In Vivo HSC Gene Therapy for Hemoglobinopathies: A Proof of Concept Evaluation in Rhesus Macaques

Current gene therapy or genome editing studies for hemoglobinopathies require highly sophisticated medical facilities to perform hematopoietic stem cell collections / selections and genetic modifications. In addition, patients receive high-dose chemotherapy to facilitate engraftment of gene-modified cells. Thus, current gene therapy protocols will not be accessible to most patients suffering from hemoglobinopathies. Here we describe a highly portable and scalable approach using in vivo hematopoietic stem cell (HSC) gene therapy to potentially overcome these limitations. The central idea of our in vivo HSC gene therapy approach is to mobilize HSCs from the bone marrow, and while they circulate at high numbers in the periphery, transduce them with an intravenously injected HSC-tropic, helper-dependent adenovirus HDAd5/35++ gene transfer vector system. Transduced cells return to the bone marrow where they persist long-term. Transgene integration is either achieved by a Sleeping Beauty transposase (SB100x) in a random pattern or by homology-directed-repair into a safe genomic harbor site. Currently, an in vivo selection system (involving the mgmtP140K gene/low-dose O6BG/BCNU) is employed to achieve 80-100% marking levels in peripheral blood cells. We demonstrated safety and efficacy of our approach in mouse models for thalassemia intermedia, Sickle Cell Disease, and hemophilia A, where we achieved a phenotypic correction. We now present data in 3 rhesus macaques. We show that treatment with G-CSF/AMD3100 resulted in efficient HSC mobilization into the blood circulation and subsequent intravenous injection of the HDAd5/35++ vector system (total 1-3 x1012 vp/kg, in two doses) was well tolerated. The longest follow up thus far is 24 weeks after in vivo HSC transduction with a human-gamma-globin expressing HDAd5/35++ vector. After in vivo selection with O6BG plus low dose (10 to 20 mg/m2) of BCNU, a dose that is up to 100-fold lower than what is used for autologous transplantation protocols, gamma-globin marking in peripheral red blood cells rose to ~90% and was stable for the duration of the study (see Figure). gamma-globin levels in red blood cells were ~18% of adult alpha1-globin (by HPLC). No abnormalities in genome and transcriptome analyses of animal #1 were found at the time of scheduled necropsy. We show that a new prophylaxis regimen (dexamethasone, IL-6R, IL-1bR antagonists, saline bolus IV) was able to mitigate all side effects associated with intravenous HDAd5/35++ vector administration. Analysis of day 3 bone marrow showed 30% transduced HSCs. Vector DNA biodistribution studies demonstrated very low or absent transduction of most tissues (including testes and CNS). Analysis of bone marrow showed efficient, preferential HSC transduction and re-homing of transduced CD34+/CD90+ cells to the bone marrow. At week 4, about 5% of progenitor colony-forming cells demonstrated stable transduction with integrated vector, and this frequency increased after starting the in vivo selection. The level of human mgmtP140K mRNA expression in PBMCs also increased after in vivo selection. In summary: Using a new and optimized prophylaxis regimen intravenous delivery of HDAd5/35++ was very well tolerated without any cytokine activation. We saw efficient transduction of HSCs and efficient in vivo selection of transduced progenitors with low dose O6BG/BCNU. This is the first proof-of-concept study that in vivo HSC gene therapy could be feasible in humans without the need of high-dose chemotherapy conditioning and without the need for highly specialized medical facilities. This approach would provide a major advance for the gene therapy and genome editing field and allow the necessary portability and accessibility to reach patients in places with limited medical resources. Figure 1 Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Umoja: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees. Lieber:Ensoma, Inc: Consultancy, Research Funding.

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Mgta-145 / Plerixafor-Mediated HSC Mobilization and Intravenous HDAd5/35++ Vector Injection into Mice Allows for Efficient In Vivo HSC Transduction and Stable Gene Marking in Peripheral Blood Cells of CD46-Transgenic and Thalassemia Mice

Blood ◽

10.1182/blood-2020-141485 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-5

Author(s):

Chang Li ◽

Sucheol Gil ◽

Kevin A. Goncalves ◽

John C. Davis ◽

Hans-Peter Kiem ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Board Of Directors ◽

Blood Cells ◽

Day Treatment ◽

Publicly Traded ◽

Advisory Committees ◽

Gamma Globin

Background. In vivo hematopoietic stem cell (HSC) gene therapy represents a simpler approach to treating hemoglobinopathies without the need for myelosuppressive conditioning and autologous HSC transplantation. We developed a helper dependent adenovirus (HDAd5/35++)-based platform that enables efficient in vivo transduction of mobilized HSCs via CD46. Transduced HSCs can be positively selected by low-dose O6BG/BCNU-treatment to achieve ~90% marking rates in peripheral blood. Initial proof-of-concept in murine models as well as in rhesus macaques demonstrates high level of g-globin expression after gene addition by a Sleeping Beauty transposase. While the current mobilization regimen-4 days of G-CSF injection followed by an injection of AMD3100/plerixafor on day 5-mobilizes HSCs from the bone marrow to the periphery, several issues exist. Despite widespread use as a mobilization agent in oncology, G-CSF is contra-indicated in patients with sickle cell disease. Additionally, G-CSF results in unselective bone marrow cell mobilization, which leads to leukocytosis and elevated numbers of cytokine-producing cells in the periphery that come into contact with HDAd particles, leading to high cytokine levels. Mobilized (committed) bone marrow cells in the periphery also sequester HDAd thus reducing the effective dose for primitive HSCs. Further, the five-day treatment regimen and high costs associated with G-CSF + plerixafor justify the development of an alternative mobilization regimen. A single-day, G-CSF-free mobilization regimen that mobilizes a high proportion of HSCs may therefore be preferred for in vivo gene therapy. Results . Here we tested HSC mobilization by truncated MGTA-145, a CXCR2 agonist, and plerixafor in the context of in vivo HSC transduction. CD46-transgenic animals were mobilized with GCSF + plerixafor (5 days) or with MGTA-145 + plerixafor (same-day treatment) and then injected one hour later with an integrating HDAd5/35++ mgmt/GFP vector. MGTA-145 + plerixafor resulted in robust mobilization of HSCs, less leukocytosis and no significant elevation of cytokines, as observed with G-CSF + plerixafor. With both mobilization regimens, after in vivo selection with O6BG/BCNU, >90% of PBMCs expressed GFP and marking rates were stable long-term. Mice were sacrificed 12 weeks after in vivo transduction and bone marrow lineage-negative cells were harvested for transplantation into secondary recipients. Stable transgene expression (>90% at week 16 after transplantation) was observed with both mobilization regimen in secondary recipients and multilineage engraftment was observed with MGTA-145 + plerixafor in primary and secondary recipients. Importantly, the mobilization with MGTA-145 + plerixafor worked efficiently in a mouse disease model for thalassemia (Hbbth3/CD46+/+). In this model, after in vivo transduction with an integrating HDAd5/35++mgmt/gamma-globin vector and in vivo selection, over 95% of peripheral red blood cells (RBCs) expressed human gamma-globin. The gamma-globin protein level reached 36% over mouse beta-globin. Phenotypic analyses showed a complete correction reflected by normal RBC morphology and absence of blood reticulocytosis, extramedullary hemopoiesis and hemosiderin deposition in spleen and liver sections. In secondary recipients of Lin- cells (harvested at week 14 from in vivo transduced Hbbth3/CD46+/+ mice), gamma-globin marking in RBCs was stable at 99% (currently at week 11 after transplantation). This demonstrates that MGTA145 + plerixafor mobilizes long-term repopulating HSCs. Conclusions . These data demonstrate that the combination of MGTA-145 and plerixafor could serve as an efficient and potentially safer one-day mobilization regimen for in vivo HSC gene therapy in patients with hemoglobinopathies. Disclosures Goncalves: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kiem:Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy. Lieber:Ensoma, Inc: Consultancy, Research Funding.

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Improved Gene Therapy for Metachromatic Leukodystrophy

Blood ◽

10.1182/blood-2021-150986 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3979-3979

Author(s):

Lucas Tricoli ◽

Adeline Vanderver ◽

Laura Adang ◽

Maxwell Chappell ◽

Laura Breda ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Metachromatic Leukodystrophy ◽

Mrna Translation ◽

Advisory Committees ◽

Patient Fibroblast

Abstract Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease (LSD) characterized by a decreased Arylsulfatatse A (ARSA) enzymatic activity. The most common form, late infantile MLD, universally results in rapid loss of neurologic function in early childhood. Ex-vivo hematopoietic stem cell (HSC) gene therapy using a lentiviral vector (LV) can improve clinical outcomes by supplying a functional copy of the ARSA cDNA (Biffi A, et al, Science 2013). Unfortunately, this approach is only successful in pre- and minimally symptomatic individuals and only a small subset of individuals are diagnosed during the limited therapeutic window. As such, the development of additional approaches targeting early symptomatic individuals are critically needed. The only clinical vector (CV) approved to treat MLD patients, PawMut6, includes the human ARSA cDNA gene under the control of the human Phosphoglycerate Kinase (PGK) promoter and includes, in the integrating transcriptional unit, the viral sequences Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence to increase titer and mRNA translation (Biffi A, et al, Science 2013). To increase expression of ARSA cDNA at single integration level, we generated several LVs that include the ARSA gene with a variety of insulators to optimize ARSA expression and enhance safety in transduced cell lines. We placed the ARSA cDNA under the control of the human Elongation Factor 1 alpha (EF1-alpha) promoter, which has been shown to promote higher transcription rates in different cell lines, compared to human PGK as shown by Jane Yuxia Qin, PLos One 2010. Our constructs carry versions of the ARSA gene with and without the 5' and 3' untranslated regions (UTR+ or UTR-) and a Traceable Codon Optimized (TCO) modified sequence to distinguish the transgene from the endogenous ARSA. An ankyrin or foamy insulator have been incorporated to minimize genotoxicity caused by integration events. The WPRE has been proven to enhance the performance of viral vectors. However, to prevent WPRE integration in the host genome, we placed it directly after the 3'-self inactivating LTR (SIN-LTR) together with a strong bovine growth hormone polyA signal (for sequence termination) (BGHpA), as shown by Breda L. et al, Mol Ther 2021. We compared the ARSA activity (normalized to vector copy number (VCN)) of our constructs to that of PawMut6, the LV currently used in clinical trial, on MLD primary patient fibroblast cultures. Our top performing vectors, TCO-EAAWP-UTR +, TCO-EAFWP-UTR - and TCO-AEAFWP-UTR - showed 2X, 10X and 4X more ARSA activity, respectively, compared to that generated using PawMut6. We also detected a superior ability of our vectors to secrete functional ARSA enzyme into the culture media of transduced primary MLD patient fibroblast cells, which is a critical modality for transfer of functional ARSA from microglia to oligodendrocytes. Extracellular vesicle isolation, purification, and immunoblot analysis has demonstrated small vesicle secretion is the primary modality by which ARSA is secreted, having significant implication for how we approach treatment of MLD. In parallel experiments on murine HSC, the TCO-AEAFWP-UTR - vector reproduced similar results, with about 4x more ARSA activity. To exclude potential toxicity, we performed bone marrow transplants on WT animals with HSCs transduced at up to 13 copies per genome. Mice transplanted with high VCN transduced bone marrow did not show signs of bone marrow failure or distress; more extensive evaluation of these animal models is ongoing. Clonogenic assays and secondary transplants are in progress. Upon completion of the in-vivo studies in WT mice, at least two of our best vector candidates will be utilized on a MLD mouse model (ARSA-KO) that we generated using CRISPR-Cas9. Analysis will include pathological sections of the CNS, brain lysate collection and sulfatase activity assays. Our studies are currently focused on completing in-vivo validation and toxicity assays to move our best vector to the pre-clinical and IND application. The accumulated data on our novel vectors imply new mechanistic considerations for treatment of MLD and demonstrate utility as a strong approach for treating early symptomatic patients. Disclosures Vanderver: Homology: Research Funding; Takeda: Research Funding; Ionis and Illumina Inc: Research Funding; Biogen: Research Funding; Eli Lily and Company: Research Funding; Orchard Therapeutics: Research Funding; Gilead Sciences Inc: Research Funding. Adang: MEGMA: Consultancy; Orchard Therapeutics: Consultancy; Takeda Pharmaceuticals: Consultancy. Rivella: Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Forma Theraputics: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.

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Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.133.133 ◽

2010 ◽

Vol 116 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Patricia Maiso ◽

AbdelKareem Azab ◽

Yang Liu ◽

Yong Zhang ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Mechanism Of Action ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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Dynamic Regulation of the Level of Hypoxia In the Bone Marrow Regulates Cell Dissemination In Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.4035.4035 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4035-4035

Author(s):

Abdel Kareem A. Azab ◽

Phong Quang ◽

Feda Azab ◽

Brian Thompson ◽

Patricia Maiso ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Progression ◽

Board Of Directors ◽

Tumor Burden ◽

Secondary Antibody ◽

Advisory Committees ◽

Hypoxic Conditions ◽

Mild Hypoxia

Abstract Abstract 4035 INTRODUCTION: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis, implying that progression of MM occurs through continuous interaction between the BM and MM cells, which controls the ability of MM cells to egress out of the BM and home into new BM niches. We have previously shown that the CXCR4/SDF1 axis as well as Rho GTPases downstream of the receptor was important for chemotaxis, adhesion, homing and egress of MM cells. However, the driving force for MM cells to leave the BM and metastasize to other BM sites is not well understood. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and are associated with poor patient prognosis, cell motility, associated angiogenesis and metastasis. In this study, we tested the role of hypoxia in the dissemination of MM cells in vivo, as well as regulation of the retention/egress of MM cells in and out of the BM. METHODS: To test the effect of hypoxia on induction of MM egress, MM1s-GFP+/Luc+ cells were injected into 12 SCID mice, and then mice with different stages of tumor development (based on the tumor size detected by bioluminescence) were treated with the hypoxia marker pimonidazole. Blood was drawn and BM was obtained from the femur. Mononuclear cells were then fixed, permeabilized, and stained with antibodies against pimonidazole, followed with an APC- secondary antibody, PE-mouse-anti-human CXCR4, and anti-cadherin antibody followed by an Alexa-Fluor-594 secondary antibody. MM cells in BM and peripheral blood were identified by gating on cells with high GFP signal. To confirm the effects of severe hypoxia found in vivo compared to physiologic mild hypoxia found in the BM, we tested the effect of mild hypoxic conditions (6% O2) and severe hypoxic conditions (0.5% O2) on MM expression of cadherins and CXCR4, as well on functional adhesion of MM cells to stromal cells and chemotaxis. RESULTS: Twelve mice with different stages of MM tumor progression were used. A bi-phasic correlation between tumor progression and the percent of hypoxic cells in BM was found, showing that severe hypoxic conditions in the BM correlated with tumor burden. The correlation between the tumor burden and the number of circulating cells was not linear; however, a direct linear correlation was observed between the number of circulating MM cells and hypoxia in the BM. Moreover, hypoxia in BM correlated directly with the expression of CXCR4 and negatively correlated with the expression of cadherins in MM cells isolated from the BM. To test the effect of the severe hypoxic conditions induced by tumor progression compared to mild hypoxic conditions found physiologically in the BM, we tested the effect of 0.5% O2 (severe hypoxia) and 6% O2 (mild hypoxia) compared to normoxia (21%) on MM cell adhesion to BMSCs, as well as on chemotaxis in response to SDF1, as well as expression of CXCR4 and cadherins. We found that severe hypoxic conditions decreased MM expression of cadherins and adhesion to BMSCs, as well as increased expression of CXCR4 and chemotaxis to SDF1 compared to cells in normoxia. In contrast, mild hypoxic conditions did not alter the expression of CXCR4 and cadherins, adhesion of MM cells to BMSCs, or chemotaxis of MM to SDF1 compared to normoxic cells. CONCLUSION: Hypoxia in the BM directly correlates with the number of circulating MM cells, and with changes in expression of cadherins and CXCR4 in vivo. Severe hypoxic conditions, but not mild hypoxic conditions, induce hypoxic responses in MM cells. Based on these findings, further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy in MM are warranted. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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A Phase II Study of Pomalidomide, Daily Low Dose Oral Cyclophosphamide, and Dexamethasone in Relapsed/Refractory Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4520.4520 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4520-4520

Author(s):

Ajai Chari ◽

Hearn Jay Cho ◽

Samir Parekh ◽

Kenneth Lau ◽

Gillian Morgan ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Low Dose ◽

Advisory Committees ◽

Common Grade ◽

Metronomic Therapy ◽

Grade 3 ◽

Study Therapy

Abstract Background A treatment option for patients with relapsed/refractory multiple myeloma (RRMM) is pomalidomide(pom) and dexamethasone (dex), with an overall response rate (ORR) of 33% and median progression free survival (PFS) of 4.2 months. Adding the alkylatingagent cyclophosphamide(Cy) to pom and steroids improves ORR and PFS. Baz et al (Blood, 26 May 2016) combined daily pom with weekly dosing ofCy anddex (PCD), with an ORR of 64.7% and a median PFS of 9.5 months, although grade 3/4 neutropenia increased from 31% to 52%. In our experience, compared to weekly Cy, low dose daily oral Cy is better tolerated with less myelosuppression. Palumbo et al (Blood, 17 Oct 2013) in fact combined pom with alternate day dosing ofCy and prednisone, with an ORR of 51% and a median PFS of 10.4 months and a grade 3/4 neutropenia rate of 42%. However, importantly, granulocyte stimulating factor (G-CSF) and platelet transfusion support wereprohibited, resulting in a lower maximum tolerated dose of pom of 2.5 mg (vs 4 mg in the Baz) and therefore, the rates of neutropenia cannot be compared between the two studies. In the present study, we explored PCD at the doses/schedule shown in table 1 with hematologic support even in patients with baselinecytopenias. This type of metronomic therapy has demonstrated efficacy in refractory B cell malignancies, possibly because the anti-angiogenic effects of metronomic therapy may be synergetic with conventional anti-neoplastic agents. Methods This was an open label, single arm, and single center phase 2study. The primary objective was to evaluate the best ORR. Secondary objectives were to evaluate safety, clinical benefit response (CBR), PFS, and overall survival (OS). Inclusion criteria included lenalidomide refractory, pom naïve RRMM patients with at least 2 prior lines of therapy. Patients were required to have measurable disease, adequate performance status, Cr <3 mg/dL, normal hepatic function, and ANC > 1000/uL and platelets > 50,000/uL if bone marrow plasma cells were < 50%, otherwise >30,000/uL. G-CSF and platelet support were permitted during screening and study treatment if needed. Each drug was administered at the doses and schedule shown in Table 1. Results Overall, 28 evaluable patients with progressive disease (PD) at screening have been enrolled. The median age is 66 (57% > 65 yr) with a median of 3 lines of prior therapy over 5 years since diagnosis. 3 (11%) had ANC<1.5 and 2 (7%) hadplts<50,000/µL at study entry.High-risk molecular findings were present in 13 patients (46%), including 3 with del p53 and 6 with gain of 1q21 by FISH (2 with concurrentt(4;14) and 2 with concurrent del p53). With 8 patients still on study therapy, responses include 3 complete responses (CR), 7 very good partial responses (VGPR), 9 partial responses (PR), 3 minor responses (MR), 5 stable disease (SD), and 1 PD, for an ORR of 67%, CBR (i.e. MR or better) of 78% and a median PFS of approximately 14.5 months. The median OS has not been reached. The most common grade 3/4 toxicity (regardless of drug attribution) was neutropenia with 20 (71%) of subjects experienced grade 3/4 neutropenia. Importantly, there was only 1 episode of febrile neutropenia during study therapy. Grade 3/4 thrombocytopenia was seen in 25% of subjects, and 3/4 anemia seen in 18%. The most common grade 3/4 non-hematologic toxicity was pulmonary disease with Grade 3 lung infections occurring in 21% of subjects (3 viral, 2 bacterial, 1 unknown) and 1 additional grade 3 URI. Of note, all of these admissions occurred at local hospitals and none of these occurred in the setting of neutropenia. One additional pt hadpneumonitisattributed to pom requiring study discontinuation. Grade 3rashwas also observed in 14% of subjects leading to pom dose reductions. Correlative data from peripheral blood and bone marrow aspirates taken at baseline, Cycle 3 Day 15, and at disease progression from all patients will be updated at the time of conference. These include PCD-associated changesin gene expression, clonal evolution and immune microenvironment during therapy and on progression. Conclusions With toxicities similar to those in other studies, the ORR of 67% and PFS of 14 months in our study of PCD compares very favorably to pomdexas well as other triplet regimens containingCy. Disclosures Chari: Takeda: Consultancy, Research Funding; Array Biopharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Research Funding; Amgen Inc.: Honoraria, Research Funding. Cho:Genentech Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Agenus, Inc.: Research Funding; Ludwig Institute for Cancer Research: 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, Research Funding. Catamero:Celgene: Honoraria, Speakers Bureau. Verina:Celgene: Speakers Bureau. Jagannath:Bristol Myer Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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Anti-CD74 Antibody hLL1 Is Associated with Lymphoma and Chronic Lymphocytic Leukemia Cell Effects In Vivo

Blood ◽

10.1182/blood.v118.21.1796.1796 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1796-1796

Author(s):

Felipe Samaniego ◽

Jillian F Wise ◽

Luis Fayad ◽

Michelle Fanale ◽

Fredrick Hagemeister ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Bone Marrow Involvement ◽

Leukemia Cell ◽

B Cell Lymphoma ◽

Cancer Development ◽

Antigenic Peptides ◽

Advisory Committees

Abstract Abstract 1796 Background: CD74 is the invariant chain of the MHCII complex and has an established role as a chaperone protein, which also prevents premature loading of antigenic peptides to the MHC. More recently, CD74 has been implicated as having a role in cancer development by its mediating the prosurvival signaling of the ligand macrophage migration inhibitory factor (MIF) stimulation and it's associated higher level expression in chemoresistant cancers. Blocking of CD74, the receptor for MIF, or its downstream target, a4b1 integrin has been reported to block the homing of CLL cells to bone marrow. The hLL1 antibody (Milatuzumab; Immunomedics, Inc, Morris Plains, NJ) shows promising anti-cancer activity, with eradiation of lymphoma and myeloma xenografts in mice through an undefined mechanism of action. Because of this apparent central role in cancer development, we examined CLL cells of patients (pts) treated with humanized anti-CD74 antibody hLL1. Study Design: Adults with relapsed non-Hodgkin's lymphoma or CLL, who had at least one prior therapy, were enrolled on this phase I-II trial. Patient criteria; ANC 1000, hemoglobin 9, platelets 50,000, adequate organ function, ECOG performance of 1–2, provided informed consent. The hLL1 antibody, in 4 or 8 mg/kg dosages, was administered intravenously over 4 hours, two or three times per wk for 4 wks. Results: Seven pts (4 male) with a median age 65 (range 57–72) with a median 2 (range 1–4) prior chemotherapy treatments were enrolled. Histologies included showed 1 pts with large B cell lymphoma, 5 with pts with SLL/CLL (5) and 1 with marginal zone lymphoma. All pts completed therapy except one who received 5 of 8 doses of planned therapy; 2 pts received dose escalated hLL1 at 8 mg/kg. The hLL1 was well-tolerated with some infusion related side effects, which responded to medications. All other patients maintained adequate hematologic indices, which did not prevent completing the initial therapy plan. Tumor responses were: one partial response, 1 progression, 1 non-evaluable, and 4 with stable disease. Pts' tumor cells were analyzed by reverse-phase protein analysis for activation status of 120 signaling proteins. In 4 of 4 pts with CLL, hLL1 therapy was associated with consistently lower phosphorylated levels of Akt T308 and Notch3; but whether this effect was caused by other concurrent medications could not be ruled out. All the pts who had elevated lymphocyte counts (with bone marrow involvement) showed a rise in lymphocyte counts (Figure) during treatment with hLL1 therapy and then returned to baseline or below baseline after completing infusion. Interestingly, the blocking of CD74 has been shown to interfere with the homing of CLL cells to bone marrow through the binding of CD74-dependent a4b1 integrin. The binding of CLL a4b1 integrin to stroma is a potent cell survival stimulus. To ascertain this mechanisms of CD74 hLL1 antibody induced release of lymphocytes, we analyzed migration of CLL cells in transwell towards MIF. CLL cells without hLL1 exposure had a 2–3 fold cell number migration towards MIF indicating these cells have an intact CD74 receptor mechanism. This analysis suggests that hLL1 targets CLL cells in vivo and interferes with homing of CLL. Follow up analyses will examine whether CLL cells of hLL1-treated pts downregulate a4b1 integrin mediate binding and alter binding to bone marrow stroma. Uncovering such actions are important steps in the elucidation of hLL1's mechanisms of action and for the development of targeted therapies for hematopoietic cancers. Disclosures: Fanale: Novartis Corporation: Honoraria, Research Funding; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; MedImmune: Research Funding; Millenium: Research Funding. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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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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HIF-1α Is Not Essential For The Establishment Of MLL-Leukaemic Stem Cells

Blood ◽

10.1182/blood.v122.21.3767.3767 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3767-3767

Author(s):

Deniz Gezer ◽

Amelie V Guitart ◽

Milica Vukovic ◽

Chithra Subramani ◽

Karen Dunn ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Myeloid Leukaemia ◽

Progenitor Cells ◽

Board Of Directors ◽

Advisory Committees ◽

Hypoxic Conditions ◽

Significant Difference

Abstract Haematopoietic stem cells (HSCs) reside in hypoxic niches in the bone marrow (BM) and sustain long-life haematopoiesis. HSCs are largely quiescent, self-renew, undergo apoptosis and generate progenitor cells, which differentiate to multiple blood lineages. The strict regulation of the balance between these fate decisions is essential for haematopoiesis and their dysregulation in HSCs and progenitor cells can result in leukaemic transformation. HSCs and leukemic stem cells (LSCs) are suggested to share the same niche and are in need to adapt to hypoxic conditions. Hypoxia-inducible-factor-1α (HIF-1α) is a key mediator of cellular responses to hypoxia and is important for the maintenance of HSC functions under stressful conditions. Furthermore, in chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) HIF-1α is essential for LSC maintenance and ablation or knockdown of HIF-1α leads to exhaustion of established LSCs. The aim of this study was to investigate the requirement for HIF-1α in the generation of pre-LSCs and the establishment of LSCs. To investigate the role of HIF-1α in the generation of pre-LSCs we retrovirally transduced haematopoietic stem and progenitor cells (HSPCs) from either WT or HIF1-αfl/fl Vav-iCre with MLL-ENL retroviruses. Next we performed serial re-plating assays under normoxic and hypoxic conditions to generate pre-LSCs. Surprisingly, WT and HIF-1α deficient HSPCs generated comparable numbers of colonies in normoxia and hypoxia (Fig. 1a). In addition no significant difference was found in the immunophenotypic profile of colonies (Figure 1b). Furthermore, microscopic examination indicated that colonies of all genotypes were dense consistent with their transformed shape (Fig. 1c). WT and HIF-1α-deficient pre-LSCs cultured under normoxia and hypoxia had similar cloning efficiency, which is known to directly correlate with the numbers of LSCs in vivo (Fig. 2). These results indicate that HIF-1α is dispensable for the generation of pre-LSCs. To test the role of HIF-1α in establishment of LSCs from pre-LSCs we transplanted pre-LSCs into lethally irradiated mice together with support BM and monitored the mice for disease development. No significant difference was found in disease latency (Fig. 3a) or frequency of LSCs in peripheral blood, bone marrow or spleens (Fig. 3b) indicating that pre-LSCs lacking HIF-1α can efficiently generate LSCs that cause aggressive AML. In conclusion, we provide genetic evidence that HIF-1α is dispensable for the generation of pre-LSCs and the establishment of LSCs from pre-LSCs. These surprising findings, together with published results indicating that HIF-1α is essential for maintenance of LSCs, imply that HIF-1α has different roles at different stages of leukaemic transformation. Further studies are required to explain the distinct roles of HIF-1α in different stages of leukaemogenesis. Disclosures: Ratcliffe: RedOx: Founder Other. Holyoake:Novartis: 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; Ariad: Membership on an entity’s Board of Directors or advisory committees.

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Evaluation of Minnelide As a Potential Therapeutic Agent for Preventing the Relapse of AML

Blood ◽

10.1182/blood-2019-123567 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5159-5159

Author(s):

Pooja Roy ◽

Bhuwan Giri ◽

Vanessa Tonin Garrido ◽

Dujon Brandon Edwards ◽

Justin M. Watts ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Board Of Directors ◽

Tumor Burden ◽

Fold Change ◽

Water Soluble ◽

Stem Cell Markers ◽

Advisory Committees ◽

Cell Markers

BACKGROUND: Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults which requires multiple phases and combinations of chemotherapeutic agents. Despite this complex regimen, most patients either fail to achieve remission or relapse. Triptolide, a diterpenoid triepoxide compound and Minnelide its water-soluble prodrug have shown significant efficacy in decreasing leukemic burden in preclinical animal models. In the current study, we evaluated the potential of Minnelide to prevent recurrence of AML via its effect on leukemic stem cells and in the bone marrow. METHODS: To determine the effect of triptolide on the stemness of AML cells, two chemotherapy-resistant cell lines (THP-1 and KG-1) were treated overnight with triptolide at a dose of 2.5nM and 25nM. The colonies formed per well were subsequently measured. Stem cell markers (CD47, CD95, CD126 and TIM3) were also measured after treatment with various doses of triptolide (5nM, 10nM, 25nM). We also carried out in-vivo experiments in which luciferase-tagged THP-1 cells were intravenously injected into NSG mice. Positively implanted mice were then treated intraperitoneally with either saline or Minnelide (0.15mg/kg/day) for 30 days. RESULTS: Triptolide treated cells had significantly reduced number of colonies per well in a colony formation assay, indicating decreased clonogenicity. In addition, treatment with triptolide reduced expressed of stem cell markers CD47 and CD126 at a concentration of 20nM (fold change of 0.3 and 0.55 for CD47 and fold change of 0.14 and 0.66 for CD126 for THP-1 and KG-1 respectively). In-vivo, Minnelide was able to successfully reduce tumor burden as evidenced by serial measurements of radiance (ROI) with IVIS. Furthermore, bone marrow histology of Minnelide treated mice resembled the bone marrow of non-diseased animals. Analysis with flow cytometry supported our findings showing a significant reduction of human CD45RA positive cells in the bone marrow of Minnelide treated mice. CONCLUSION: Minnelide is not only successful in reducing tumor burden but it is potentially an effective therapy for preventing relapse of AML. This is evidenced by the reduction in stemness of AML cells treated with triptolide and through the reduction in tumor burden in the bone marrow of mice treated with low doses of Minnelide. FIGURE 1: Bone marrow from Minnelide treated mice had a significantly reduced infiltration of myeloid cells compared to saline treated mice and even resembled negative controls. Figure 1 Disclosures Watts: Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Banerjee:Minneamrita Therapeutics LLC: Consultancy. Saluja:Minneamrita Therapeutics, LLC: Other: Co-founder and the Chief Scientific Officer.

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Spatial Transcriptomics Reveals DPP4 As Novel Marker of a More Proliferative Phenotype in Early AML Progression

Blood ◽

10.1182/blood-2021-151917 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3310-3310

Author(s):

Christa Haase ◽

Karin Gustafsson ◽

Shenglin Mei ◽

Jelena Milosevic ◽

Shu-Chi Yeh ◽

...

Keyword(s):

Bone Marrow ◽

Disease Progression ◽

Board Of Directors ◽

Hematologic Malignancy ◽

New Technology ◽

Publicly Traded ◽

Advisory Committees ◽

Privately Held

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy with poor prognosis for which the standard-of-care chemotherapy treatment regimen has remained virtually unchanged over the past 40 years. We have employed "Image-Seq", a new technology that was developed in our laboratory, to study spatial variations in early leukemia progression in a mouse model of HoxA9-Meis1 AML. We visualized leukemia cells with differing proliferative phenotype using intravital microscopy, captured these cells under image guidance from individual bone marrow microenvironments and studied their differential expression by single-cell RNA sequencing. This analysis identified DPP4 as a key upregulated gene in AML cells from more proliferative bone marrow compartments and associated DPP4 expression with a cell cluster enriched in progenitor cell markers for HoxA9-Meis1 AML, including Flt3, Itgb7 and Ddx4. Strikingly, DPP4 is not expressed in vitro, and its expression in vivo (as quantitated by FACS analysis) correlated with disease progression and marked a more proliferative phenotype both at the 1-week and 2-week time-points during disease progression. Disclosures Sykes: Clear Creek Bio: Current equity holder in publicly-traded company; SAFI Biosolutions: Consultancy, Current equity holder in publicly-traded company; Keros Therapeutics: Consultancy. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; FOG Pharma: Consultancy; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Editas Medicines: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: sponsored research; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

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