The BET Inhibitor INCB054329 Primes AML Cells for Venetoclax-Induced Apoptosis

Abstract Bromodomain and extra-terminal (BET) inhibitors may be efficacious for treatment of acute myeloid leukemia because they attenuate the expression of critical oncogenes including MYC and BCL2. These BET inhibitors (BETi) disrupt the transcriptional elongation process by displacing BET family members BRD2,3, and 4 off of chromatin, and causing RNA polymerase promoter-proximal pausing. We used precision nuclear run-on transcription sequencing (PROseq) to directly measure the effects of INCB054329, a potent BETi, on RNA polymerase II pausing and elongation. We found dramatic reductions on the elongation of key oncogenes such as MYC and BCL2 within 15 min of adding the drug. These effects became more significant over time, eventually affecting nearly two thousand genes. By four hours after drug addition, we found a loss of ribosomal gene expression and a loss of mitochondrial gene expression that is characteristic of genes regulated by MYC, suggesting that these were secondary to turning off MYC expression. When we examined the potential of the BETi INCB054329 for therapeutic efficacy in AML using Alamar Blue assays, which measure cellular redox potential, we noted marked growth inhibition of AML cell lines. However, growth assays and measurements of apoptosis using Annexin V staining found that BETi induced minimal apoptosis and cells were largely cytostatic. BrdU incorporation assays showed that INCB054329 caused the cells to accumulate in the G0/G1 phase of the cell cycle. Metabolic studies indicated that INCB054329 treatment for 48 hours caused disruption of mitochondrial respiration rate and severely reduced glycolytic capacity. Taken together, the growth inhibition, cell cycle arrest and reduced metabolic rate suggests that INCB054329 promoted quiescence in AML cells, but that this is reversible, consistent with the clinical experience of single-agent treatment of hematologic malignancies with BETi. MLL fusion proteins enhance transcription by stimulating RNA polymerase elongation, suggesting INCB054329may provide a therapeutic option to reverse this effect. However, the cell cycle arrest suggested that a second compound may be needed to trigger cell death. We first performed in vivo studies with INCB054329 using a systemic AML xenograft model of MV4-11 cells that express MLL-AF4. Engrafted NSGS mice received INCB054329 in 3 different doses (vehicle vs 10, 30 and 75mg/kg q.d) daily. During treatment, the kinetics of MV-4-11 expansion was monitored via flow cytometry for the detection of human AML in the blood. At approximately 4 weeks after transplant, the vehicle mice became moribund, and all experimental groups were sacrificed for analysis of chimerism. Significant decreases in leukemic expansion were evident in the bone marrow (vehicle vs75mg/kg, p<.001) and spleen (vehicle vs. 75mg/kg, p <.001) of treated mice. As BETi decreases expression of BCL2, we posited that BH3 directed therapy with the BCL-2 inhibitor venetoclax (VEN) could be enhanced by INCB054329. In vitro, we found that the combination of INCB054329 and VEN resulted in significant growth inhibition and apoptosis of treated AML cells. This finding prompted us to test the combination of INCB054329 with VEN in vivo. Mice engrafted with human AML cells received INCB054329 (50mg/kg q.d), VEN (25mg/kg q.d) or the combination. Four weeks after transplant, analyses by flow cytometric measurement of human CD45 of combination treated mice revealed significant decreases of AML cells in the bone marrow (vehicle vs. BRDi/VEN p = 0.004) and spleen (vehicle vs.BRDi/VEN, p = 0.001). Further studies are underway to test this combination in both VEN sensitive and resistant AML primary xenograftmodels. These preliminary data suggest that INCB054329 may serve as a non-cytotoxic priming agent for BH3 directed therapy, and the combination of INCB054329 +VEN may provide a potent therapy in a variety of genetically distinct subtypes of AML. Disclosures Stubbs: Incyte: Employment. Liu:Incyte: Employment. Rathmell:Calithera: Research Funding. Hiebert:Incyte: Research Funding. Savona:Boehringer Ingelheim: Consultancy; Celgene: Consultancy, 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.

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Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.1815.1815 ◽

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.

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

Blood ◽

10.1182/blood.v114.22.3830.3830 ◽

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.

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MCT1 As Molecularly Validated Predictive Marker for Lenalidomide-Maintenance Therapy in Multiple Myeloma

Blood ◽

10.1182/blood-2019-130734 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3187-3187

Author(s):

Jacob Stroh ◽

Anja Seckinger ◽

Michael Heider ◽

Ruth Eichner ◽

Martina Emde ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Research Funding ◽

Maintenance Treatment ◽

Xenograft Model ◽

Predictive Marker ◽

High Dose ◽

Advisory Committees

Introduction: Lenalidomide-based maintenance therapy is the currently approved standard of care for multiple myeloma (MM) patients after high-dose melphalan and autologous stem cell transplantation (HD-Mel), which significantly prolongs progression-free (PFS) and overall survival (OS). For patients with del17p bortezomib based maintenance treatment is considered overcoming adverse prognosis of this aberration. Predictive markers of response to lenalidomide maintenance have remained elusive. We have previously shown that IMiDs exert their anti-MM activity via destabilization of MCT1 and CD147 and combined overexpression reduces response to lenalidomide-treatment in vitro and in an in vivo MM xenograft model (Eichner et al. Nature Medicine 2016). Methods: CD138-purified myeloma cell samples of 654 patients receiving high-dose melphalan therapy and autologous stem cell transplantation and either bortezomib (n=101), thalidomide (n=98) or lenalidomide (n=455) maintenance treatment were assessed by gene expression profiling (GEP) using U133 2.0 plus DNA microarrays, 316 by RNA-sequencing (RNA-seq). Expression of CD147 and MCT1 were assessed and correlated with PFS and OS data. Gene expression based risk scores, including UAMS70-gene, Rs-score and gene expression based proliferation index were assessed alongside routine iFISH-analysis. Survival curves and median time to progression were computed with nonparametric survival estimates for censored data using the Kaplan-Meier method. Difference between the curves were tested using the G-rho Log-rank test. Landmark analysis was performed by defining an alternative start point (landmark) at 12 months. In vitro, CD147 and MCT1 were lentivirally overexpressed in MM1S cells, which were subjected to lenalidomide or bortezomib treatment and proliferation analysis. Xenografted MM-tumors were followed by 18FDG-PET and analyzed by immunohistochemistry. Results: Patients with high gene expression levels of MCT1 showed significantly reduced PFS (31.9 vs. 48.2months in MCT1high vs. MCT1low,P=.03) and OS (75.9 months vs. not reached (NR) months in MCT1high vs. MCT1low; P=.001) in case of lenalidomide maintenance. Likewise, patients with thalidomide maintenance showed reduced PFS (34.8 vs. 43.7 months in MCT1high vs. MCT1low, P=.23) and significantly shorter OS (83.6 months vs. not reached (NR) months in MCT1high vs. MCT1low;P=.03). For bortezomib based maintenance, MCT1 expression had no significant impact on PFS (39.8 months vs. 32.6 months in MCT1high vs. MCT1low) and OS (125.8 months vs. 129.8 months in MCT1high vs. MCT1low). No association with other prognostic factors was found. As still differences between MCT1high vs. MCT1lowexpression myeloma cells might be attributed to undiscerned molecular factors and for functional validation, we lentivirally overexpressed CD147 and MCT1 in human myeloma cell lines. Overexpression of MCT1 significantly reduced cytotoxicity of lenalidomide, while no change was observed in MM cells treated with bortezomib. We subsequently validated our results in vivo. Functional investigations in the mechanism of MCT1 impact on cellular survival are ongoing. Conclusion: Taken together MCT1 expression as potential predictive marker for response to IMiD-based maintenance treatment. Both PFS and OS were significantly reduced in patients with high gene expression levels of MCT1. In vitro and in vivo (xenograft model), MCT1 overexpression reduced sensitivity to lenalidomide unlike bortezomib treatment. Disclosures Salwender: Bristol-Myers Squibb: Honoraria, Other: Travel or accommodations; Janssen Cilag: Honoraria, Other: Travel or accommodations; AbbVie: Honoraria; Celgene: Honoraria, Other: Travel or accommodations; Sanofi: Honoraria, Other: Travel or accommodations; Takeda: Honoraria, Other: Travel or accommodations; Amgen: Honoraria, Other: Travel or accommodations. Bertsch:Sanofi: Other: travel support; Celgene: Other: travel support. Goldschmidt:Chugai: Honoraria, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Molecular Partners: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; John-Hopkins University: Research Funding; MSD: Research Funding; Mundipharma: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Weisel:Takeda: Consultancy, Honoraria; GSK: Honoraria; Sanofi: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno: Consultancy; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Adaptive Biotech: Consultancy, Honoraria. Scheid:Celgene: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Takeda: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Bassermann:Celgene: Consultancy, Research Funding.

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

Blood ◽

10.1182/blood-2021-150699 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3472-3472

Author(s):

Isabel Weinhäuser ◽

Diego A Pereira-Martins ◽

Jacobien R Hilberink ◽

Luciana Yamamoto Almeida ◽

Douglas RA Silveira ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Cell Population ◽

Research Funding ◽

Poor Prognosis ◽

Ex Vivo ◽

M2 Macrophages ◽

Advisory Committees

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

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Specific Targeting of KRAS Using a Novel High-Affinity KRAS Antisense Oligonucleotide in Multiple Myeloma

Blood ◽

10.1182/blood-2019-124391 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3104-3104 ◽

Cited By ~ 1

Author(s):

Antonio Sacco ◽

Cinzia Federico ◽

Katia Todoerti ◽

Bachisio Ziccheddu ◽

Arianna Giacomini ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Research Funding ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Wild Type ◽

Down Regulation ◽

Advisory Committees

INTRODUCTION. The multiple myeloma (MM) mutational landscape has identified KRAS as the most recurring somatic variant, observed in around 26% of cases, therefore KRAS may represent an important therapeutic target. Despite several attempts to develop a targeted therapeutic for KRAS mutant cancers, either direct KRAS enzymatic inhibition, or inhibition of MAPK- and PI3K- downstream effector cascades have not been successful. Therefore, there is a need to develop novel therapeutic approaches that may target the KRAS mutational event in MM. We have studied AZD4785, a novel, potent and selective high affinity 2'-4' constrained ethyl residues containing therapeutic antisense oligonucleotide (ASO) targeting KRAS, both in vitro and in vivo. METHODS. AZD4785 productive uptake was assessed by measuring KRAS knockdown at both the mRNA and protein level. Molecular mechanisms underlying AZD4785-dependent anti-MM activity were studied, interrogating the transcriptome profiling of AZD4785-treated MM cells. Anti-MM activity of AZD4785 was assessed in vitro in the context of primary MM patients' derived bone marrow stromal cells (BMSCs). Endpoints included evaluation of cell proliferation, cytotoxicity, cell cycle modulation, apoptosis, MM cell migration and adhesion; modulation of MAPK-, PI3K-, apoptotic-signaling. KRAS-mutated (MM1S; KMS20); -wild type (U266; KMS11) MM cell lines; BM MM patients' and peripheral blood healthy donor derived cells were tested. A non-targeting ASO (ASO-ctrl) was used as control. Synergism between AZD4785 and bortezomib, in modulating MM growth was tested. AZD4785-dependent modulation of tumor growth was studied in vivo in a subcutaneous MM.1S.-Luc model and a disseminated GFP/Luc-MM.1S model (BLI); MM cell dissemination to distant BM niches was studied ex vivo, using confocal laser scanning microscopy. RESULTS. AZD4785 led to specific dose-dependent inhibition of KRAS mRNA and protein expression, in KRAS-mutant, -wild-type cell lines and MM patient-derived CD138+ cells; without affecting NRAS and HRAS content. Wide mRNA transcriptome was performed using AZD4785 treated MM.1S cells vs control: GSEA showed down-regulation of MAPK, cell cycle, TP53 signaling pathways (FDR<0.25; P<0.05) in AZD4785-treated MM cells. Functionally, AZD4785 significantly impaired proliferation and survival of KRAS-mutant MM cells in a dose- and time-dependent manner even in the presence of patients' derived BM-MSCs. Cell growth of KRAS-wild type MM cells was not significantly affected. AZD4785 did not target healthy donors' derived PBMCs. Consistently with the effect on cell growth, AZD4785-treated KRAS mutant MM cells showed S-phase down-regulation, increased of G0/G1 phase and increased apoptotic rate, supported by up-regulation of cleaved-caspase-3, -PARP and BIM. The efficacy of AZD4785 in targeting MM cells within the context of the BM milieu was tested, revealing AZD4785-dependent impairment of MM cell adhesion and migration towards primary BM-MSCs, supported by inhibition of paxillin, cofilin, Src. Protein studies showed inhibition of both MAPK (phospho(p)-ERK1/2, p-MEK, p-RSK90, p-CRAF), and PI3K-Akt signaling pathways, selectively in AZD4785-treated KRAS mutant cells. AZD4785-dependent anti-MM activity was potentiated by the combinatory use of bortezomib, resulting in a significantly higher inhibition of MM cell proliferation, induction of apoptosis, and cell cycle arrest. AZD4785 exerted in vivo down-regulation of KRAS and anti-tumour activity in MM models, being more efficacious when used in combination with bortezomib, in terms of both inhibition of tumor growth and MM cell BM niches colonization, as evaluated by using in vivo whole body-bioluminescence imaging and ex vivo confocal laser scanning microscopy, respectively. CONCLUSION. Taken together, these data suggest that AZD4785 may represent a novel therapeutic approach for targeting mutant KRAS in MM, either alone or in combination with proteasome inhibitors; and warrant further development. Disclosures Giacomini: Fondazione Cariplo: Research Funding. Belotti:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Revenko:Ionis Pharmaceuticals: Employment. MacLeod:Ionis Pharmaceuticals: Employment. Willis:AstraZeneca: Employment. Cai:AstraZeneca: Employment. Hauser:AstraZeneca: Employment. Rooney:AstraZeneca: Employment. Ambrose:AstraZeneca: Employment. Staniszewska:AstraZeneca: Employment. Hanson:AstraZeneca: Employment. Rossi:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees. Ronca:Associazione Italiana per la Ricerca sul Canctro (AIRC): Research Funding. Bolli:GILEAD: Other: Travel expenses; JANSSEN: Honoraria; CELGENE: Honoraria. Moschetta:AstraZeneca: Employment. Ross:AstraZeneca: Employment. Roccaro:Celgene: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding.

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Citron Rho Interacting Kinase (CRIK) Regulates Survival in IL-6 Dependent Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.1825.1825 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1825-1825

Author(s):

Hai T. Ngo ◽

Alexey A. Leontovich ◽

Aldo M. Roccaro ◽

Abdel Kareem Azab ◽

Judith M. Runnels ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Protein Expression ◽

Board Of Directors ◽

Research Funding ◽

Protein Microarray ◽

Advisory Committees ◽

Inhibition Of Proliferation

Abstract Abstract 1825 Poster Board I-851 Purpose Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. We have previously demonstrated that Citron Rho Interacting Kinase (CRIK) is overexpressed in primary multiple myeloma (MM) cells, as compared to the normal plasma cell counterpart, using an antibody-based protein microarray technique. We therefore sought to investigate the functional role of CRIK in MM cells. Methods We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM.1S, RPMI8226, and INA6 MM cell lines were used in this study. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by thymidine uptake, MTT, PI and Annexin/PI staining and flow cytometric analysis, respectively. Results Overexpression of CRIK has been confirmed in primary CD138+ tumor cells isolated from bone marrow of 12 patients with MM, as compared to normal plasma cells obtained from healthy donors. We found that CRIK-knockdown exerted an anti-proliferative and pro-apoptotic effect only in IL-6-dependent MM cell line INA6; in contrast, no effect on proliferation and survival was observed in MM1.S and RPMI8226. Indeed, INA6 CRIK-knockdown cells were characterized by a reduction in the proliferation rate, associated with decreased S-phase and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity was also demonstrated in CRIK knockdown cells compared to scramble probe transfected or non-transfected cells. We also showed that CRIK knockdown led to cytokinesis in INA6, indicating a possible mechanism for inhibition of proliferation of these cells. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Conclusion In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of IL-6 dependent MM cells. Moreover, CRIK protein expression correlated with poor survival in patients with MM. Disclosures Anderson: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Mechanisms Underlying the Synergistic Interaction of Filanesib with Pomalidomide and Dexamethasone (FPD) in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.1801.1801 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1801-1801 ◽

Cited By ~ 1

Author(s):

Susana Hernández-García ◽

Laura San-Segundo ◽

Lorena González-Méndez ◽

Montserrat Martín-Sánchez ◽

Luis A Corchete ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Board Of Directors ◽

Research Funding ◽

Annexin V ◽

Triple Combination ◽

Advisory Committees ◽

Cell Cycle Profile

Abstract Introduction: Filanesib (ARRY-520) is a novel inhibitor of the "kinesin spindle protein" (KSP), which has demonstrated efficacy in heavily pretreated patients with refractory MM, (Lonial et al, ASH 2013). Our preliminary studies demonstrated synergy with standard anti-MM agents, especially with pomalidomide and dexamethasone. This set the stage for a recently activated trial being run by the Spanish MM group investigating FPD in relapsed MM patients. In this abstract we investigate the mechanisms underlying the synergy of the combination. Methods: In vitro action of FPD was evaluated in MM cell lines by MTT assay, bioluminescence, Annexin V staining, cell cycle profile analysis and TMRE staining by flow cytometry. Synergy was quantified with the Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice (The Jackson Laboratory, Bar Harbor, ME, USA). The mechanism of action was analyzed by Western blot, flow cytometry, genomic techniques, immunohistochemistry and immunofluorescence techniques. Results: The triple combination of FPD resulted in clear synergy in multiple myeloma cell lines (MM1S, OPM2, and RPMI8226) with combination indices between 0.4-0.7, and abrogated the effect of the soluble cytokines IL-6 and IGF-I and the protective effect of the adhesion of plasma cells to BMSCs, HS-5 and TERT cells. FPD caused cell cycle arrest in G2/M and specific apoptosis of cells arrested in these proliferative phases (with apoptosis percentage of 5, 23, 58 and 88 for control, poma+dexa, filanesib and FPD, respectively) demonstrated by flow cytometry with DRAQ5 and Annexin-V. Thus, FPD and filanesib in monotherapy treatments induced a similar effect on the cell cycle profile (arrest in G2/M) with a concordant increase of cyclin B1 and phosphorylated Histone H3. Although a secondary increase of KSP protein levels would be expected, pomalidomide and dexamethasone induced a decrease of the levels of this protein, which was still present in the triple combination (FPD). This fact could be contributing to the potentiation observed with the combination. Attending to apoptosis mechanism, proapoptotic stimulus from the extrinsic and intrinsic apoptotic pathways were promoted by pomalidomide and dexamethasone and filanesib, and converged in the triple combination. In this regard, a decrease of MCL-1 (antiapoptotic protein) and a significant increase of the proapoptotic BCL2 family members of the intrinsic pathway like NOXA and BIMEL BIML, BIMS(this last one being the most potent proapoptotic isoform), tBID (extrinsic pathway) and Bax protein were observed. We confirmed that all these proteins were translocated into the mitochondria, resulting in a decrease of the mitochondrial membrane potential by TMRE, increase of permeability and a release of cytochrome C and AIF. These results were confirmed in vivo in a model of subcutaneous plasmacytoma in small (70 mm3) and large (2000 mm3) tumors. In this model we observed a significant reduction of tumor growth, which was correlated with a statistically significant improvement in survival. Changes induced by FPD in the gene expression profile were concordant with the in vitro results as several overexpressed genes belonging to the previous pathways were identified, such as spindle assembly checkpoint (CENP-E and CENP-F) and apoptosis (BCL2L11, gene that codifies BIM protein). Furthermore, IHC of tumors treated with FPD showed more apoptosis by TUNEL and a significant increase of monopolar spindles (2, 0, 53 and 140 per 10 high-power fields, for control, poma+dexa, filanesib and FPD, respectively). Conclusions: The synergy observed with filanesib in combination with pomalidomide and dexamethasone is the result of several coincidental mechanisms: a potentiation of the KSP inhibition with a subsequent increase in monopolar spindle formation and a simultaneous activation of the intrinsic and extrinsic pathways of apoptosis. In this regard, NOXA, BIM, BAX and tBID are probably the central players that, through different mechanisms, inhibit antiapoptotic proteins (MCL-1, BCL2 and BCL-XL) and promote mitochondrial outer membrane permeabilization and the release of apoptogenic factors such us cytochrome C and AIF. This work was funded in part by the company Array BioPharma. Disclosures Tunquist: Array BioPharma: Employment. Mateos:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Ocio:Jassen: Honoraria; Celgene: Honoraria, Research Funding; Pharmamar: Consultancy, Research Funding; MSD: Research Funding; Novartis: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Array BioPharma: Consultancy, Research Funding.

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In Vivo Assessment of Intracellular Dynamics Comparing Injection Versus Oral Azacitidine in a Phase IIb Investigator Initiated Clinical Trial

Blood ◽

10.1182/blood-2019-123921 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4247-4247

Author(s):

Ashwin Unnikrishnan ◽

Xin Ying Lim ◽

Swapna Joshi ◽

Andrea C. Nunez ◽

Lachlin Vaughan ◽

...

Keyword(s):

Cell Cycle ◽

Clinical Trial ◽

Dna Methylation ◽

Bone Marrow ◽

High Risk ◽

Board Of Directors ◽

Research Funding ◽

Dna Demethylation ◽

Advisory Committees

Introduction: 5'-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML). Response is associated with improved survival. However, only half of patients respond, and these responses are rarely durable. We recently reported that primary AZA resistance is associated with a molecular signature of cell cycle quiescence within bone marrow (BM) hematopoietic progenitor cells (Unnikrishnan et al, Cell Reports, 20:572-585 (2017)). As DNA incorporation of the deoxyribonucleic form of AZA (5-aza-2′-deoxycytidine, DAC) occurs during DNA replication, cell cycle quiescence is predicted to lead to less DAC in DNA and concomitantly less DNA demethylation. We recently developed a quantitative multi-parameter assay, AZA-MS (Unnikrishnan, Vo et al, Leukemia 32:900-910 (2018)), to measure the intracellular dynamics of AZA in patients. Using AZA-MS, we reported data supporting the predicted resistance model. CC486 is an oral formulation of AZA. A 28-day cycle of CC486 involves 21 continuous days (21/28) versus the standard 7/28 subcutaneous (SC) injection AZA scheme. Whether levels of in vivo DAC incorporation into DNA during a cycle of CC486 are comparable with that of SC AZA is unknown. AZA-MS provides us with a unique opportunity to empirically assess the in vivo intracellular dynamics of SC versus oral AZA. Study Design and Methods: To directly assess in vivo DAC incorporation and concomitant DNA demethylation with SC AZA and CC486 in the same patient, we initiated a phase II clinical trial (NCT03493646; Fig A). MDS (IPSS; intermediate-2 or high-risk), CMML (bone marrow [BM] blasts 10-29%) and AML (20-30%) patients were recruited for six cycles of SC AZA (75mg/m^2/day for 7/28 days) followed by six cycles of CC486 (100mg bid for 21/28 days in C7-C8 and 150mg bid for 21/28 in C9-C12). Clinical response was assessed at the end of C6 and C12 using International Working Group criteria. Clinical responders and non-responders to SC AZA at C6 received CC486 from C7 onwards. From each patient, 36 peripheral blood (PB) samples and five BM samples were collected over the study period. DNA, RNA and intracellular fractions were isolated from the PB MNCs, for intracellular DAC/AZA measurements by AZA-MS (primary endpoint; Fig A). BM MNCs were utilised for AZA-MS as well as flow cytometry-based cell cycle measurements (secondary endpoint). Results: 31 of 42 consented patients have commenced treatment since trial opening (Fig B-C). We applied the AZA-MS assay on the longitudinal PB and BM samples collected from the seven patients who had completed six months AZA and commenced CC486 as at 26th June 2019 (Fig D). DAC incorporation into DNA and DNA methylation levels were quantified within the same cells, in addition to measuring other parameters (Fig E). As represented by patient 61213-005 (Fig F) who had a complete response (CR) at cycle 6, after 7 days of injection AZA we observed robust incorporation of DAC within PB MNCs (left panel, Fig F) together with concomitant DNA demethylation (right panel, Fig F). DAC levels diminished upon cessation of AZA within a cycle, with corresponding increases in DNA methylation. There were quantitatively higher levels of DAC incorporated in DNA during SC AZA cycles versus CC486. The trend observed is also appreciated from 2.3x higher area under the curve (AUC) measurements in 61213-005 during the SC AZA cycle. DAC incorporation was higher at C9/10 (CC486 150mg bid 21/28) than at C7/8 (CC486 100mg bid 21/28) without appreciable changes in DNA demethylation. During SC AZA cycles, higher DAC levels (top panel, Fig G) and greater DNA methylation (lower panel, Fig G) were seen in the BM MNCs. In a non-responding patient at cycle 6 (61290-002, SD), we saw less DAC incorporation and DNA demethylation (Fig H). We also observed a positive correlation between baseline proportions of cycling BM cells (LIN-CD34+CD38+) and the amount of DAC incorporated in BM MNCs at C1 day 8 (Fig I). Conclusion: AZA-MS can be used to reliably measure in vivo DAC incorporation and concomitant DNA demethylation in PB MNCs and inform appropriate CC486 dosing. Figure Disclosures Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fong:Astellas: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau. Roncolato:St. George Hospital: Employment. Enjeti:Roche: Honoraria, Speakers Bureau; Bayer and Sanofi: Honoraria, Speakers Bureau; Astellas: Consultancy; Novartis: Consultancy; Abbvie: Consultancy. Hertzberg:BMS: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Polizzotto:Janssen: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; ViiV: Research Funding. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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CD62L Expression Level Dictates the Cell Fate of Myeloid Progenitors in Mice and Humans

Blood ◽

10.1182/blood-2020-134753 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 26-27

Author(s):

Yusuke Ito ◽

Fumio Nakahara ◽

Yuki Kagoya ◽

Mineo Kurokawa

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Board Of Directors ◽

Transcriptome Analysis ◽

Research Funding ◽

Advisory Committees ◽

Differentiation Potential ◽

Cell Assay ◽

Colony Forming Cell

Hematopoietic cells are hierarchically differentiated from hematopoietic stem cells through several progenitors. Recent studies showed that each progenitor population has significant heterogeneity and some of the subsets have skewed differentiation potential. However, it has not been elucidated how and when common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) acquire different fates. We hypothesized that progenitor cells with skewed differentiation potential had acquired a part of gene expression profiles of more differentiated cells. By analyzing publicly available single-cell RNA sequencing data of human and murine CMPs and GMPs, we thoroughly explored surface markers with heterogeneous expression patterns and identified CD62L as a candidate to refine the differentiation potential of CMPs and GMPs. First, at CMP level, we clarified that CD62L-low CMPs had genuine CMP potential, whereas CD62L-high CMPs were mostly restricted to GMP potentials in both mice and humans. CD62L expression on CMPs was widely distributed and when divided into low, middle, and high fraction, colony forming-cell assay showed that murine CD62L-high CMPs mostly differentiated into CD11b-positive granulocytes and macrophages (97.4 ± 1.7%), whereas CD62L-low CMPs produced many erythroid and megakaryocytic colonies (38.7 ± 3.3%), and human CMPs had similar tendency. Also, liquid culture assay showed that murine CD62L-low CMPs differentiated into both GMPs (43.3 ± 4.1%) and megakaryocyte-erythrocyte progenitors (MEPs) (20.6 ± 3.7%), while CD62L-high CMPs mainly produced GMPs (82.8 ± 1.7%) and the proportion of MEPs was only 1.3 ± 0.1%. As for in vivo kinetics, we transplanted CD62L-low and high CMPs from GFP-expressing mice into irradiated wild-type mice, thus donor-derived platelets can be detected as GFP-positive. On day 7 after transplantation of each 15,000 cells, GFP-positive platelets from CD62L-low CMPs accounted for 9.8 ± 2.7% of all platelets, meanwhile CD62L-high CMPs accounted for only 0.20 ± 0.14%, which reinforced our hypothesis. Moreover, we performed transcriptome analysis using data of murine and human CMPs and focused on several transcription factors. Gata1, Klf1, Tal1 and Gfi1b are important transcription factors for erythroid and megakaryocytic differentiation, and these expressions were exclusively high in CD62L-low CMPs. On the other hand, Spi1 and Irf8 are important for differentiation into granulocytes and monocytes, and these expressions were higher in CD62L-high CMPs, which further corroborated the role of CD62L as a marker for refining the differentiation fate of CMPs. Second, at GMP level, we found that part of CD62L-neg GMPs possess remaining CMP potential and CD62L-low GMPs were skewed to granulocyte differentiation. CD62L expression on GMPs was mostly positive, but when we defined lowest 10% of GMPs as CD62L-neg GMPs, colony-forming cell assay in mice showed that part of CD62L-neg GMPs produced erythroid and megakaryocytic colonies (3.9 ± 2.6%), which suggested that this subset still possesses CMP potential. In vivo transplantation experiment using GFP-positive mice showed that CD62L-neg GMPs produced GFP-positive platelets slightly (0.02 ± 0.01%), but no platelets from CD62L-positive GMPs. Also, single-cell RNA-seq data revealed that part of CD62L-neg GMPs had CMP-specific gene expression pattern, suggesting that the bona fide GMPs were restricted to CD62L-positive GMPs. Next, we divided GMPs into CD62L-low and high GMPs, and performed colony-forming cell assay. CD62L-low GMPs produced granulocyte colony (CFU-G) 73.2 ± 6.1% and macrophage colony (CFU-M) 17.4 ± 3.3%, whereas CD62L-high GMPs produced CFU-G 46.6 ± 1.7% and CFU-M 42.9 ± 2.7%, which suggested that CD62L-low GMPs were granulocyte-skewed population. Also, we performed in vivo transplantation assay using Ly5.1 and Ly5.2 mice. On day 5 after transplantation, murine CD62L-low GMPs produced more neutrophils (87.6 ± 3.1%) in spleen than bulk GMPs (78.9 ± 1.9 %), which further confirmed this hypothesis. In summary, our in vitro and in vivo experiment and transcriptome analysis revealed that CMPs and GMPs had high heterogeneity. CD62L expression level refines the definition of CMPs and GMPs in both mice and humans, and elucidates the differentiation mechanism of myeloid cells in more detail. Disclosures Nakahara: Bristol-Myers Squibb Company: Honoraria; Eisai Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria. Kagoya:NIPPON SHINYAKU CO.,LTD.: Research Funding; Bristol-Myers Squibb Company: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding. Kurokawa:Eisai: Research Funding, Speakers Bureau; Teijin: Research Funding; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Shire Plc: Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Sanwa-Kagaku: Consultancy; Pfizer: Research Funding; Otsuka: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau.

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Sequential Study By RNA-Seq Shows a Reduction in Inflammation and a Cell Cycle Activation in T-Lymphocytes from Myelodysplastic Syndrome with Del(5q) after Lenalidomide Treatment in Patients Included in the "Sintra-REV" Clinical Trial

Blood ◽

10.1182/blood-2020-140703 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 26-27

Author(s):

Mónica Del Rey ◽

Luis A Corchete ◽

Teresa González ◽

Félix López Cadenas ◽

Eva Lumbreras ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Clinical Trial ◽

T Lymphocytes ◽

Myelodysplastic Syndrome ◽

Board Of Directors ◽

Inflammatory Cytokines ◽

Research Funding ◽

Advisory Committees ◽

Cell Cycle Pathway

Introduction: Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 [del(5q)]. The clinical efficacy of lenalidomide in MDS patients has been extensively reviewed and although the mechanisms of action in del(5q) clone have been previously described, in vivo sequential studies of modulatory effect on T lymphocytes are lacking. Our study was conducted in patients included in the Sintra-REV Clinical Trial: Lenalidomide (Revlimid) phase III, multicenter, randomized, double-blind study versus placebo in patients with low-risk MDS (low and intermediate IPSS-1) with del(5q), with anemia (HB≤12gr/dl) and without transfusion needs. Aim: The aim of this study was to explore the effect of lenalidomide in T-lymphocytes in MDS patients with del(5q) and without transfusion dependence. Materials and Methods: Sequential study was carried out in 26 samples from 13 paired MDS patients with del (5q). Seven out 13 were treated with lenalidomide and achieved a major erythroid and cytogenetic response. Peripheral blood (PB) samples were collected before and one month after treatment in treated-patients and at the same time points for non-treated patients. CD3+ cells were collected from PB samples and total RNA was isolated. SureSelect Strand Specific RNA library (Agilent Technologies) was applied to study changes in RNA levels. Raw reads were aligned against the Human genome GRCh37 using the STAR aligner. Counts were assigned to Ensembl gene IDs through HTseq using its UNION version. Differential gene expression was determined with DESeq2, considering as statistically significant those genes with FDR < 0.05. Pathway over-representation analysis (ORA) was conducted in the Webgestalt suite. Results: 332 genes were differentially expressed in CD3+ lymphocytes one month after lenalidomide treatment in our cohort of patients; 199 of them were over-expressed after the administration of this drug (Fig 1a). Of note, none of them were observed in non-treated patients after one month. The ORA revealed significant differences in the gene expression profile of sixteen cytokines and enrichment of genes of the cell cycle pathway (35 genes). The most relevant up-regulated cytokines were: IL10, TNFSF10, IFNGand IL6. These data explain lenalidomide-induced activation of an antileukemic immune response and secretion of anti-inflammatory cytokines. Although lenalidomide has been reported to reduce the expression of IL6 secreted by myeloid cell derived from MDS clon, we have observed upregulation of this gene in T-lymphocytes. Moreover, our study showed a downregulation of MBP6 that may help to correct the anemia and also attenuate inflammation signaling in MDS patients with del(5q) (Fig 1b). In addition, the most represented up-regulated genes related to cell cycle pathway were: cyclines (CCNB1, CCNB2, CDK1), centromere genes (CENPE, CENPM, CENPU), kinesin family members (KIF18A, KIF23, KIF2C), BUB1 mitotic checkpoint genes (BUB1, BUB1B), and genes involved in cell division (CDC6, CDC7,CDC25A). It has been described that lenalidomide inhibits CDC25A selectively in the del(5q) clone resulting in G2/M arrest and apoptosis. By contrast, our study showed that this gene was upregulated in T-lymphocytes promoting cell cycle and proliferation of these cells (Fig 1b). Conclusions: The immunomodulatory properties of lenalidomide can be summarized in two: a) regulation of antileukemic and anti-inflammatory cytokines production, b) activation of cell cycle and proliferation in T cells. To our knowledge, this is the first report describing RNA expression profiles in PB CD3+ lymphocytes collected from lenalidomide-treated del(5q) patients, contributing to overall understanding of lenalidomide action. Disclosures Sanz: Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees. Fenaux:Abbvie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Diez-Campelo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Lenalidomide was administered in anemic but not transfusion-dependence patients with low-risk MDS and del(5q)

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