Biodistribution and Efficacy of L-Annamycin in a Novel Imageable AML Mouse Model

Annamycin (Ann) is an anti-tumoral anthracycline whose anti-leukemia activity is relatively unaffected by P-glycoprotein-related multidrug resistance. Unlike for the related doxorubicin (DOX), Ann accumulates in multidrug resistant cell lines, which is accompanied by DNA damage and apoptosis. In preclinical toxicology studies, in contrast to DOX, free Ann displayed a greatly reduced cardiotoxicity, while L-Ann appeared to be non-cardiotoxic. A liposomal formulation of Ann, termed L-Annamycin (L-Ann), is currently evaluated in patients with acute myeloid leukemia (AML). Anti-leukemia activity of Ann was demonstrated in several leukemia models as judged by circulating blast cytoreduction and extension of overall survival. However, the efficacy of L-Ann in the microenvironment of the bone marrow and other organ tissues remains unclear. In the current study, we assessed the anti-AML efficacy of Ann in a novel AML model that allows visualizing the dynamics of individual AML cells in vivo by two-photon microscopy. In this model, mouse AML cells bearing the MLL/ENL-FLT3/ITD[p53-/-] mutations co-express high levels of the cyan fluorescent protein mTurquoise2. Upon intravenous infusion of several tens of thousands cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease reliably develops within 2 weeks. Using host mice expressing appropriate fluorescence reporter genes, the bright cyan fluorescence enables sensitive intravital imaging of individual AML cells in the context of organ architecture. Using this model in Thy1-RFP reporter mice expressing red fluorescence in all organ tissues with the blood flow marked by BSA-AF647 fluorescence, we evaluated AML cellularity reduction in the bone marrow and other organs after a single dose of L-Ann as well as in response to chronic treatment. In addition, we assessed the localization of the surviving AML cells at a high spatial resolution. We evaluated the in vivo organ biodistribution of intravenously infused L-Ann in C57BL6 mice by flow cytometry and two-photon microscopy based on the intrinsic fluorescence of the drug. In addition, we visualized the intracellular compartmentalization of L-Ann using confocal microscopy. Consistent with in vitro findings, we observed a rapid and deep reduction of AML blasts in the peripheral blood after a single dose of L-Ann in a dose-dependent manner (1-4 mg/kg). This reduction was strongly correlated with prolongation of animal survival from 14 days (vehicle) to 37 days (L-Ann 4 mg/kg once weekly started on day 10). In vitro and intravital microscopy revealed a distinct pattern of L-Ann distribution in organ tissues, which correlated in part with the local index of AML cellularity reduction and residual disease localization. Interestingly, in addition to the expected uptake of Ann in the cell's nucleus, Ann was also accumulated in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. Administration of L-Ann early in the course of AML resulted in occasional complete responses some of which associated with resistance to AML re-challenge, suggesting capacity for anti-AML immune memory induction. This study confirms the efficacy of the drug in the model setting of syngeneic, immune-competent AML. Besides reinforcing the rationale for further development of Annamycin in AML, this study demonstrates a highly advantageous AML mouse model that is highly informative in studies of AML pharmacology, minimum residual disease (MRD), microenvironment and immunology. Disclosures Fokt: Moleculin Biotech, Inc.: Equity Ownership, Research Funding. Andreeff:Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees. Priebe:Moleculin Biotech, Inc.: Consultancy, Equity Ownership, Research Funding. Zal:VueBio.com: Equity Ownership; BioLineRx: Research Funding; Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding; NIH-CTEP: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding.

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Overcoming Drug Resistance in Myeloma By Synchronized Delivery of Therapeutic and Bone Marrow Disrupting Agents By Nanoparticles Targeting Tumor-Associated Endothelium

Blood ◽

10.1182/blood-2018-99-118251 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1931-1931

Author(s):

Cinzia Federico ◽

Barbara Muz ◽

Jennifer Sun ◽

Kinan Alhallak ◽

Justin King ◽

...

Keyword(s):

Bone Marrow ◽

Drug Resistance ◽

Tumor Progression ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Equity Ownership ◽

Dual Drug

Abstract Proteasome inhibitors (PIs) have improved the treatment of multiple myeloma (MM) and prolonged patient survival, but several challenges remain to overcome drug-resistance and toxicity. Bone marrow microenvironment (BMM) drives tumor progression and PIs-resistance in MM; and agents that inhibit the interaction between MM and BMM have been shown to re-sensitize MM cells to therapy. However, the synchronized in vivo delivery of BMM-targeting agents with PIs has been a challenge so far. Nanoparticles offer a valuable platform to encapsulate drugs, and if functionalized, they can facilitate specific delivery to tumor, thus improving treatment efficacy and reducing off-target effects. Within the BMM, the endothelium plays a relevant tumor promoting role. By analyzing the expression of an array of markers in normal and in MM-related endothelium, we found high levels of P-selectin expression on MM-activated endothelial cells (ECs) than normal cells and on ECs collected from the BM of either MM patients or MM-bearing mice compared to their respectively healthy BMMNCs. We next sought to develop lipid nanoparticles (LNPs) targeting the MM-related endothelium, loaded with both PI and BMM-targeting agent for synchronized delivery and reversal of the BMM-induced drug resistance. At this aim, we developed targeted LNPs towards P-selectin by decorating their surface with P-selectin-glycoprotein-ligand-1 (PSGL-1). PSGL-1-targeted LNPs showed specific binding to recombinant P-selectin than identically non-targeted particles, and to MM-associated endothelium compared to healthy endothelium, both in vitro and in vivo. To reverse BMM-induced resistance, LNPs were loaded with bortezomib (BTZ) together with a BMM disrupting agent, ROCK-inhibitor (Y-27632) that inhibits the downstream signaling of the RhoA GTPase pathway, known to be instrumental to the interaction of MM cells with BMM. Consequently, we tested the effect of synchronized delivery of BTZ and Y-27632 in the same LNP on MM cell survival in co-culture with the BMM in vitro. While Y-27632-loaded LNPs did not affect cell proliferation, LNPs loaded with both Y-27632 and BTZ enhanced responsiveness of MM cells to BTZ, compared to BTZ-loaded LNPs, thus overcoming the BMM-induced resistance. Mechanistically, we observed more significant inhibition of PI3K and MAPK signaling, decrease of pRb and up-regulation of p21 and induction of pro-apoptotic pathway (caspase-3, caspase-9 and PARP) by drug-loaded LNPs, compared to free drugs. In addition, drug-loaded LNPs were able to decrease adhesion and impair the migration of MM cells to ECs. We also investigated the in vivo efficacy of BTZ/Y-27632-loaded PSGL-1-targeted LNPs in a humanized murine model of MM. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls (vehicle, BTZ and Y-27632 alone or in combination as free drugs, or encapsulated in non-targeted or in PSGL-1-targeted LNPs) demonstrating that both P-selectin targeting and combination of Y-27632 with BTZ reverses the BMM-induced drug resistance and enhances the efficacy of therapy in vivo. Altogether, our data demonstrate the ability of PSGL-1-decorated LNPs to specifically target MM-BMM; to efficiently encapsulate and deliver drugs to tumor tissue; to overcome BMM-induced drug resistance in vitro and in vivo, to reduce tumor growth and prolong overall survival. This study provides the preclinical basis for future clinical trials using MM-BMM-targeted nanomedicine able to enhance the effect of PIs or other drugs for the treatment of MM. Disclosures Roccaro: GILEAD: Research Funding; AMGEN: Other: Advisory Board. Vij:Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: 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; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Azab:Cellatrix LLC: Equity Ownership, Other: Founder and owner; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner; Ach Oncology: Research Funding; Glycomimetics: Research Funding.

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Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽

10.1182/blood-2018-99-118367 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1442-1442

Author(s):

Xiangmeng Wang ◽

Po Yee Mak ◽

Wencai Ma ◽

Xiaoping Su ◽

Hong Mu ◽

...

Keyword(s):

Progenitor Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Single Agent ◽

Advisory Committees ◽

Equity Ownership ◽

Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

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LNA Anti-MicroRNA-155: A Novel Therapeutic Strategy in Waldenstrom Macroglobulinemia and Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.2728.2728 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2728-2728

Author(s):

Yong Zhang ◽

Christopher P. Rombaoa ◽

Aldo M Roccaro ◽

Susanna Obad ◽

Oliver Broom ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Tumor Cells ◽

Stromal Cells ◽

Research Funding ◽

Mrna Levels ◽

Advisory Committees ◽

Qrt Pcr

Abstract Abstract 2728 Background. We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods. WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results. The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn't exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary. A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures: Roccaro: Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial: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; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: 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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Long-Term Survival in Adult Patients with Relapsed/Refractory B-Precursor Acute Lymphoblastic Leukemia (ALL) Who Achieved Minimal Residual Disease (MRD) Response Following Anti-CD19 BiTE® Blinatumomab

Blood ◽

10.1182/blood.v124.21.2287.2287 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2287-2287 ◽

Cited By ~ 4

Author(s):

Gerhard Zugmaier ◽

Nicola Goekbuget ◽

Andreas Viardot ◽

Matthias Stelljes ◽

Svenja Neumann ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Residual Disease ◽

Advisory Committees ◽

Term Survival ◽

Long Term Survival ◽

Equity Ownership

Abstract Introduction: Relapsed/refractory (r/r) B-precursor ALL in adults has an unfavorable prognosis with a median overall survival of 4–8 months and a 5-year survival of <10%. Long-term follow-up data are presented from an exploratory phase 2 study with blinatumomab, an investigational bispecific T-cell engager (BiTE®) antibody construct that directs cytotoxic T-cells to CD19-expressing target cells (Topp MS et al. Blood 2012;120(21):670). Methods: The primary endpoint was hematologic complete remission (CR) or CR with partial hematologic recovery (CRh*) within 2 cycles of blinatumomab. Secondary endpoints included rate of minimal residual disease (MRD) response (defined as < 10-4), overall survival (OS), and relapse-free survival (RFS). Blinatumomab was administered by continuous intravenous infusion for 28 days followed by a 14-day treatment-free interval. Responding patients had the option to receive 3 additional cycles of treatment or to proceed to allogeneic hematopoietic stem cell transplantation (aHSCT). Results: 36 patients were treated; 25 (69%) responded, with 15 (42%) achieving CR and 10 (28%) CRh*. MRD response was achieved in 22 (88%) of these 25 patients with CR or CRh*. Thirteen patients with CR or CRh* proceeded to aHSCT after blinatumomab treatment. In addition, one patient with hypocellular bone marrow and MRD response after the first cycle underwent aHSCT. Follow-up for RFS is 22.4 months; median RFS is 8.8 months. Median follow-up for OS is 30.2 months; median OS is 12.9 months. Ten patients (28%) are alive at 29.7 months (Figure). We analyzed the characteristics of the 10 living long-term survivors, defined as OS of 2 years or longer, seven of whom were relapse-free. The age of these 10 patients at the time of first infusion ranged from 21 to 72 years; the blast count at screening ranged from 8% to 97% (median, 56%). Four of the 10 patients alive had received aHSCT prior to blinatumomab treatment. Of the six patients without a prior aHSCT, two were primary refractory; two had the first relapse within 12 months and two after 12 months post first diagnosis. In the 10 surviving patients blinatumomab treatment induced CR in seven patients, CRh* in two patients, and blast-free hypo-cellular bone marrow in one patient. All 10 surviving patients had an MRD response following blinatumomab treatment. The patient with hypocellular bone marrow received a transplant after the first cycle before potential recovery of blood counts qualifying for CR/CRh* could occur. Seven of the surviving patients underwent aHSCT after blinatumomab, including four patients who received a second aHSCT after they had already received an aHSCT prior to blinatumomab. One of the three patients who did not undergo aHSCT after CRh* had grade 4 cytokine release syndrome requiring resuscitation after 1 day of blinatumomab treatment and has remained in ongoing remission for 22 months without any further treatment aside from 5 cycles of blinatumomab. Another one of these three patients, who had a grade 3 neurologic event on day 2 of cycle 2, has remained in ongoing remission for 34 months without any further treatment aside from 5 cycles of blinatumomab. The third of these three patients had two CD19-positive relapses after CR following blinatumomab treatment. The patient was retreated with 3 cycles of blinatumomab, resulting twice in CR and MRD response. Two of the 10 surviving patients relapsed after blinatumomab and aHSCT; one patient with a CD 19-negative relapse achieved another hematologic remission by chemotherapy. Summary: These data show that patients with r/r ALL, who achieved MRD response and received subsequent aHSCT following blinatumomab immunotherapy may achieve long-term survival longer than 2 years. Studies with a larger sample size are warranted to confirm these data. Two patients with grade 3 or 4 toxicities showed long-term survival without aHSCT after blinatumomab. Figure Figure. Disclosures Zugmaier: Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Off Label Use: This presentation will discuss the off-label use of blinatumomab, as this agent is not approved for use by the FDA, EMA or any other regulatory authorities.. Goekbuget:Amgen Inc.: Consultancy, Honoraria, Research Funding. Viardot:Amgen Inc.: Membership on an entity's Board of Directors or advisory committees, Travel support Other; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel support, Travel support Other; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Travel support Other. Horst:Amgen Inc.: Honoraria, Research Funding. Brueggemann:Amgen Inc.: Consultancy, Research Funding. Holland:Amgen Inc.: Employment, Equity Ownership. Schmidt:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Mergen:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Bargou:Amgen Inc.: Consultancy, Honoraria. Topp:Amgen Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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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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Predictors of T Cell Expansion and Clinical Responses Following B-Cell Maturation Antigen-Specific Chimeric Antigen Receptor T Cell Therapy (CART-BCMA) for Relapsed/Refractory Multiple Myeloma (MM)

Blood ◽

10.1182/blood-2018-99-119665 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1974-1974 ◽

Cited By ~ 2

Author(s):

Adam D. Cohen ◽

J. Joseph Melenhorst ◽

Alfred L. Garfall ◽

Simon F Lacey ◽

Megan Davis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Advisory Committees ◽

Car T ◽

Equity Ownership

Abstract Background: Relapsed/refractory (rel/ref) MM is associated with progressive immune dysfunction, including reversal of CD4:CD8 T cell ratio and acquisition of terminally-differentiated T cell phenotypes. BCMA-directed CAR T cells have promising activity in MM, but the factors that predict for robust in vivo expansion and responses are not known. In a phase 1 study of CART-BCMA (autologous T cells expressing a human BCMA-specific CAR with CD3ζ/4-1BB signaling domains) in refractory MM patients (median 7 priors, 96% high-risk cytogenetics), we observed partial response (PR) or better in 12/25 (47%) (Cohen et al, ASH 2017, #505). Recently, we demonstrated in CLL pts receiving CD19-directed CAR T cells that certain T cell phenotypes prior to generation of the CAR T product were associated with improved in vivo expansion and clinical outcomes (Fraietta et al, Nat Med 2018). We thus sought to identify pre-treatment clinical or immunological features associated with CART-BCMA expansion and/or response. Methods: Three cohorts were enrolled: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART cells. Phenotypic analysis of peripheral blood (PB) and bone marrow (BM) mononuclear cells, frozen leukapheresis aliquots, and phenotype and in vitro kinetics of CART-BCMA growth during manufacturing were performed by flow cytometry. CART-BCMA in vivo expansion was assessed by flow cytometry and qPCR. Responses were assessed by IMWG criteria. Results: Responses (≥PR) were seen in 4/9 pts (44%, 1 sCR, 2 VPGR, 1 PR) in cohort 1; 1/5 (20%, 1 PR) in cohort 2; and 7/11 (64%, 1 CR, 3 VGPR, 3 PR) in cohort 3. As of 7/9/18, 3/25 (12%) remain progression-free at 11, 14, and 32 months post-infusions. As previously described, responses were associated with both peak in vivo CART-BCMA expansion (p=0.002) as well as expansion over first month post-infusion (AUC-28, p=0.002). No baseline clinical or MM-related characteristic was significantly associated with expansion or response, including age, isotype, time from diagnosis, # prior therapies, being quad- or penta-refractory, presence of del 17p or TP53 mutation, serum hemoglobin, BM MM cell percentage, MM cell BCMA intensity, or soluble BCMA concentration. Treatment regimen given before leukapheresis or CART-BCMA infusions also had no predictive value. We did find, however, that higher CD4:CD8 T cell ratios within the leukapheresis product were associated with greater in vivo CART-BCMA expansion (Spearman's r=0.56, p=0.005) and clinical response (PR or better; p=0.014, Mann-Whitney). In addition, and similar to our CLL data, we found that a higher frequency of CD8 T cells within the leukapheresis product with an "early-memory" phenotype of CD45RO-CD27+ was also associated with improved expansion (Spearman's r=0.48, p=0.018) and response (p=0.047); Analysis of manufacturing data confirmed that higher CD4:CD8 ratio at culture start was associated with greater expansion (r=0.41, p=0.044) and, to a lesser degree, responses (p=0.074), whereas absolute T cell numbers or CD4:CD8 ratio in final CART-BCMA product was not (p=NS). In vitro expansion during manufacturing did associate with in vivo expansion (r=0.48, p=0.017), but was not directly predictive of response. At the time of CART-BCMA infusion, the frequency of total T cells, CD8+ T cells, NK cells, B cells, and CD3+CD56+ cells within the PB or BM was not associated with subsequent CART-BCMA expansion or clinical response; higher PB and BM CD4:CD8 ratio pre-infusion correlated with expansion (r=0.58, p=0.004 and r=0.64, p=0.003, respectively), but not with response. Conclusions: In this study, we found that CART-BCMA expansion and responses in heavily-pretreated MM patients were not associated with tumor burden or other clinical characteristics, but did correlate with certain immunological features prior to T cell collection and manufacturing, namely preservation of normal CD4:CD8 ratio and increased frequency of CD8 T cells with a CD45RO-CD27+ phenotype. This suggests that patients with less dysregulated immune systems may generate more effective CAR T cell products in MM, and has implications for optimizing patient selection, timing of T cell collection, and manufacturing techniques to try to overcome these limitations in MM patients. Disclosures Cohen: Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Janssen: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Kite Pharma: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Seattle Genetics: Consultancy. Melenhorst:Parker Institute for Cancer Immunotherapy: Research Funding; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Garfall:Amgen: Research Funding; Kite Pharma: Consultancy; Bioinvent: Research Funding; Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Vogl:Karyopharm Therapeutics: Consultancy. Pruteanu:Novartis: Employment. Plesa:Novartis: Research Funding. Young:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy, Patents & Royalties, Research Funding; CRC Oncology: Consultancy; Incysus: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Cure Genetics: Consultancy. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AbbVie, Inc: Research Funding; Janssen: Consultancy. Milone:Novartis: Patents & Royalties.

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Bion-1301: A Novel Fully Blocking APRIL Antibody for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2112.2112 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2112-2112 ◽

Cited By ~ 1

Author(s):

John Dulos ◽

Driessen Lilian ◽

Marc Snippert ◽

Marco Guadagnoli ◽

Astrid Bertens ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cell ◽

Board Of Directors ◽

Clinical Development ◽

Advisory Committees ◽

Equity Ownership ◽

Parental Antibody

Abstract A PRoliferation Inducing Ligand (APRIL, TNFSF13), is a ligand for the receptors BCMA and TACI. APRIL serum levels are enhanced in patients diagnosed with Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), and Colorectal Carcinoma correlated with poor prognosis. Our anti-APRIL antibody blocked CLL survival and inhibited mouse B1 hyperplasia in vivo (Guadagnoli et al., 2011). APRIL is produced by cells in the bone marrow niche, including myeloid-derived cells, osteoclasts and plasmacytoid dendritic cells. APRIL critically triggers BCMA in vitro and in vivoto drive proliferation and survival of human MM cells (Tai et al., 2016). Importantly, APRIL induces resistance to lenalidomide, bortezomib and other standard-of-care drugs. Furthermore, APRIL drives expression of PD-L1, IL-10, VEGF and TGFβ forcing an immunosuppressive phenotype on BCMA+ cells. As MM survival, resistance to treatment and the immunosuppressive phenotype can be blocked by neutralizing APRIL (Tai et al., 2016), development of an antibody blocking APRIL provides a novel avenue for the treatment of MM. A novel mouse anti-human APRIL antibody hAPRIL.01A (Guadagnoli et al., 2011) initially discovered using Aduro's B-Select platform, was humanized and further engineered enhancing its stability (designated as BION-1301). The antibody binds to recombinant human APRIL with a KDof 0.4 ± 0.15 nM determined by BioLayer Interferometry and an EC50 of 0.29 ± 0.05 nM by ELISA. The epitope of BION-1301 was mapped to the BCMA and TACI binding site explaining its fully blocking capacity. Blocking potency (IC50) was 1.61 ± 0.78 nM (BCMA) and 1.29 ± 0.89 nM (TACI) respectively, corroborated by potent and complete blockade of APRIL-induced cytotoxicity of BCMA-Fas and TACI-Fas Jurkat transfectants. In vitro, BION-1301 suppressed APRIL-induced B-cell IgA and IgG class switching in a dose-dependent fashion. In vivo, BION-1301 was shown to suppress human APRIL induced T cell-independent B cell responses to NP-Ficoll. Biophysical and functional experiments indicated that BION-1301 recapitulated all characteristics of the mouse parental antibody hAPRIL.01A. To support the clinical development of BION-1301, quantitative assays were developed using several mouse-anti-human APRIL antibodies and shown to detect free and complexed APRIL in human blood samples. Results obtained with assays demonstrate that APRIL can be quantified reproducibly in human sera and overcome the drawbacks of previous assays, such as requirement of polyclonal sera, Ig adsorption, interference by human serum and reduced sensitivity. In conclusion, we have generated and functionally characterized a novel humanized APRIL neutralizing antibody, designated BION-1301. The mechanism-of-action and anti-tumor activity described for the parental antibody hAPRIL.01A in vitro and in vivo strongly support the development of BION-1301 as a single agent or in combination with lenalidomide, bortezomib, and suggest a rationale for combination with checkpoint inhibitors. BION-1301 is expected to enter clinical development in 2017. References:Guadagnoli M, Kimberley FC, Phan U, Cameron K, Vink PM, Rodermond H, Eldering E, Kater AP, van Eenennaam H, Medema JP. Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-cell lymphomas. Blood. 2011 Jun 23;117(25):6856-65Tai YT, Acharya C, An G, Moschetta M, Zhong MY, Feng X, Cea M, Cagnetta A, Wen K, van Eenennaam H, van Elsas A, Qiu L, Richardson P, Munshi N, Anderson KC. APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment. Blood. 2016 Jun 23;127(25):3225-36 Disclosures Dulos: Aduro Biotech Inc.: Equity Ownership. Lilian:Aduro Biotech Inc.: Equity Ownership. Snippert:Aduro Biotech Inc.: Equity Ownership. Guadagnoli:Aduro Biotech Inc.: Equity Ownership. Bertens:Aduro Biotech Inc.: Equity Ownership. David:Aduro Biotech Inc.: Equity Ownership. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Eenennaam:Aduro Biotech Inc.: Equity Ownership. Elsas:Aduro Biotech Inc.: Equity Ownership.

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ACY-241, a Novel, HDAC6 Selective Inhibitor: Synergy with Immunomodulatory (IMiD®) Drugs in Multiple Myeloma (MM) Cells and Early Clinical Results (ACE-MM-200 Study)

Blood ◽

10.1182/blood.v126.23.3040.3040 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3040-3040 ◽

Cited By ~ 8

Author(s):

Ruben Niesvizky ◽

Paul G. Richardson ◽

Nashat Y. Gabrail ◽

Sumit Madan ◽

Andrew J. Yee ◽

...

Keyword(s):

Clinical Trial ◽

Combination Therapy ◽

Board Of Directors ◽

Research Funding ◽

Selective Inhibitor ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Histone deacetylase (HDAC) enzymes are attractive therapeutic targets in oncology, but non-selective HDAC inhibitors have led to dose-limiting toxicities in patients, particularly in combination with other therapeutic agents. Ricolinostat (ACY-1215), a first-in-class orally available HDAC inhibitor that is 11-fold selective for HDAC6, synergizes in vitro and in vivo in models of MM and lymphoma with bortezomib (Santo, Blood, 2012; Amengual, Clin Cancer Res, 2015) or carfilzomib (Mishima, Br J Haematol, 2015; Dasmahapatra, Mol Cancer Ther, 2014). Furthermore, ricolinostat has demonstrated an excellent safety and tolerability profile in phase I trials as an oral liquid formulation (Raje, Haematologica, 2014, Suppl 1). We have now identified ACY-241 as a structurally related and orally available selective inhibitor of HDAC6 that is undergoing clinical evaluation in tablet form. In combination with ricolinostat, the immunomodulatory (IMiD®) class of drugs, including lenalidomide (Len) and pomalidomide (Pom), exhibit striking anti-myeloma properties in a variety of MM models (Quayle, AACR, 2014) and have demonstrated clinical activity in MM patients (Yee, ASH, 2014). In support of our ongoing development of ACY-241, we show here that combination with either Len or Pom leads to synergistic decrease in MM cell viability in vitro. Time course studies demonstrated cell cycle arrest followed by progressive induction of apoptosis after prolonged exposure to Len or Pom. Notably, the addition of ACY-241 to either Len or Pom resulted in synergistic increases in apoptosis of MM cells. At the molecular level, treatment with IMiDs reduced expression of the critical transcription factors MYC and IRF4, which was further reduced by combination treatment with ACY-241. Current studies are exploring the molecular mechanism underlying this effect, which may be a consequence of low level inhibition of HDAC1, 2, and 3 by ACY-241. Prolonged treatment with ACY-241 plus Pom was well tolerated in vivo with no evidence of toxicity, and the combination resulted in a significant extension of survival in a xenograft model of MM. Given the comparable tolerability profiles of ricolinostat and ACY-241 and the similar preclinical activity in combination with IMiDs, a clinical trial (NCT02400242) is currently evaluating ACY-241 in combination with Pom and low-dose dexamethasone in MM patients. Predicated upon the clinical experience with ricolinostat and the non-clinical pharmacokinetics of ACY-241, we designed an expedited first-in-human phase 1a/1b clinical trial of a single cycle of ACY-241 monotherapy followed by ACY-241 in combination with Pom and dexamethasone in MM patients. A merged monotherapy/combination trial design was chosen to grant patients access to combination therapy with an established regimen while enabling insight into the safety, pharmacokinetics, and pharmacodynamics of ACY-241 monotherapy. Patients with relapsed or relapsed-and-refractory MM previously treated with at least two cycles of Len and a proteasome inhibitor were eligible for this trial. The first patient was enrolled in June 2015. This patient tolerated monotherapy well and pharmacokinetics showed maximal plasma levels of ACY-241 in the micromolar range, consistent with predictions. An update on enrollment, pharmacokinetic and pharmacodynamic profiles as well as safety of monotherapy and combination therapy will be provided. Disclosures Niesvizky: Celgene: Consultancy, Speakers Bureau. Richardson:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Gabrail:Onyx: Honoraria, Speakers Bureau; BI: Honoraria, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Honoraria, Speakers Bureau. Madan:Onyx: Speakers Bureau; Celgene: Speakers Bureau. Quayle:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Almeciga-Pinto:Acetylon Pharmaceuticals, Inc: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Houston:Acetylon Pharmaceuticals, Inc: Employment. Hayes:Acetylon Pharmaceuticals, Inc: Employment. Van Duzer:Acetylon Pharmaceuticals, Inc: Employment. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Trede:Acetylon Pharmaceuticals, Inc: Employment. Raje:Acetylon: Research Funding; Celgene Corporation: Consultancy; BMS: Consultancy; Amgen: Consultancy; Millenium: Consultancy; AstraZeneca: Research Funding; Novartis: Consultancy; Onyx: Consultancy; Eli Lilly: Research Funding; Takeda: Consultancy.

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Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Blood ◽

10.1182/blood.v126.23.1329.1329 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1329-1329

Author(s):

Jessica Leonard ◽

Joelle Rowley ◽

Brandon Hayes-Lattin ◽

Jeffrey W. Tyner ◽

Marc Loriaux ◽

...

Keyword(s):

Older Adults ◽

Cell Line ◽

Board Of Directors ◽

Research Funding ◽

Mononuclear Cells ◽

Annexin V ◽

Advisory Committees ◽

Equity Ownership

Abstract Introduction: Treatment of adult Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) remains a challenge. While the addition of the targeted tyrosine kinase inhibitors (TKI) to standard cytotoxic therapy has greatly improved upfront treatment, treatment related mortality in older adults remains high. A novel induction regimen combines the targeted dual Abl/Src TKI Dasatinib (Sprycel, BMS) with a corticosteroid. After the first 21 days of induction the corticosteroids are tapered due to significant toxicities, particularly in older adults. Unfortunately, remaining on TKI monotherapy renders patients susceptible to the development of TKI resistance and thus identifying targeted agents that could enhance the activity of TKIs is urgently needed. Recently a novel and selective inhibitor of BCL-2, ABT-199 (Venetoclax, AbbVie) has shown impressive activity against other lymphoid malignancies including CLL and NHL. Here we describe the pre-clinical and in vivo efficacy of ABT-199 in combination with dasatinib in Ph+ ALL and propose its potential use in future clinical trials. Methods: Drug efficacy in vitro was determined using the Ph+ ALL cell line SupB15, primary Ph+ ALL sample (12-149), the dasatinib sensitive Pre-B ALL cell line RCH and the CML cell line K562. Cells were treated with dasatinib, ABT199 or in combination for 72 hours. Cell viability was assessed with the colorimetric MTS assay and apoptosis was assessed with annexin V staining. Expression of the BCL family proteins BCL-2 and MCL-1 were assessed via immunoblot. Immunodeficient NSG mice were injected with 12-149, then one week later treated with vehicle, 5 mg/kg dasatinib, 5 mg/kg ABT-199, or the combination daily for 5 days each week. Peripheral blood was obtained every 1-2 weeks to assess for engraftment as defined by the presence of >10% human CD45+ cells in the peripheral blood. Once engrafted, mice were euthanized and examined. Mononuclear cells were extracted and assessed for BCL2 and MCL1 expression. Statistical methods were performed using Calcusyn and PRISM. Results: Susceptibility to BCL2 inhibition: Of the dasatinib sensitive cells tested, SupB15 and 12-149 cells were susceptible to ABT-199 while RCH and K562 cells were not. The ALL cells expressed BCL-2 while the CML cell line expressed BCLx. SupB15 expressed low levels of the antiapoptotic protein MCL1 while RCH cells had relatively higher levels. siRNA of MCL-1 rendered the RCH cells sensitive to inhibition by ABT-199. In SupB15 cells, treatment with ABT-199 alone led to upregulation of MCL-1 at 24h which was prevented by the combination of dasatinib + ABT199. Synergy in Ph+ ALL: The calculated IC50 of dasatinib and ABT199 in SupB15 were 8.8nM and 5.9nM, respectively. The IC50 of equimolar combination was 0.42nM, and synergistic with combination index (CI) values between 0.15 and 0.49. Primary Ph+ ALL xenograft cells showed a similar pattern of synergy to the dasatinib + ABT199 combination. Combination treatment also greatly increased apoptosis as measured by Annexin V staining. Xenograft Studies: Animals were treated with a ten-fold lower dose of dasatinib and ABT199 from prior published data. There was no significant difference in time to engraftment or disease burden between vehicle or single agent ABT-199. In contrast, less than one half of the animals treated with dasatinib engrafted by 90 days while none of the animals treated with both dasatinib and ABT-199 engrafted. Most intriguing was the decrease in disease burden as measured by splenic size in the combination group compared to all other groups (P<0.0001, one-way ANOVA). Analysis of BCL-2 family proteins from mononuclear cells isolated from untreated animals confirmed upregulation of BCL-2 and relatively low levels of MCL-1. Animals treated with ABT-199 had greatly upregulated levels of MCL-1, while those treated with dasatinib or the combination did not. Conclusions: The combination of ABT-199 with dasatinib synergistically targets Ph+ ALL cells both in vitro and in vivo, laying the foundation for further evaluation in vivo for adult Ph+ ALL. As demonstrated by others, malignancies that are particularly susceptible to BCL targeting are those which display high BCL-2 expression and a low MCL-1: BCL-2 ratio. Combined targeted therapies may offer the potential for greater and longer responses without the morbidity associated with cytotoxic chemotherapy, particularly in older adults. Disclosures Tyner: Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Array Biopharma: Research Funding; Constellation Pharmaceuticals: Research Funding. Druker:Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Novartis Pharmaceuticals: Research Funding; Sage Bionetworks: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Henry Stewart Talks: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotide Pharmaceuticals: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy.

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