Establishing a Novel Pipeline That Combines  in-Silico Prediction with in-Vitro and  Ex-Vivo Validation to Discover Secondary Drug Combinations Against Relapsed and/or Refractory Multiple Myeloma

Abstract Multiple myeloma (MM) is the second-most common hematological malignancy in the US. MM is an incurable, age-dependent plasma cell neoplasm with a 5-year survival rate of less than 50%. Extensive inter-individual variation in response to standard-of-care drugs like proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), drug resistance, and dose-limiting toxicities are critical problems for the treatment of MM. Clinical success in anti-myeloma treatment, therefore, warrants continuous development of novel combination therapy strategies with the explicit goal to improve the therapeutic efficacy by concomitantly targeting multiple signaling pathways. Previously, we have reported the development of an in-house computational pipeline called secDrug that applies greedy algorithm-based set-covering computational optimization method followed by a regularization technique to predict secondary drugs that can be repurposed as novel synergistic partners of standard-of-care drugs for the management of refractory/ resistant MM. Top among these secondary drugs (secDrugs) were the HSP90 inhibitor 17-AAG. In this study, we used 17-AAG as a proof of principle to establish a pipeline that integrates our in silico predictions with in vitro and ex vivo validation as well as multi-omics technologies to identify, validate, and characterize therapeutic agents that could be used either alone or in combination with standard-of-care drugs for the treatment of R/R MM patients (Figure 1). To screen and validate our in silico prediction results, we performed in vitro cytotoxicity assays using 17-AAG on a panel of human myeloma cell lines (HMCLs; in vitro model systems) that captures a wide range of biological and genetic heterogeneity representing the complexities encountered in clinical settings. These cell lines include HMCLs representing innate sensitive/resistance, >10 pairs of parental and clonally-derived PI- and IMiD-resistant pairs (P vs VR or LenR; representing acquired/emerging resistance/relapse), NRAS mutants which leads to the constitutive activation of oncogenic Ras signaling, and CRISPR-edited HSP90 knockdown cell line. Our results showed that 17-AAG has high synergistic activity in combination with PI in inducing apoptosis even in innate and acquired PI-resistant HMCLs and significantly reduces the effective dose of PI required to achieve IC 50 (Chou-Talalay's Dose Reduction Index or DRI 7±1.4). Moreover, 17-AAG+IMiD showed synergistic cell killing activity in clonally-derived IMiD resistant HMCL. Further, 17-AAG induced cell death was comparable with Hsp90 knockdown as evident from the cytotoxicity assay using PI and 17-AAG in combination in RPMI8226-wild type and RPMI-HSP90AA1 knocked down cell line. Notably, 17-AAG was strikingly effective against the NRAS-mutant cell line indicating an additional niche (NRas mutant myeloma) where 17-AAG could be most effective. Next, we performed RNA sequencing to elucidate the molecular mechanisms behind 17-AAG drug action, drug synergy, 17-AAG-induced cell death. Our gene expression profiling (GEP) followed by Ingenuity Pathway Analysis (IPA) analysis revealed protein ubiquitination, aryl hydrocarbon receptor signalling pathway as the top canonical pathways. 17-AAG induced apoptosis via mitochondrial mediated pathway in myeloma. 17-AAG exerts its cytotoxic effect by activating intrinsic pathway of apoptosis which we further confirmed through the increase in reactive oxygen species generation and decrease in mitochondrial membrane potential. 17-AAG was also effective in reducing the expression of hallmarks of MM such as p65/NF-kB, IRF4, c-Myc. Finally, we performed mass cytometry (CyTOF; Cytometry by time of flight) on primary bone-marrow cells (PMCs) from myeloma patients for further validation of proteomic signatures at the single-cell level. CyTOF analysis confirmed 17-AAG-induced cell death and key changes in MM-specific proteomic markers. 17-AAG treated PMCs showed elevated cleaved caspase levels and down-regulation of IRF4 and phospho-STAT3. GEP and CyTOF results were confirmed using immunoblotting assays. Together, our study demonstrates a unique pipeline for drug repositioning that has the potential to revolutionize clinical decision-making by minimizing the number of drugs required for discovering successful combination chemotherapy regimens against drug-resistant myeloma. Figure 1 Figure 1. Disclosures Kumar: BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Beigene: Consultancy; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche-Genentech: Consultancy, Research Funding; Bluebird Bio: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

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CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽

10.1182/blood-2018-99-118926 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3199-3199 ◽

Cited By ~ 2

Author(s):

Subhashis Sarkar ◽

Sachin Chauhan ◽

Arwen Stikvoort ◽

Alessandro Natoni ◽

John Daly ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Advisory Committees ◽

Cd38 Expression ◽

Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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The Fully Human Anti-CD47 Antibody SRF231 Has Dual-Mechanism Antitumor Activity Against Chronic Lymphocytic Leukemia (CLL) Cells and Increases the Activity of Both Rituximab and Venetoclax

Blood ◽

10.1182/blood-2018-99-110181 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4393-4393 ◽

Cited By ~ 1

Author(s):

Rebecca Valentin ◽

Marisa O. Peluso ◽

Timothy Z. Lehmberg ◽

Ammar Adam ◽

Li Zhang ◽

...

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Ex Vivo ◽

Employment Equity ◽

Advisory Committees ◽

Isotype Control ◽

Equity Ownership

Abstract Background CD47 is over-expressed by many tumor types and protects tumor cells from destruction via tumor-intrinsic and -extrinsic means. The fully human anti-CD47 monoclonal antibody (mAb) SRF231 has previously been shown to block the "don't eat me" CD47/signal regulatory protein alpha (SIRPα) interaction and induce macrophage-mediated phagocytic uptake of CD47-expressing tumor cells, either alone or in the presence of anti-CD20 mAb. Furthermore, SRF231 inhibited tumor growth in preclinical models of aggressive non-Hodgkin lymphoma (Holland P, et al. ASH 2016). Here, we explored the activity of SRF231 against CLL cells for the first time, both as monotherapy and in combination with rituximab or venetoclax (VEN). Methods Peripheral blood mononuclear cells from 24 CLL patients were evaluated for CD47 surface expression by flow cytometry. Primary CLL or Jurkat target cells were treated ex vivo with SRF231 or isotype control and evaluated in phagocytosis and cell death assays. Human monocyte-derived macrophages were cocultured with fluorescently-labeled target tumor cells and exposed to SRF231 and/or rituximab (commercial supply). BH3 profiling was performed by gently permeabilizing primary CLL cells and measuring the release of cytochrome C (Cyto-C) in response to BH3-only peptides by flow cytometry. Priming for apoptosis was measured by Cyto-C release in response to BIM BH3 peptide, and pro-survival protein dependencies were measured by response to specific BH3-only sensitizer peptides. Statistical analyses were by unpaired and paired t-test with a two-tailed nominal p ≤ 0.05 considered as significant. In vivo antitumor activity was assessed using tumor xenograft studies in CB17 SCID mice. Mice with established, subcutaneous Ri-1 tumors were randomized and treated with either isotype control, SRF231, VEN (Medkoo), or combination of SRF231 and VEN. Results CD47 was expressed in all primary CLL cells (n = 24, median mean fluorescence intensity [MFI] 7913, range 3575-18,329) with a slightly higher expression in unmutated CLL (U-CLL) vs mutated CLL (M-CLL) samples (U-CLL median MFI = 9106, n = 8 vs M-CLL, median MFI = 7713, n = 14, 2 unknown, p = 0.047). Primary CLL cells were significantly more susceptible to phagocytosis upon ex vivo treatment with SRF231 in combination with rituximab (median % increase in phagocytosis over isotype control of 32.28% in the combination vs 11.78% with rituximab alone, n = 24, p < 0.0001). Upon coculture of Jurkat cells with macrophages, SRF231 not only induced phagocytosis (EC50, 332 ± 65 ng/mL, n = 3), but also induced cell death of non-phagocytosed target tumor cells (EC50, 295 ± 43 ng/mL, n = 3). While soluble SRF231 did not induce significant target tumor cell killing, immobilized SRF231 induced Jurkat cell and primary CLL cell death (median % alive of 34.6% in SRF231 treated cells vs 64.4% in controls, n = 24, p < 0.0001). To assess the mechanism of cell death induction, tumor cells were pretreated with a pan-caspase inhibitor, Z-VAD-FMK, which revealed that SRF231-mediated tumor cell death is caspase-independent. In primary CLL cells, BH3 profiling confirmed that SRF231 did not alter mitochondrial priming for apoptosis or pro-survival Bcl-2 family protein dependencies. Pre-treatment with the phospholipase C (PLC) inhibitor U73122 prior to SRF231 exposure partially blocked the ability of SRF231 to kill CLL cells (median % alive of 45.4% in pre-treated cells vs 25.4% in controls, n = 6, p = 0.0029). In addition to these in vitro studies, SRF231 displayed profound antitumor activity in a xenograft model of B-cell lymphoma as a single agent, and led to complete and durable tumor regression in combination with VEN. Conclusion Ex vivo treatment of primary CLL cells with SRF231 led to dual antitumor effects of tumor cell-extrinsic plus -intrinsic mechanisms by augmenting rituximab-induced phagocytosis and inducing tumor cell death. SRF231 induced death of tumor cells through a caspase-independent mechanism that depends at least partially on PLC. In vivo, SRF231 in combination with VEN led to complete and durable tumor regression in a xenograft model. SRF231 is currently being evaluated across multiple tumor types in a Phase 1 clinical trial (NCT03512340). Disclosures Valentin: Roche: Other: Travel reimbursement; AbbVie: Other: Travel reimbursement. Peluso:Surface Oncology: Employment, Equity Ownership. Adam:Surface Oncology: Employment, Equity Ownership. Zhang:Surface Oncology: Employment, Equity Ownership. Armet:Surface Oncology: Employment, Equity Ownership. Guerriero:GSK: Research Funding; Eli Lilly: Research Funding. Lee:Surface Oncology: Employment, Equity Ownership. Palombella:Surface Oncology: Employment, Equity Ownership. Holland:Surface Oncology: Employment, Equity Ownership. Paterson:Surface Oncology: Employment, Equity Ownership. Davids:Surface Oncology: Research Funding; Celgene: Consultancy; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Consultancy, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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3D-Tissue Engineered Bone Marrow (3DTEBM) Culture Retrospectively Predicts Treatment Clinical Outcomes of Multiple Myeloma Patients

Blood ◽

10.1182/blood-2018-99-118888 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1987-1987

Author(s):

Amanda Jeske ◽

Feda Azab ◽

Pilar De La Puente ◽

Barbara Muz ◽

Justin King ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

In Vitro Models ◽

Advisory Committees ◽

Treatment Plans ◽

Equity Ownership

Abstract Background: Multiple Myeloma (MM) is the second most common hematological malignancy, and continues to be a fatal disease even with the development of novel therapies. Despite promising preclinical data in standard tissue culture models, most drugs fail in clinical trials and show lower efficacy in patients. This highlights the discrepancy between the current in vitro models, the pathophysiology of the disease in the patients, and the urgent need for better in vitro models for drug development and improved prediction of efficacy in patients. We have previously developed a patient-derived 3D-Tissue Engineered Bone Marrow (3DTEBM) culture model, which showed superior properties for proliferation of primary MM cells ex vivo, and better recapitulated drug resistance. The long-term goal of this study is to use the 3DTEBM model as a tool to perform drug screens on BM aspirates of MM patients and prospectively predict the efficacy of different therapies in individual patients, and help treatment providers develop personalized treatment plans for each individual patient. In the current study, we used the 3DTEBM model to, retrospectively, predict clinical responses of MM patients to therapy, as a proof of concept. Methods: We used whole-BM, viably frozen tissue banked samples from 20 MM patients with clear clinical response patterns of complete remission, and either very good partial response (sensitive) or progressive disease (non-sensitive). The BM aspirates were used to develop a 3DTEBM that represents each individual patient. The patient-derived 3DTEBM cultures were treated ex vivo with the same therapeutic regimen that the patient received in the clinic for 3 days. The treatment ex vivo was based on combinations at different concentrations which mimic the steady state concentrations (Css) of each drug. The efficacy of the treatment ex vivo was evaluated by digestion of the 3DTEBM matrix, extraction of the cells, and analysis for prevalence of MM cells in the treatment groups compared to the non-treated controls. Patients were defined "sensitive" if the effect reached 50% killing in the range of 10xCss. The ex vivo sensitivity data was then correlated with the clinical response outcomes. Results: We found that the 3DTEBM was predictive in approximately 80% of the cases (in about 85% of the combination therapy cases, and in about 70% of the single therapy cases). Broken down by individual drug, it was predictive in 80% of the cases treated with Bortezomib, 78% Lenalidomide, 84% Dexamethasone, 100% Daratumumab, 50% Carfilzomib, 50% Pomalidomide, and 100% Doxorubicin. Conclusions: The 3DTEBM is a more pathophysiologically relevant model which predicts clinical efficacy of drugs in multiple myeloma patients, retrospectively. This data provides the bases for future studies which will examine the ability of the 3DTEBM model to predict treatment efficacy, prospectively, for development of personalized treatment plans in individual multiple myeloma patients. Disclosures Jeske: Cellatrix LLC: Employment. Azab:Cellatrix LLC: Employment. De La Puente:Cellatrix LLC: Other: Co-founder. Vij:Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Azab:Ach Oncology: Research Funding; Cellatrix LLC: Equity Ownership, Other: Founder and owner; Glycomimetics: Research Funding; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner.

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Engineered iPSC-Derived NK Cells Expressing Recombinant CD64 for Enhanced ADCC

Blood ◽

10.1182/blood-2020-139760 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-11

Author(s):

Kate Dixon ◽

Robert Hullsiek ◽

Kristin Snyder ◽

Zachary Davis ◽

Melissa Khaw ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Target Cells ◽

Advisory Committees ◽

High Affinity ◽

Adcc Assay

Natural killer (NK) cells are innate cytotoxic lymphocytes. They target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and also recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). While ADCC by NK cells is a key mechanism of several clinically successful therapeutic monoclonal antibodies (mAbs), most patients exhibit or acquire resistance to mAb therapies. ADCC by human NK cells is exclusively mediated by the IgG Fc receptor, CD16A (FcγRIIIA). Studies have demonstrated that increasing the binding affinity between CD16A and therapeutic mAbs can augment their clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we are interested in enhancing the ADCC potency of NK cell-based therapies for various malignancies. CD64 is the only high affinity FcγR family member and binds to the same IgG isotypes as CD16A (IgG1 and IgG3) but with > 30-fold higher affinity. CD64 (FcγRI) is normally expressed by certain myeloid cells but not by NK cells. We generated a recombinant version of this receptor consisting of the extracellular region of CD64 and the transmembrane and intracellular regions of human CD16A, referred to as CD64/16A (figure 1A). An important feature of CD64/16A is that due to its high affinity state, soluble monomeric anti-tumor mAbs can be pre-adsorbed to engineered NK cells expressing the recombinant FcγR, and these pre-absorbed mAbs can be switched or mixed for universal tumor antigen targeting (figure 1B). The engineered NK cells used in our study were derived from genetically edited and clonally derived induced pluripotent stem cells (iPSCs) through a series of stepwise differentiation stages (figure 2). Engineered iPSC-derived NK (iNK) cells can be produced in a uniform and clinically scalable manner (figure 2). In Figure 3, using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against SKOV3 cells, an ovarian adenocarcinoma cell line, in the presence of the anti-HER2 therapeutic mAb trastuzumab (Herceptin) or anti-EGFR1 therapeutic mAb cetuximab (Erbitux), when either added to the assay or pre-adsorbed to the iNK cells (figure 3). Considering the high affinity state of CD64, we examined the effects of free IgG in human serum on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis System, ADCC was evaluated in the presence or absence of 5% human AB serum, in which free IgG was approximately 50-fold higher than the IgG saturation level of the CD64/16A receptors on iNK cells (data not shown). Despite the high levels of excess free IgG, iNK-CD64/16A cells mediated efficient ADCC when Herceptin was either added to the assay or pre-adsorbed to the cells (figure 4). ADCC assays were also performed with Raji cells, a Burkitt lymphoma cell line, as target cells and the therapeutic mAb rituximab (Rituxan). iNK-CD64/16A cells were added with or without pre-adsorbed Rituxan and the assay was performed in 10% AB serum. Again, iNK-CD64/16A cells mediated effective target cell killing in the presence of serum IgG (figure 5), demonstrating that saturating levels of free IgG did not prevent ADCC. To determine if we can further optimize the function of recombinant CD64, we engineered CD64 with the transmembrane regions of CD16A or NKG2D and signaling/co-signaling domain from CD28, 2B4 (CD244), 4-1BB (CD137), and CD3ζ (figure 6). CD64/16A signals by non-covalent association with the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling adapters CD3ζ and FcRγ found in the cell membrane, whereas the other recombinant CD64 constructs use ITAM and non-ITAM regions to mediate their signaling. The various recombinant CD64 constructs were initially expressed in NK92 cells (lacks expression of endogenous FcγRs) (figure 7). Using the Delfia® ADCC assay system, we examined the function of each recombinant CD64 construct and found all combinations are able to effectively induce ADCC (figure 8). We are in the process of generating iNK cells with these constructs and testing their ability to kill hematologic and solid tumors in vitro and in vivo. Our goal is to utilize this docking approach to pre-absorb mAbs to iNK cells for adoptive cell therapy. The mAbs would thus provide tumor-targeting elements that could be exchanged as a means of preventing tumor cell escape by selectively and easily altering NK cell specificity for tumor antigens. Figure Disclosures Lee: Fate Therapeutics, Inc.: Current Employment. Chu:Fate Therapeutics: Current Employment. Abujarour:Fate Therapeutics, Inc: Current Employment. Dinella:Fate Therapeutics: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding.

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BCL-2, a Therapeutic Target for High Risk Hypodiploid B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.280.280 ◽

2016 ◽

Vol 128 (22) ◽

pp. 280-280 ◽

Cited By ~ 5

Author(s):

Ernesto Diaz-Flores ◽

Evan Q. Comeaux ◽

Kailyn Kim ◽

Kyle Beckman ◽

Kara L. Davis ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Advisory Committees ◽

Chromosomal Number ◽

Patient Derived Xenograft

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. Specific genetic subsets, including hypodiploid ALL, are associated with particularly high rates of relapse. Despite the poor outcomes of hypodiploid B-ALL with traditional therapeutic approaches, there have been no known effective alternative therapies or novel candidates tested to improve outcome. We hypothesized that new therapeutic targets could by identified by integrated biochemical and genomic profiling, combined with functional drug assays in order to determine which pathways play an essential role in transformation. For biochemical profiling, we analyzed multiple pathways commonly deregulated in leukemias using phosphoflowcytometry (including receptor tyrosine kinases, JAK/STAT, MAPK, PI3K, PTEN, Bcl-2 survival and pro-apoptotic family members and p53). We subjected hypodiploid cell lines (NALM-16, MHH-CALL2) and patient derived xenograft samples in vitro to inhibitors against each of these pathways (PP2:Src family;Ruxolitinib: JAK/STAT; PD235901/CI1040: MAPK; GDC-0941, PI-90, PI-103, p110 (a, b, g, d): PI3K isoform specific; PP-242:mTOR; ABT-263/ABT-737: Bcl-2/Bcl-xl, and ABT-199: Bcl-2 specific). We found that the Bcl-2 inhibitors (ABT-263, ABT-737 and ABT-199) and to a lesser extent PI3K pathway inhibitors GDC-0941 and PP-242, but not the MAPK or RTK inhibitors, efficiently reduced proliferation of hypodiploid cells. However, only ABT-263/ABT-199 induced high levels of apoptosis at nanomolar concentrations. Based on the consistent efficacy observed with ABT-199 against hypodiploid patient-derived cells and cell lines in culture, we selected eight cryopreserved, previously xenografted (F3 generation) hypodiploid patient samples (4 low hypodiploid, chromosomal number between 32 and 39; and 4 Near Haploid, chromosomal number between 24 and 31) and three non-hypodiploid patient samples (Ph-positive,Ph-Like and Erg+) for a preclinical trial in immunodeficient mice. Each patient sample was engrafted into six mice, which were randomized to receive vehicle or ABT-199 daily over 60 days (Figure 1). Treatment started when the peripheral blood (PB) human CD45 count reached 15%. A rapid decrease in PB blasts was noted at 7 days (Figure 1). Eighty-five percent of the hypodiploid xenografts survived 60 days with either undetectable or low levels of leukemia in the PB. In contrastPh+ andPh-Like xenografts died within 10-20 days regardless of treatment. Importantly, hypodiploid leukemic blasts gradually emerged after discontinuing ABT-199 after 60 days. Additionally, despite low or undetectable levels of leukemic blasts in PB and reduced levels in bone marrow and spleen, all mice had high percentages of leukemic cells in the liver (Figure 2). In conclusion we have identified the survival protein Bcl-2 as a promising molecular target in hypodiploid B-ALL. ABT-199 for dramatically reduced leukemia cells in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. However, the liver represented a protective niche for these leukemias. In addition, our biochemical characterization of the organ infiltrating blasts collected from mice on trial indicate that the sensitivity of hypodiploid ALL to ABT-199 relies not only on high levels of Bcl-2 and deficiency for other survival proteins such as Bcl-xl but also on high levels of proapoptotic proteins, providing two different signatures that correlate with response to ABT-199. Using genome editing (CRISPR/Cas9) we interrogated the necessity for individual proapoptotic genes, including PUMA, NOXA, and BAD, for ABT-199-induced cell death. This study provides encouraging preclinical data that Bcl-2 may be a promising target for the treatment of hypodiploid B-ALL. Our studies identify signature biomarkers that correlate with drug response and identify essential proteins mediating ABT-199-induced cell death. Importantly, this report also identifies the limitations of using ABT-199 as single drug, and provides the rationale for using combinatorial therapies in order to improve the efficacy of the drug. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

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L-Stereoisomer RNA Oligonucleotide Anti-SDF-1 (Nox-A12) Disrupts the Interaction of Multiple Myeloma Cells with the Bone Marrow Milieu In Vivo, Leading to Enhanced Sensitivity to Bortezomib

Blood ◽

10.1182/blood.v118.21.887.887 ◽

2011 ◽

Vol 118 (21) ◽

pp. 887-887

Author(s):

Aldo M Roccaro ◽

Antonio Sacco ◽

Phong Quang ◽

AbdelKareem Azab ◽

Patricia Maiso ◽

...

Keyword(s):

Bone Marrow ◽

Confocal Microscopy ◽

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Ex Vivo ◽

In Vivo Confocal Microscopy ◽

Advisory Committees

Abstract Abstract 887 Background. Stomal-cell-derived factor 1 (SDF-1) is known to be involved in bone marrow (BM) engrafment for malignant tumor cells, including CXCR4 expressing multiple myeloma (MM) cells. We hypothesized that de-adhesion of MM cells from the surrounding BM milieu through SDF-1 inhibition will enhance MM sensitivity to therapeutic agents. We therefore tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vivo and in vitro. Methods. Bone marrow (BM) co-localization of MM tumor cells with SDF-1 expressing BM niches has been tested in vivo by using immunoimaging and in vivo confocal microscopy. MM.1S/GFP+ cells and AlexaFluor633-conjugated anti-SDF-1 monoclonal antibody were used. Detection of mobilized MM-GFP+ cells ex vivo has been performed by flow cytometry. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence detection, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12 followed by bortezomib. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–200nM) treated primary MM BM stromal cells (BMSCs), in presence or absence of bortezomib (2.5–5nM), were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software (combination index: C.I. according to Chou-Talalay method). Results. We first showed that SDF-1 co-localizes in the same bone marrow niches of growth of MM tumor cells in vivo. NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BM stromal cells in vitro. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the PB of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 (50–100 nM) and bortezomib (2.5–5 nM) was observed (C.I.: all between 0.57 and 0.76). These findings were validated in vivo: tumor burden detected by BLI was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 followed by bortezomib was observed as compared to bortezomib alone treated mice (P <.05). Similarly, NOX-A12 + bortezomib combination induced significant inhibition of MM cell homing in vivo, as shown by in vivo confocal microscopy, as compared to bortezomib used as single agent. Conclusion. Our data demonstrate that the SDF-1 inhibiting Spiegelmer NOX-A12 disrupts the interaction of MM cells with the BM milieu both in vitro and in vivo, thus resulting in enhanced sensitivity to bortezomib. Disclosures: Roccaro: Roche:. Kruschinski:Noxxon Pharma AG: Employment. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Advisory Board, Research Funding.

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a Combination of Ex Vivo and Computational Models Predicts Clinical Response in MM Treatment Combinations of Proteasome Inhibitors, Imids, Nuclear Export Inhibitors and Alkylating Agents

Blood ◽

10.1182/blood.v128.22.3291.3291 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3291-3291

Author(s):

Ariosto Silva ◽

Maria Silva ◽

Timothy Jacobson ◽

Mark B Meads ◽

Allison Distler ◽

...

Keyword(s):

Clinical Response ◽

Board Of Directors ◽

In Silico ◽

Nuclear Export ◽

Research Funding ◽

Ex Vivo ◽

Proteasome Inhibitors ◽

Tumor Burden ◽

Advisory Committees ◽

Genetics Research

Abstract Introduction: Multiple myeloma is a heterogeneous plasma cell neoplasm that remains all but incurable despite significant advances in treatment. We anticipate that the ability to overcome this hurdle resides in personalized strategies designed to specifically recognize, target, and anticipate dynamic tumor subpopulations with variable drug response profiles within an individual. To this end, we have developed a novel multi-disciplinary approach using organotypic drug screening and mathematical modeling to assess drug sensitivity of the different subpopulations within the tumor burden of individual patients and, in turn, provide accurate predictions of clinical outcome to anti-myeloma therapy. Material and methods: We have used a novel combination of ex vivo drug sensitivity assay and mathematical models to predict clinical response of 48 MM patients (11 newly diagnosed and 37 relapsed, 18 females and 30 males, median age 64.5, range 45-77) treated with a combination of proteasome inhibitors and IMIDs (37), nuclear export and topo2 isomerase inhibitors (10), and high dose melphalan (1). MM cells (CD138+) were extracted from fresh bone marrow aspirates and seeded in an ex vivo co-culture model with human stroma in 384-well plates. These cells were exposed to a number of chemotherapeutic and experimental agents (up to 31) for a period of 4 days, during which viability was assessed continuously using bright field imaging and digital image analysis. A mathematical model was used to interpolate the dose response dynamics to each drug, and combined with drug and regimen-specific pharmacokinetic data, generate predictions of clinical response to each individual drug. We have then validated ex vivo-based predictions with actual outcome 90 days post-biopsy. In patients treated with combinations, the mathematical model combined the effect of each single drug assuming additivity. Results: To examine the accuracy of the predicted in silico responses, we have assessed the model according to three increasingly strict standards of accuracy: (A) The model correctly predicted 32 out of 32 responders (100%) and 14 out of 16 non-responders (88%), with an overall accuracy of 96%; (B) According to IMWG stratification, the model correctly stratified 14 out of 16 patients as stable or progressive disease (PD/SD, 88%, the remaining 2 incorrectly predicted as MR/PR), 15 our of 18 as minimal or partial response (MR/PR, 83%, the remaining 3 incorrectly predicted as VGPR/CR), and 10 out of 14 patients as very good partial response or complete response (71%, the remaining 4 incorrectly classified as MR/PR), with an overall accuracy of 81%; (C) The 48 patients from this study provided a total of 120 measures of tumor burden (M-spike or SFLC) within the 90-day post-biopsy period. The direct correlation between tumor burden measures and model predictions led to a Pearson r=0.5547 (P<0.0001) and the correlation line [Actual]=0.8282*[Model]+16.27, where [Model] and [Actual] are the predicted and actual tumor burdens as a percentage of tumor burden measure at the moment of treatment initiation. From the 14 non-responders, the model predicted that 2 would have had a VGPR/CR and 3 would have had a MR/PR if treated with drugs differing from those given clinically. Intriguingly, from the 13 patients who received 3-agent therapies with matched in silico drug testing, only 2 had response to all agents, 6 had a response to 2 agents, 4 responded to only 1 of the 3 agents and 1 responded to none of the agents. Next, we examined the 18 patients with a 2-drug match between in silico and actual treatment. From these, 1 patient model responded to 2 drugs, 12 responded to only 1 drug, and 5 responded to none. From the 17 patients with a single agent match between treatment and in silico, 8 had a response and 9 had no response. In summary, among the drugs tested both ex vivoand actually administered to patients, 48% had some predicted clinical benefit, while 52% of agents had none, and could theoretically be removed without affecting clinical outcome. Conclusion: We observed an excellent correlation between in silicopredicted and clinically observed responses in 48 MM patient specimens. Our data suggest that this model may provide critical insight in the selection the appropriate therapeutic agents and number of agents to combine for a given individual. Further validation is required to better define the role of this approach as a clinical decision support tool. Figure Figure. Disclosures Baz: Novartis: Research Funding; Signal Genetics: Research Funding; Karyopharm: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda/Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Merck: Research Funding. Shain:Signal Genetics: Research Funding; Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Speakers Bureau.

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Assessment of Minimal/Measurable Residual Disease Testing in Acute Myeloid Leukaemia By Molecular Methods in an Interlaboratory Study

Blood ◽

10.1182/blood-2021-152259 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3461-3461

Author(s):

Stuart Scott ◽

Richard Dillon ◽

Christian Thiede ◽

Sadia Sadiq ◽

Ashley Cartwright ◽

...

Keyword(s):

Cell Line ◽

Board Of Directors ◽

Research Funding ◽

Residual Disease ◽

Standard Of Care ◽

Negative Sample ◽

Research Support ◽

Advisory Committees ◽

Log Reduction ◽

Measurable Residual Disease

Abstract Background Minimal/measurable residual disease (MRD) testing is increasingly utilised and accepted as standard of care to manage a range of different haematological malignancies. It's use as a surrogate outcome in clinical trials of new therapies is being explored, where it has the potential to accelerate drug assessment and approval. The phenotypic and genetic heterogeneity of acute myeloid leukaemia (AML) has limited the use of MRD in this context; however, the European LeukaemiaNet (ELN) MRD working group have recently published consensus guidelines to standardise both flow cytometric and molecular genetic MRD testing. To assess the accuracy of testing and concordance between laboratories, crucial to patient safety, external quality assessment (EQA)/proficiency testing (PT) is required. Aims To determine the performance of molecular methods for measuring of MRD using the t(8;21)(q22:q22) RUNX1-RUNX1T1, inv(16)(p13q22) CBFB-MYH11, t(15;17)(q24.1;q21.2) PML-RARA and NPM1 Type A markers in an international interlaboratory study. Methods A total of 12 batches of lyophilised EQA material were manufactured. These consisted of three batches of samples for each marker all containing 9x10^6 cells: an MRD 'high' sample; an MRD 'low' sample; and an MRD 'negative' sample. The t(8;21)(q22:q22) RUNX1-RUNX1T1 positive samples were manufactured using the KASUMI-1 cell line, the inv(16)(p13q22) CBFB-MYH11 positive samples using the ME-1 cell line; t(15;17)(q24.1;q21.2 PML-RARA positive samples using the NB4 cell line and the NPM1 Type A (NM_002520.6:c.860_863dup) positive samples using the OCI-AML3 cell line. MRD positive samples were diluted with HL60 cells to achieve the desired MRD level. MRD negative samples were manufactured using the HL60 cell line. The samples were shipped at ambient temperature to the 29 laboratories in 12 countries. Participants were asked to test the blinded samples with their in-house assay and report % normalised ratio of the relevant marker alongside additional methodological and technical data. Results For t(8;21) RUNX1-RUNX1T1, all participants who returned results (n=23) classified the MRD 'high' and MRD 'low' samples as positive and the MRD 'negative' sample as negative. The robust mean log reduction between the MRD 'high' and MRD low sample was 2.7 (range 2.5-2.9). For inv(16) CBFB-MYH11, all participants who returned results (n=22) classified the MRD 'high' sample as positive, 21/22 (95.5%) classified the MRD 'low' sample as positive and 21/22 (95.5%) classified the MRD negative sample as negative. The robust mean log reduction between the MRD 'high' and MRD 'low' sample was 3.16 (range 2.8-4.2). For t(15;17) PML-RARA, all participants who returned results (n=22) classified the MRD 'high' sample as positive, 21/22 (95.5%) classified the MRD 'low' sample as positive and 21/22 (95.5%) classified the MRD negative sample as negative. The robust mean log reduction between the MRD 'high' and MRD 'low' sample was 2.1 (range 1.4-2.4). For NPM1, all participants who returned results (n=23) classified the MRD 'high' as positive, 21/23 (91.3%) classified the MRD 'low' sample as positive and, 17/23 (73.4%) classified the MRD negative sample as negative. The robust mean log reduction between the MRD 'high' and MRD 'low' sample was 3.8 (range 3.2-4.2). Summary/Conclusion The majority of participants in this study were able to detect and accurately quantify MRD when assessing the t(8;21)(q22:q22) RUNX1-RUNX1T1, inv(16)(p13q22) CBFB-MYH11, t(15;17)(q24.1;q21.2) PML-RARA and NPM1 markers, at levels that would be expected within a clinical trial or standard of care setting. A high proportion of participants reported false positive results in the NPM1 marker negative sample. This would have significant consequences clinically, with NPM1 marker false-positivity potentially committing patients to unneeded additional chemotherapy and/or transplant with the attendant risk of morbidity and mortality which highlights the need for ongoing EQA in this area. UK NEQAS LI will work with laboratories advocating they undertake a root cause analysis process to identify the source(s) of error contributing to false positive NPM1 marker results and support their subsequent corrective actions; sharing any educational findings with all participants. Figure 1 Figure 1. Disclosures Scott: Novartis: Research Funding; Biorad: Research Funding. Dillon: Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events; Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Jazz: Other: Education events; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees. Whitby: Roche: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Other: Teaching.

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Tumor Associated Macrophages Promoted Fatal Patient Derived Acute Promyelocytic Leukemia In Vivo

Blood ◽

10.1182/blood-2021-150699 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3472-3472

Author(s):

Isabel Weinhäuser ◽

Diego A Pereira-Martins ◽

Jacobien R Hilberink ◽

Luciana Yamamoto Almeida ◽

Douglas RA Silveira ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Cell Population ◽

Research Funding ◽

Poor Prognosis ◽

Ex Vivo ◽

M2 Macrophages ◽

Advisory Committees

Abstract With immune therapies on the rise, an in-depth understanding of the immunological changes in leukemic bone marrow (BM) niches becomes indispensable. Being an crucial part of the tumor microenvironment (TME) in solid tumours, tumour-associated macrophages are often associated with poor prognosis (Bruni et al. 2020). Yet, in acute myeloid leukaemia (AML) the role of macrophages has not been thoroughly studied. The expression of the M2-markers CD163 and CD206 in the AML BM cell population predicted poor clinical outcome. We identified that this expression emerges from a more mature (CD45 midSSC highHLA-DR +CD14 +CD16 +/-) myeloid cell population (hereafter called AML-associated macrophages - AAM) and not from the leukemic blasts. By employing flow cytometry analysis (FACS) we noted a decrease in the expression of the M1-marker (CD80) and an increase of the M2-markers CD163/CD206on AAM (n=70) compared to healthy donors (HD, n=10). Unsupervised clustering based on the CD163/CD206 levels detected on AAM generated 4 distinct clusters, whereby patients within the CD163 low/CD206 low cluster displayed better overall survival than the other clusters. In vitro, the co-culture of HD-derived M1 macrophages and AML primary/cell lines reduced AML growth via apoptosis induction and cell cycle arrest, while M2-macrophages promoted AML survival and phagocytosis/drug-resistance when treated with FLT3/BCL2 inhibitors. Primary AML cells were also able to repolarize M1- into M2-macrophages, suggesting that leukemic cells actively remodel their microenvironment. Next, we evaluated the impact of M2-macrophages on leukemogenesis in a patient derived xenograft (PDX) model, using the notoriously difficult to engraft primary Acute Promyelocytic Leukaemia (APL) cells (n=7 patient samples). Intra-BM injection of M2-macrophages and retro-orbital transplant of primary APL cells induced full-blown APL in NSGS mice. More strikingly, ex vivo culture of APL cells on M2-macrophages (48h) was sufficient to "train" these cells to engraft and induce fatal APL. Maintenance of self-renewal was shown in a secondary transplant and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays. To identify the biological changes acquired by leukemic blasts, we performed RNA sequencing comparing AML/APL samples at diagnosis to cells that were "trained" (48 h) on M2-macrophages or on MS5 mesenchymal BM stromal cells. Gene ontology and gene set enrichment analysis on the genes up-regulated upon M2 co-culture were significantly enriched for cell migration, cell cycle progression and oxidative phosphorylation (OXPHOS) signatures. In line with our RNAseq data, we noted improved in vivo homing of primary APL cells to the BM within 18 h post-transplant upon ex vivo M2 co-culture compared to diagnosis (n=7 APL blasts). Concurrently, we detected increased levels of surface protein expression Integrin-α4 (CD49d) and -α5 (CD49e) on APL/AML blast cells after M2 exposure. The CD49d expression remained high in primary and secondary transplants. Using seahorse measurements, we confirmed the increased respiration capacity (basal and maximum) of primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5.FACS analysis revealed that M2-macrophages were able to transfer more mitochondria than MS5 cells to primary AML cells, which could underlie the observed increase in OXPHOS mitochondrial metabolism. Treatment with Etomoxir (50 µM), prevented the gain in functional respiration when AML blast were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures, suggesting increased fatty acid oxidation to drive the OXPHO-like state. Finally, we noted that training on M2 macrophages significantly increased colony formation and endowed the cells with long term proliferation in liquid cultures for over 30 days. Overall, we reveal that the frequency of M2-macrophages is up-regulated in a subgroup of AML patients representing a group with poor prognosis. M2 macrophages can support leukemic growth and therapy-resistance, and support fatal APL in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter adhesion, homing and metabolic characteristics of leukemic blasts to allow efficient engraftment and fatal leukemogenesis. Our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions. Disclosures Silveira: BMS/Celgene: Research Funding; Servier/Agios: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding. Mota: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Technopharma: Speakers Bureau; Bristol Myer Squibb: Speakers Bureau; Bayer: Speakers Bureau; Pfizer: Speakers Bureau; AstraZeneca: Speakers Bureau; Astellas: Speakers Bureau; Ipsen: Speakers Bureau; Amgen: Speakers Bureau.

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Development and Exploitation of a Fully Human and Modular Organotypic Bone Marrow Niche Model to Study the Role of Stroma-Produced Factors in Human MDS

Blood ◽

10.1182/blood-2020-142782 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 23-23

Author(s):

Alexander Schaeffer ◽

Ewelina Czlonka ◽

Irene Tirado-González ◽

Thomas Böse ◽

Jennifer Beauvarlet ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Standard Of Care ◽

Bone Marrow Niche ◽

Advisory Committees ◽

Bone Marrow Biopsies ◽

Primary Bone ◽

Primary Bone Marrow

Background: Myelodysplastic syndromes (MDS) are a heterogenous group of stem cell driven disorders primarily affecting the elderly and characterized by inefficient production of mature blood cells and a high risk (30%) of evolution to secondary acute myeloid leukemia. Despite tremendous progress in the past decade, treatment options for MDS patients remain limited, and primarily address disease symptoms, rather than altering disease course. This points to the urgent need to better understand the pathogenesis of this heterogenous group of syndromes to develop new therapies that address disease vulnerabilities. However, this effort has been largely hampered by the limited availability of model systems that allow the exploration of MDS biology in a fully humanized setting. In recent years, studies from our lab and others, have highlighted the crucial role niche cells play in human MDS, hence reinforcing the notion that MDS is a disease of a tissue rather than hematopoietic cells alone. Therefore, exploration of MDS biology requires the further development of fully human MDS models in which both constituents of the disease, namely hematopoietic and niche cells, are present. Methods: To address this issue we successfully isolated endothelial cells (ECs) and mesenchymal stromal cells (MSC) from bone marrow biopsies obtained from MDS patients or healthy age matched controls, and subsequently utilized them to develop fully human 2D and 3D organotypic niche models, which were successfully used to support normal and MDS HSPCs expansion ex-vivo. The 3D system makes use of a collagen scaffold, as this protein makes up for 90% of the matrix proteins in the bone. Importantly, MSC and EC cultures could be successfully established from several independent donors and immortalized to generate primary cell lines that can be used to reproducibly establish these ex-vivo systems in a robust manner. Moreover, we could show that these niche cells were easily amenable to genetic editing using CRISPR-Cas9 technology as well as modified to carry fluorescent reporter proteins for tracking cellular interactions using live cell imaging and confocal microscopy. Results: In this work, we successfully isolated human mesenchymal and endothelial cells, from primary bone marrow biopsies (MDS and healthy) and established fully human 2D and 3D organotypic co-cultures ex-vivo. Of note, although bone marrow ECs represent an essential component of the hematopoietic niche, they have so far been omitted in previously described human bone marrow niche models, owing to the notorious difficulties in isolating and expanding this cell type from primary bone marrow biopsies. Therefore, we established immortalized EC lines (iECs) that faithfully recapitulate the morphological, phenotypic and functional features of primary bone marrow ECs. When cultured at defined ratios and under defined conditions, MSCs instructed ECs and iECs to form of vessel-like structures that mimic the meshwork observed in vivo and are typically escheated by aSMA positive cells that stabilize the structures. Genetic manipulation of the cellular components of the niche also allowed to explore the functional relevance of a specific ECM protein, which we previously identified to be significantly upregulated in MSCs isolated from MDS patients, namely the Secreted Protein Acidic and Rich in Cysteine (SPARC). SPARC ablation triggered enhanced proliferation of MDS derived HSPCs and sensitized them treatment with 5-Azacytidine, a standard of care hypomethylating agent used for the treatment of MDS patients. Additional studies are underway to further understand the underlying molecular mechanisms and define a potential druggable target that could sensitize MDS cells to standard of care treatment. Besides gene targeting studies, these organotypic models are also being used to evaluate the relative fitness of MDS and healthy stem/progenitor cells in healthy versus patient derived niches, to explore the contribution of niche components to the establishment of the progressive clonal dominance observed in MDS. Disclosures Bönig: Terumo BCT: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kiadis: Honoraria; Bayer: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fresenius: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Uniqure: Research Funding; medac: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Healthineers: Current equity holder in publicly-traded company; Chugai: Honoraria, Research Funding; Erydel: Research Funding; Miltenyi: Honoraria, Research Funding; Polyphor: Research Funding; Sandor-Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stage: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Platzbecker:Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Götze:Celgene: Research Funding. Medyouf:Bergenbio: Consultancy, Research Funding.

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