scholarly journals Improved Gene Therapy for Metachromatic Leukodystrophy

Blood ◽  
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.

Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1815-1815
Author(s):  
Patricia Maiso ◽  
Yi Liu ◽  
Abdel Kareem Azab ◽  
Brittany Morgan ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Stromal Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Cycle Arrest

Abstract Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other 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 and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight 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, immunochemistry, 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. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. 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: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Interactions with a "Humanized" Mesenchymal Bone Marrow Stromal Niche In Vivo Modify the Patterns of Essential Genes for Myeloma Cells: Therapeutic Implications

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-40
Author(s):  
Ryosuke Shirasaki ◽  
Sondra L. Downey-Kopyscinski ◽  
Ricardo De Matos Simoes ◽  
Olga Dashevsky ◽  
Sara Gandolfi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Broad Spectrum ◽  
Research Funding ◽  
Advisory Committees ◽  
Stromal Compartment ◽  
Oncology Research

Background: The biology and treatment response of human multiple myeloma (MM) cells in vivo is influenced by interactions with mesenchymal bone marrow stromal cells (BMSCs). For several key BMSC-derived cytokines (including IL-6) only the human, not murine, form optimally interacts with their respective receptor(s) on human MM cells. To better simulate the treatment responses of human MM cells in the BM, "humanized" BM-like niches in vivo have been engineered with biocompatible ceramic scaffolds "functionalized" via osteogenic differentiation of human mesenchymal BMSCs and implanted subcutaneously in immunocompromised mice. Aim: To determine if the patterns of genetic dependencies elucidated through in vitro CRISPR-based functional genomic studies are recapitulated when human MM cells are grown in mice within BM-like scaffolds with "humanized" mesenchymal stromal compartment. Methods: Cas9+ MM cell lines KMS11 and XG7 were transduced with a library of 1372 single-guide RNAs (sgRNAs) targeting 184 genes of interest (4 sgRNAs/gene), including 89 genes preferentially essential for MM cell lines compared to other neoplasms in vitro; broad-spectrum oncogenic targets (e.g. KRAS, MYC); tumor suppressor genes (e.g. PTEN); genes with limited in vitro essentiality in MM cells, but significantly higher expression vs. non-MM lines (e.g. ZFP91, ZBP1); and 155 olfactory receptor (OR) genes (typically not expressed or biologically inactive in tumor cells) as "DNA cutting" control sgRNAs. MM cells transduced with this focused CRISPR knockout (KO) library were injected into "humanized" scaffolds implanted subcutaneously (s.c.) in NSG mice. Tumors were collected when mice reached criteria for euthanasia and next-generation sequencing quantified the changes in sgRNA distribution at the end vs. start of experiment. Results: A large majority of genes defined by in vitro CRISPR KO screens as MM-preferential dependencies were also essential for MM cells in BM-like scaffolds. Among 57 MM-preferential dependencies in vitro which were plausible dependencies for KMS11 cells (e.g. CERES scores <-0.4), 50 genes were essential for KMS11 cells in BM-like scaffolds in vivo (average log2fold change<-1.0 and depletion of 3+ of 4 sgRNAs relative to the 99% confidence interval for control sgRNAs). These genes included key transcription factors/cofactors (e.g. IRF4, PRDM1, POU2AF1, RELB, MAF); epigenetic regulators (e.g. CARM1, MTA2, DOT1L); kinases upstream of NFkappaB (CHUK, IKBKB); ER regulators (e.g. SYVN1). Most "core-essential" or broad-spectrum oncogenic dependencies (e.g. MYC, CFLAR, CDK7 on both lines; KRAS in XG7) of this sgRNA library remained essential in vivo; while PTEN KO cells were enriched. Notably, several genes had more pronounced essentiality in vivo vs. invitro (e.g. BCL2, PIM2); or converted from non-essential in vitro to essential in vivo. For instance, among 95 genes of this library which are not likely dependencies in vitro (CERES scores >-0.4) for KMS11 cells, 29 genes exhibited in vivo essentiality for both KMS11 and XG7 cells: several of these latter "in vivo dependencies" are recurrently essential for other MM lines in vitro (e.g. ZBTB38, UBE2J1, TCF3, DNAJB11), while also others have limited if any in vitro essentiality (e.g. ZBP1, PYGO2) across MM despite significantly higher transcript levels vs. other neoplasias. Notably, several genes with increased essentiality in the "humanized" BM scaffolds vs. in vitro also had stronger in vivo dependency in the BM scaffolds vs. when growth of the same MM cells as s.c. plasmacytomas (e.g. BCL2, PIM2, UBE2J1, SYVN1, ALG9, AMFR). Co-culture with BMSCs or IL-6 treatment induces several of these transcripts in MM cells suggesting that increased dependency of MM on these genes in the "humanized" BM model is due at least partly to its human cytokines. Conclusions: This study provides evidence for significant impact of the "humanized" BM-like niche on the patterns of genetic dependencies for human MM cells. Most genes preferentially essential for MM cells in vitro remain essential for their in vivo growth in the "humanized" BM model. Notably, several genes that do not meet criteria for dependency in vitro show such metrics in "humanized" BM scaffolds, but not sc plasmacytomas. These observations highlight important implications of the "humanized" BM-like in vivo model for current and future efforts to define and prioritize therapeutic targets for MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Janssen/Johnson & Johnson: Research Funding; Arch Oncology: Research Funding; TEVA: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; EMD Serono: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 133-133 ◽  
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.

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

Blood ◽  
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.

scholarly journals Dissecting Signaling Network Responses to PAK4 Allosteric Modulators (PAMs) in Cell Subsets within Primary Human Acute Myeloid Leukemia Samples

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3686-3686
Author(s):  
Paul Brent Ferrell ◽  
William Senapedis ◽  
Alexander Cook ◽  
Erkan Baloglu ◽  
Yosef Landesman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Stem Cells ◽  
Allosteric Modulators ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28). Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21). Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK. Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. Disclosures Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals In Vitro and inVivo Activity of Melflufen in Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3100-3100 ◽  
Author(s):  
Ken Flanagan ◽  
Muntasir M Majumder ◽  
Romika Kumari ◽  
Juho Miettinen ◽  
Ana Slipicevic ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Plasma Cell ◽  
Light Chain ◽  
Research Funding ◽  
Plasma Cells ◽  
Al Amyloidosis ◽  
Advisory Committees

Background: Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils and deposit on vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell directed therapeutics, aimed at preferentially eliminating the clonal population of amyloidogenic cells in bone marrow are expected to reduce production of toxic light chain and alleviate deposition of amyloid thereby restoring healthy organ function. Melphalan flufenamide ethyl ester, melflufen, is a peptidase potentiated alkylating agent with potent toxicity in myeloma cells. Melflufen is highly lipophilic, permitting rapid cellular uptake, and is subsequently enzymatically cleaved by aminopeptidases within cells resulting in augmented intracellular concentrations of toxic molecules, providing a more targeted and localized treatment. Previous data demonstrating multiple myeloma plasma cell sensitivity for melflufen suggests that the drug might be useful to directly eliminate amyloidogenic plasma cells, thereby reducing the amyloid load in patients. Furthermore, the increased intracellular concentrations of melflufen in myeloma cells indicates a potential reduction in systemic toxicity in patients, an important factor in the fragile amyloidosis patient population. To assess potential efficacy in amyloidosis patients and to explore the mechanism of action, we examined effects of melflufen on amyloidogenic plasma cells invitro and invivo. Methods: Cellular toxicity and apoptosis were measured in response to either melflufen or melphalan in multiple malignant human plasma cell lines, including the amyloidosis patient derived light chain secreting ALMC-1 and ALMC-2 cells, as well as primary bone marrow cells from AL amyloidosis patients, using annexin V and live/dead cell staining by multicolor flow cytometry, and measurement of cleaved caspases. Lambda light chain was measured in supernatant by ELISA, and intracellular levels were detected by flow cytometry. To assess efficacy of melflufen in vivo, the light chain secreting human myeloma cell line, JJN3, was transduced with luciferase and adoptively transferred into NSG mice. Cell death in response to melflufen or melphalan was measured by in vivo bioluminescence, and serum light chain was monitored. Results: Melflufen demonstrated increased potency against multiple myeloma cell lines compared to melphalan, inducing malignant plasma cell death at lower doses on established light chain secreting plasma cell lines. While ALMC-1 cells were sensitive to both melphalan and melflufen, the IC50 for melphalan at 960 nM was approximately 3-fold higher than melflufen (334 nM). However, ALMC-2 cells were relatively insensitive to melphalan (12600 nM), but maintained a 100-fold increase in sensitivity to melflufen (121 nM). Furthermore, while 40% of primary CD138+ plasma cells from patients with diagnosed AL amyloidosis responded to melflufen treatment in vitro, only 20% responded to melphalan with consistently superior IC50 values for melflufen (Figure 1). Light chain secreting cell lines and AL amyloidosis patient samples were further analyzed by single cell sequencing. We further examined differential effects on apoptosis and the unfolded protein response in vitro in response to either melflufen or melphalan. This is of particular interest in amyloidosis, where malignant antibody producing plasma cells possess an increased requirement for mechanisms to cope with the amplified load of unfolded protein and associated ER stress. As AL amyloidosis is ultimately a disease mediated by secretion of toxic immunoglobulin, we assessed the effects of melflufen on the production of light chain invitro, measuring a decrease in production of light chain in response to melflufen treatment. Finally, we took advantage of a recently described adoptive transfer mouse model of amyloidosis to assess the efficacy of melflufen and melphalan in eliminating amyloidogenic clones and reducing the levels of toxic serum light chain in vivo. Conclusions: These findings provide evidence that melflufen mediated toxicity, previously described in myeloma cells, extends to amyloidogenic plasma cells and further affects the ability of these cells to produce and secrete toxic light chain. This data supports the rationale for the evaluation of melflufen in patients with AL amyloidosis. Figure 1 Disclosures Flanagan: Oncopeptides AB: Employment. Slipicevic:Oncopeptides AB: Employment. Holstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy. Lehmann:Oncopeptides AB: Employment. Nupponen:Oncopeptides AB: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.

A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3830-3830
Author(s):  
Gullu Gorgun ◽  
Elisabetta Calabrese ◽  
Teru Hideshima ◽  
Jeffrey Ecsedy ◽  
Giada Bianchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Research Funding ◽  
Thymidine Incorporation ◽  
Aurora A Kinase ◽  
Advisory Committees

Abstract Abstract 3830 Poster Board III-766 Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures: Ecsedy: Millennium Pharmaceutical: Employment. Munshi: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. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

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

Blood ◽  
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.

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

Blood ◽  
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.

scholarly journals Development of Astatine-211 (211At)-Based Anti-CD123 Radioimmunotherapy for Acute Leukemias and Other CD123+ Hematologic Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3341-3341
Author(s):  
George S. Laszlo ◽  
Johnnie J. Orozco ◽  
Allie R. Kehret ◽  
Margaret C. Lunn ◽  
Donald K. Hamlin ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Hematologic Malignancies ◽  
Advisory Committees ◽  
Leukemia Cell Lines ◽  
Efficacy Studies

Abstract Background: Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia. Of current interest are alpha-particle emitting radionuclides as they deliver a very large amount of radiation over just a few cell diameters, enabling efficient and selective target cell kill. So far, alpha-emitters including astatine-211 (211At) have been primarily explored with monoclonal antibodies (mAbs) targeting CD45 or CD33 but their broad display on non-malignant target-expressing cells can lead to marked "on-target, off tumor cell" toxicities. To overcome this limitation, we developed a novel form of 211At-based RIT targeting CD123. CD123 is displayed widely on acute leukemia cells, including underlying leukemic stem cells, but is expressed only on a discrete subset of normal hematopoietic cells and is virtually absent on non-blood cells. Methods: We immunized BALB/c mice with peptides consisting of the extracellular domain of human CD123 to generate anti-CD123 mAbs. Flow cytometry-based assays with human acute leukemia cell lines were used to characterize binding of hybridoma supernatants and mAbs to CD123. mAbs were conjugated with isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10), a boron cage molecule for subsequent astatination, and were then labeled with 211At. In vivo leukemia cell targeting ("biodistribution") and efficacy studies were conducted in immunodeficient NOD-Rag1 null IL2rɣ null/J (NRG) mice xenografted with MOLM-13 cells, a CD123+ human acute myeloid leukemia cell line. Results: Based on initial hybridoma screening studies, we selected 4 mAbs (10C4, 5G4, 11F11, and 1H8) for further characterization. Phenotyping studies with CD123+ and CD123- human acute leukemia cell lines (including CD123+ cell lines in which CD123 was deleted via CRISPR/Cas9) confirmed specific binding of all mAbs to human CD123 (binding intensity: 10C4&gt;5G4=11F11=1H8), with 10C4 yielding a higher median fluorescence intensity than the widely used commercial anti-CD123 mAb clones, 7G3 and 6H6 (Figure 1). In vitro internalization with a panel of human acute leukemia cell lines studies demonstrated uptake of all mAbs by CD123+ target cells with a kinetic slower than that for anti-CD33 antibodies (typically, 30-50% of the anti-CD123 mAb internalized over 2-4 hours). All 4 anti-CD123 mAbs could be conjugated to B10 and subsequently labeled with 211At. Unlike a non-binding 211At-labeled control mAb, 211At-labeled anti-CD123 mAbs showed uptake at MOLM-13 flank tumors in NRG mice carrying MOLM-13 xenografts. After additional leukemia cell targeting studies to optimize the dosing of 10C4, we conducted proof-of-concept efficacy studies in NRG mice injected intravenously with luciferase-transduced MOLM-13 cells (disseminated leukemia model). Animals were either untreated or treated with 10 µCi, 20 µCi, or 40 µCi of 211At-labeled 10C4-B10 mAb (9-11 animals/group). This was followed by the infusion of bone marrow cells from donor mice as stem cell support 3 days later. As shown in Figure 2 and Figure 3, 211At-10C4-B10 led to a dose dependent decrease in tumor burden. Further, the treatment significantly prolonged survival compared to untreated animals (median survival: 49 days [40 µCi of 211At] vs. 31 days [10 µCi of 211At] vs. 21 days [Ctrl]; P&lt;0.0001 for Ctrl vs. 10 µCi, P&lt;0.004 for 10 µCi vs. 40 µCi), demonstrating potent in vivo anti-leukemia efficacy of a single dose of 211At-CD123 RIT. Conclusion: Our data support the further development of 211At-CD123 RIT for the treatment of patients with acute leukemia and other CD123+ hematologic malignancies. Figure 1 Figure 1. Disclosures Green: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; JANSSEN Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document