction from the TECAM pilot study underwent intracoronary infusion of autologous BMMC 9 +/- 3.1 days after onset of symptoms. We compared angiographic changes from baseline to 9 months of follow-up in the distal non-stented segment of the IRA, as well as in the contralateral coronary artery, with a matched control group. A subgroup of 15 treated patients underwent additional IVUS within the distal segment of the IRA. No difference between stem cell and control group were found regarding changes in minimum lumen diameter (0.006 +/- 0.42 vs 0.06 +/- 0.41 mm, P = ns) and the percentage of stenosis (-2.68 +/- 12.33% vs -1.78 +/- 8.75%, P = ns) at follow-up. Likewise, no differences were seen regarding changes in the contralateral artery (minimum lumen diameter -0.004 +/- 0.54 mm vs -0.06 +/- 0.35 mm, P = ns). In the intravascular ultrasound substudy, no changes were demonstrated comparing baseline versus follow-up in maximum area stenosis and plaque volume. In this pilot study, analysis of a subgroup of patients found that intracoronary injection of unfractionated BMMC in patients with acute ST-elevation myocardial infarction was not associated with accelerated atherosclerosis progression at mid term. Prospective, randomised studies in large cohorts with long-term angiographic and intravascular ultrasound follow-up are necessary to determine the safety of this therapy.

Disseminated tumor cells in primary breast cancer: Evaluation of the percentage of breast cancer stem cells in bone marrow aspirates of patients receiving neoadjuvant chemotherapy

505 Background: Disseminated tumor cells (DTC) in the bone marrow (BM) are related to poor prognosis of primary breast cancer (PBC). Cancer-initiating stem cells (CSC) have been detected in BM of PBC patients. Downregulation of the Notch pathway leads to murine mammary stem cell expansion and its expression is an early event in breast cancer. We enumerated CSC in BM mononuclear cells (BMC) and assessed the expression of Notch-1, ER, and HER-2 to study the differences between untreated patients and those treated with neoadjuvant chemotherapy. Methods: BM was collected from 90 PBC patients at time of surgery to assess DTC (CD326+CD45-) and CSC (CD326+CD45-CD44+CD24-) by FACS analysis. BM samples were also interrogated for the presence of CSC with ALDH activity using the Aldefluor® detection kit. RNA extracted from 61 BMC was analyzed for Notch-1, ER, and HER-2 transcripts by RT-PCR; the level of each transcript was normalized to that of the housekeeping gene, GAPDH. Results: The median age of patients is 51 years old (range, 27–77 years). A total of 29 patients (32%) received neoadjuvant chemotherapy, including 8 patients with anti-HER-2 targeted therapy. BMC of neoadjuvant-treated patients had a significantly higher median percentage of CSC in BMC (P= 0.046) and a significantly higher proportion of CSC cells within the Aldefluor+ population (P= 0.013) than those of untreated patients. Median level of Notch-1 transcripts in BMC was lower in neoadjuvant-treated patients compared with that of untreated patients (P= 0.07). There was an inverse correlation between the level of Notch-1 transcripts and the percentage of CSC in BMC of untreated (Spearman's rho= -0.519, P &lt; 0.001), but not in neoadjuvant treated (Spearman's rho= -0.483, P= 0.058) patients. Conclusions: Neoadjuvant chemotherapy is associated with expansion of CSC in the BMC of patients with PBC. Untreated patients with higher level of Notch-1 transcripts in the BMC tend to have lower percentages of CSC and suggest that Notch signaling may be crucial for differentiation of CSC. The prognostic value of this finding is under investigation and gene expression profile of CSC may provide indications for novel neoadjuvant therapies. No significant financial relationships to disclose.