scholarly journals The Unfolded Protein Response Mediates Resistance in AML and Its Therapeutic Targeting Enhances TKI Induced Cell Death in FLT3-ITD + AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2246-2246
Author(s):  
Timo Jaquet ◽  
Christian Preisinger ◽  
Marlena Bütow ◽  
Stefan Tillmann ◽  
Nicolas Chatain ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Board Of Directors ◽  
Research Funding ◽  
Bone Marrow Niche ◽  
Advisory Committees ◽  
Clonogenic Potential ◽  
The Unfolded Protein Response ◽  
Support Research

Abstract Introduction: The unfolded protein response (UPR) is a stress sensing signaling network that is activated upon endoplasmic reticulum (ER) stress, a condition characterized by an accumulation of mis- and unfolded proteins in the ER. To retain a functional cell metabolism, UPR activation increases protein folding and degradation. Acute myeloid leukemia (AML) stem cells are prone to develop ER stress, due to their oncogene-driven metabolism and the bone marrow niche, where they face stressors like hypoxia or nutrient fluctuations. Our preliminary work showed enhanced UPR gene expression levels, especially of IRE1α and XBP1, in different AML subtypes. Patients with high XBP1 mRNA expression had an inferior overall survival rate compared to patients with low XBP1 mRNA expression. Aims: We studied the role of elevated UPR signaling in AML therapy resistance and assessed the therapeutic potential of IRE1α-XBP1 inhibitor STF-083010 (STF) as a new strategy in different AML subtypes, including FLT3-ITD + AML. Methods: Human MV4-11 (FLT3-ITD), RS4-11 (FLT3 wildtype; WT), NB-4 (PML-RARα), THP-1 (MLLr) cells, and murine 32D cells transduced with FLT3-ITD or FLT3 WT were analyzed via western blot and RT-PCR. Metabolic activity was assessed by MTT assay, cell death and apoptosis were measured with propidium iodide (PI) or Annexin V staining using flow cytometry. FLT3 cell surface expression was measured via flow cytometry. The clonogenic potential was determined in CFU assays, using patient-derived mononuclear and CD34 + cells. For hypoxic experiments, MV4-11 cells were cultivated under hypoxia (3 % O 2) and cells were subjected to phosphoproteomic analysis, which was performed by mass spectrometry. Conditional Mx1-Cre/XBP1 fl/fl knockout mice were generated and deletion of XBP1 was induced by IP injection of Polyinosinic-polycytidylic acid (Poly(I:C)). Bone marrow and spleen cells were analyzed via flow cytometry and RT-PCR. Results: Treatment with FLT3 TKI AC220 specifically enhanced IRE1α mRNA (9.3-fold, p<0.05) and increased IRE1α protein in 32D FLT3-ITD cells. Likewise, the percentage of dead cells was significantly elevated in 32D FLT3-ITD upon IRE1α inhibition by STF compared to 32D FLT3 WT cells. Treatment with STF prevented XBP1 splicing and reduced the metabolic activity of human AML cell lines in a dose-dependent manner. Furthermore, IRE1α inhibition significantly induced apoptosis in human MV4-11 (6-fold, p<0.05), NB-4 (8-fold, p<0.01) and THP-1 (7-fold, p<0.01) cells and reduced their clonogenic potential. The combination of STF and AC220 strongly enhanced the percentage of apoptotic cells in MV4-11 cells compared to single treatments (by 3-fold, p<0.001). This strong induction of cell death was specific for FLT3-ITD + MV4-11 cells and not observed in FLT3 WT + RS4-11 cells. Similarly, the clonogenic potential of MV4-11 cells and FLT3-ITD + AML mononuclear patient cells was significantly decreased by the combinatorial treatment, while healthy donor cells were not affected. Likewise, conditional XBP1 knockout did not significantly alter normal hematopoiesis in mice. Hypoxia further enhanced IRE1α signaling in MV4-11 cells and strongly reduced the efficacy of AC220 (normoxia: 58.4-fold induction of dead cells, p<0.01; hypoxia: 2.2-fold induction, p>0.05). Analysis of phosphoproteomics revealed a less active FLT3 signaling under hypoxia. Intriguingly, the combination of IRE1α and FLT3 inhibition overcame the resistance towards AC220 under hypoxia and significantly induced cell death. Conclusion: IRE1α-XBP1 signaling is activated in different AML subtypes including FLT3-ITD + and is further enhanced by hypoxia present in the bone marrow niche. Targeting IRE1α in FLT3-ITD + cells effectively decreases clonogenic growth and induces apoptosis. Our data demonstrate that hypoxia-mediated resistance against AC220 can be overcome by simultaneous IRE1α inhibition. Genetic deletion of XBP1 does not harm steady-state murine hematopoiesis, rendering XBP1 an excellent therapeutic target. Disclosures Koschmieder: CTI: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other; Image Biosciences: Other: Travel support; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Karthos: Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Alexion: Other: Travel support; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Abbvie: Other: Travel support; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Brümmendorf: Bristol Myers: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Repeat Diagnostics: Research Funding; Takepart Media: Honoraria.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, 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.

Role of Selectins in the Pathogenesis of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 951-951 ◽  
Author(s):  
Abdel Kareem Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
Costas M Pitsillides ◽  
John T Patton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees

Abstract Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p<0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p<0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p<0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib. CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. 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. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

COMPARISON Among Citology, Histology, and FLOW Cytometry IN Evaluating Blast VALUES IN Myelodysplatic Syndromes (MDS). Survey FROM the Italian “PIEMONTE MDS REGISTRY”

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4028-4028
Author(s):  
Alessandro Levis ◽  
Daniela Maria Gioia ◽  
Laura Godio ◽  
Mauro Girotto ◽  
Bernardino Allione ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Prognostic Value ◽  
Univariate Analysis ◽  
Laboratory Data ◽  
Prognostic Impact ◽  
Advisory Committees ◽  
Blast Count

Abstract Abstract 4028 BACKGROUND. The corner stone of the WHO classification and prognostic scores of myelodysplastic syndromes (MDS) is the blast count in bone marrow. The standard cytology evaluation of at least 500 bone marrow cells is easy to perform, but some concerns arise about reproducibility of this method. Nowadays bone marrow trephine biopsy and flow cytometry are frequently considered for the diagnosis of MDS. However there is so far paucity of data comparing cytology, histology and flow cytometry in quantifying bone marrow blasts in order to differentiate non RAEB from BAEB-I and RAEB-II cases. AIM OF THE WORK. The Aim of the work was to analyse the differences and the prognostic impact of cytology, histology and flow cytometry in differentiating non RAEB from BAEB-I and RAEB-II. PATIENTS AND METHODS. Since 1999, clinical and laboratory data from 1256 new cases of MDS were prospectively recorded into the Piemonte MDS Registry. Blast count could be performed with the three different methods: BMC (bone marrow cytology) has been performed in 844 cases, BMH (bone marrow histology) in 874 cases, and BMF (bone marrow flow cytometry) in 636. In order to quantify blasts, immune-histochemistry evaluation of CD34+ cells was used in BMH, while both CD34+ and CD117+ cells were considered in BMF. Out of the total of the 636 patients analysed by BMF only 420 had an accurate and complete registration of CD34 and CD117 positivity and were considered for the present analysis. In two hundred and thirty six cases all three evaluations were contemporary available. The concordance of each diagnostic method with the others and their prognostic value were evaluated in both univariate and multivariate analyses. A comparison between BMC and BMH was available in 571 cases, between BMC and BMF in 228 cases, and between BMH and BMF in 279 cases. RESULTS. The disagreement in classifying patients as non-RAEB or RAEB-I or RAEB-II between BMC and BMH was 156/571 (27%), with BMH over-evaluating blasts in 114/571 cases (20%) and under-evaluating blasts in 42/571 cases (7%). The disagreement between BMC and BMF was 80/228 (35%), with BMF over-evaluating and under-evaluating blast percentage in comparison to BMC in 53/228 (23%) and in 27/228 (12%) cases respectively. The disagreement between BMH and BMF was present in 113/279 (41%), with BMF over-evaluating and under-evaluating blast percentage in comparison to BMH in 44/279 (16%) and in 69/279 (25%) cases respectively. In univariate analysis all three methods of quantifing blasts and differentiating non-RAEB from RAEB-I and RAEB-II retained an important prognostic value for both leukemic evolution and survival. However when the three models were tested in multivariate analysis in order to define the best predictor of leukemic evolution, BMC retained the best predictive value. CONCLUSIONS. When BMH or BMF are used instead of BMC in order differentiate non-RAEB from RAEB-I and RAEB-II, the shift to a different WHO category is evident in at least 30% of patients and BMH and BMF do not play the same role as BMC. BMC still remain the standard method to quantify blasts for classification and prognostic evaluation of MDS. Disclosures: Off Label Use: Lenalidomide in Mantle Cell Lymphoma. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

STAT5 Is a Critical Component Of The Time-Dependent Sensitivity Of CML Cells To TKI Treatment In a Bcr-Abl-Dependent, But JAK2-Independent Manner

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2705-2705
Author(s):  
Lisa Schafranek ◽  
Eva Nievergall ◽  
Jason A. Powell ◽  
Devendra K. Hiwase ◽  
Deborah L. White ◽  
...  
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Research Funding ◽  
Time Dependent ◽  
Short Term ◽  
Advisory Committees ◽  
Independent Manner ◽  
Clonogenic Potential ◽  
Tki Treatment

Abstract Introduction Bcr-Abl1 is necessary and sufficient to cause chronic myeloid leukemia (CML) and as such CML cells are dependent on Bcr-Abl signalling for survival. Targeting CML cells with tyrosine kinase inhibitors (TKIs) commits cells to apoptotic cell death. Bcr-Abl constitutively activates STAT5, however the role of JAK-2 in the activation of STAT5 by Bcr-Abl is controversial. Recent studies of transient Bcr-Abl inhibition indicate that residual low levels of TKI are sufficient to maintain STAT5 inhibition in the absence of sustained Bcr-Abl inhibition. Therefore STAT5 is a highly sensitive measure of kinase activity. We hypothesized that sustained blockade of STAT5 is essential for the commitment of CML cells to apoptosis following inhibition of Bcr-Abl by TKIs. Aim To determine the role of STAT5 and JAK inhibition in the commitment of CML cells to apoptosis. Methods Factors required for CML cell death were examined in the setting of transient inhibition of Bcr-Abl by TKIs. Induction of apoptosis was assessed by Annexin V/7AAD and the clonogenic potential of CML progenitors assessed by CFU-GM assay. Bcr-Abl and apoptotic signaling pathways were interrogated by western blotting and flow cytometry. Dasatinib was used at 100 nM for potent inhibition of Bcr-Abl. Short term refers to 30 min exposure. Standard washout refers to 3 consecutive washes following potent TKI treatment. Optimal washout refers to 3 washes with 1 h equilibration at 37°C in drug free media between washes. Results In BCR-ABL+ cell lines short term, potent dasatinib exposure followed by optimal washout resulted in reactivation of Bcr-Abl and STAT5, inhibition of apoptosis (83% viable, n=3) and maintenance of colony formation in CML progenitors (CFU-GM: 85% of untreated n=3). Plasma concentrations of dasatinib vary between patients, however peak plasma levels occur up to 6 h after dosing and dasatinib remains available for up to 24 h. CML cell lines and CP-CML CD34+ progenitors were exposed to 100 nM dasatinib for 0.5-8 h before optimal washout. Cell death was achieved if TKI exposure by at least 4 h, with maximal cell death (15% viable, n=3, p=0.008) and reduction of colonies (30.1% of control, p=0.002) achieved after 8 h exposure. Comparison of 30 min and 8 h exposures to 100 nM dasatinib followed by optimal washout was performed to assess the critical signalling components required to induce apoptosis. Reactivation of Bcr-Abl, STAT5 and Erk occurred upon washout following both the 30 min and 8 h exposures, however the 8 h exposure resulted in the inhibition of STAT5 and loss of expression of STAT5 targets Mcl-1 and Bcl-xl, but not Bcl-2. In CP-CML CD34+ cells, prolonged inhibition of STAT5 was observed after 4 h exposure, following optimal washout, highlighting loss of STAT5 activity as potentially critical to irreversible induction of cell death. Continuous inhibition of STAT5 alone with pimozide (Pz) or the specific inhibitor N’-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide (herein referred to as STAT5i) led to minimal apoptosis (73% and 75% viable, respectively, n=3) when used alone. However, when combined with 30 min exposure to dasatinib (100 nM) STAT5 inhibition proved lethal in a proportion of cells despite optimal washout (57% viable +Pz and 59% +STAT5i). The clonogenic potential CML progenitors was also significantly reduced (12%, p=0.002 and 18% CFU, p=0.003) (Figure 1). The JAK1/2 kinase inhibitor ruxolitinib was used to assess the involvement of JAK1/2 in Bcr-Abl-dependent activation of STAT5. Similar to the observations with STAT5 inhibition, ruxolitinib had minimal effect on cell death as a sole agent (74% viable). However, in contrast to our observations with STAT5 inhibition, the addition of ruxolitinib to 30 min 100 nM dasatinib exposure did not induce additional cell death (70% viable, p=0.41, n=3). Conclusion STAT5 is a critical component of the time-dependent sensitivity of CML cells to TKI treatment in a Bcr-Abl-dependent, but JAK-independent manner. In contrast to previous studies describing JAK2 as a promising secondary target for the enhancement of TKI treatment of CML, we demonstrate that inhibition of STAT5 in conjunction with standard TKI therapy is a promising therapeutic strategy for the treatment of CML. Disclosures: Nievergall: CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding. Hughes:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding.

scholarly journals Targeting Polo-like Kinase 4 Triggers Polyploidy and Apoptotic Cell Death in TP53-Mutant Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1167-1167
Author(s):  
Edward Ayoub ◽  
Rafael Heinz Montoya ◽  
Vakul Mohanty ◽  
Wencke Walter ◽  
Tallie Patsilevas ◽  
...  
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Intellectual Property Rights ◽  
Research Funding ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Advisory Committees ◽  
Grant Support ◽  
Support Research

Abstract Background: TP53 mutations in acute myeloid leukemia (AML) are associated with complex karyotype, high incidence of minimal residual disease (MRD), and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018). While numerous novel treatment regimens, including the combination of the BCL2 inhibitor venetoclax (VEN) and hypomethylating agents (HMA), have emerged as partially effective treatments and resulted in higher remission rates in patients with TP53-mutant AML, full clearance of the mutant TP53 clone is rarely achieved and the majority of patients relapse (Short et al., 2021; Takahashi et al., 2016). The mechanisms responsible for response and relapse in TP53-mutant AML remain unclear and investigating novel mechanisms is critical to develop more effective therapies. Results: In order to shed light on the defective p53 signaling pathways underlying TP53 mutant AML, and to better understand mechanisms of resistance, we performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations using samples collected from TP53-mutant or TP53-wt high-risk AML patients. Samples were collected at diagnosis (DX) and post-treatment (POSTTX) (total number of samples n= 67, TP53-mutant=36, TP53-wt=31). Diagnostic samples include bulk AML, leukemic stem cells (LSCs), and post-treatment samples including bulk mononuclear cells (MNCS) and patient specific MRD (total n= 67, DX_Bulk=15, DX_LSCs=15, POSTTX_MNCs=14, POSTTX_MRD=23). Differential gene expression analysis of TP53-mutant samples indicates a positive enrichment of the following pathways: G2/M checkpoint, MYC targets, and mitotic spindle, among others. We focused here on genes associated with TP53-mutant AML enriched pathways, and identified a key regulator of centriole biogenesis, one of E2F targets: Polo-like kinase 4 (PLK4) as a potential target highly expressed in TP53-mutant AML samples . Previous publications showed that PLK4 is transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Fischer et al., 2014; Li et al., 2005). Here we show that TP53-mutant AML samples lack the p53-dependent PLK4 repression and have higher levels of PLK4 compared to TP53-wt AML. To test the rigor of this finding, we interrogated the Munich Leukemia Laboratory (MLL) data base and analyzed their clinically annotated (e.g. karyotype, survival, complete blood counts, previous treatments ... etc) RNA-seq dataset of 726 AML samples (TP53-mutant=72, TP53-wt=654). TP53-mutant AML samples consistently showed significant PLK4 upregulation (p= 0.0003). We analyzed PLK4 expression and its correlation with TP53 mutations in The Cancer Dependency Map project dataset (1375 cell lines in 35 different types of cancers) (p= 0.004). Furthermore, we found significantly higher PLK4 protein levels in TP53-mutant AML MOLM13 cell lines when compared with syngeneic TP53-wt AML MOLM13 cells. Experimentally, we found that PLK4 inhibition using 25nM CFI-400945 in TP53-mutant AML MOLM13 cell lines triggers polyploidy &gt; 2-fold higher than in TP53-wt AML MOLM13 cell lines 72 hours post treatment (Fig.1A p&lt; 0.0001). Finally, we show that polyploidy is not reversible after drug removal and results in significantly increased levels of apoptotic cell death in TP53-mutant AML MOLM13 cells (Fig.1B). Conclusion: Our data suggest that TP53-mutant AML expresses higher levels of PLK4 in comparison to TP53-wt AML, and targeting PLK4 triggers polyploidy and apoptotic cell death in TP53-mutant AML. A clinical trial is ongoing testing the efficacy of PLK4 inhibition (CFI-400945) in AML (Clinical Trial ID: NCT04730258, TWT-202). Figure 1 Figure 1. Disclosures Issa: Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding. Borthakur: Takeda: Membership on an entity's Board of Directors or advisory committees; ArgenX: Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; Astex: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Konopleva: Ascentage: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Stemline Therapeutics: Research Funding; KisoJi: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Sanofi: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Andreeff: Senti-Bio: Consultancy; ONO Pharmaceuticals: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy; Breast Cancer Research Foundation: Research Funding; Oxford Biomedica UK: Research Funding; Karyopharm: Research Funding; Medicxi: Consultancy; Amgen: Research Funding; AstraZeneca: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Syndax: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

Decreased Expression of CD62L and CD54 Correlates with Poor Cytogenetic Risk Group In Myelodysplastic Syndromes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2915-2915
Author(s):  
Canan Alhan ◽  
Theresia M. Westers ◽  
Claudia Cali ◽  
Floortje L. Kessler ◽  
Monique Terwijn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Healthy Volunteers ◽  
Board Of Directors ◽  
Myelodysplastic Syndromes ◽  
Research Funding ◽  
Risk Category ◽  
Advisory Committees ◽  
Cd34 Cells

Abstract Abstract 2915 Interactions in the bone marrow (BM) between haematopoietic progenitor cells (HPC) and the BM micro environment are important for the regulation of cell adhesion, proliferation, differentiation and survival. Expression of both CD62L (L-selectin) and CD54 (ICAM-1) on HPC demonstrated to play a role in signal transduction routes for proliferation and growth regulation. Especially CD54 is involved in uncontrolled proliferation and block of apoptosis. Previously, it was described that decreased expression of CD62L in acute myeloid leukemia (AML) was associated with a poor cytogenetic risk profile and an adverse clinical outcome (Graf M et al, Eur J Haematol 2003) Myelodysplastic syndromes are a group of clonal HPC disorders characterized by ineffective hematopoiesis and a propensity to evolve into AML. The International Prognostic Scoring System (IPSS) provides information on both survival and risk of development of an AML. The purpose of our study was to evaluate CD62L and CD54 expression on CD34+ cells in MDS patients by flow cytometry and to assess the value of a CD62L/CD54 ratio for prognostication. Bone marrow samples of 30 newly diagnosed MDS patients (3 RA(RS)/18 RCMD(RS), the <5% blasts group; 5 RAEB-1, 4 RAEB-2, the >5% blasts group), 16 AML patients with prior MDS and 26 healthy volunteers were analyzed for CD62L and CD54 expression on CD34+ cells by using flow cytometry. An adhesion index was calculated as a ratio of the percentage and MFI of CD62L and CD54 positive cells (as was reported by Buccisano et al, Eur J Haematol 2007). The CD62L/CD54 ratio was significantly decreased in MDS with <5% blasts (median 79.09 p<0.0001) as compared to healthy volunteers (median 480.4) and even more decreased in high risk MDS (median 14.67 p<0.0001 and p=0.001 as compared to healthy volunteers and MDS with <5% blasts, respectively) and AML with prior MDS (median 12.54, p<0.0001 and p=0.009 as compared to healthy volunteers and MDS with <5% blasts, respectively). The MDS patients were assigned to the good, intermediate or poor IPSS cytogenetic risk category. Cytogenetics was available for 22 MDS patients. The CD62L/CD54 ratio was significantly lower in the cytogenetic poor risk category compared with the good risk category (median 5.4 and median 70.79 respectively, p=0.018). Moreover, a low CD62L/CD54 ratio correlated significantly with poor cytogenetics, p=0.006. In the group of MDS patients with <5% blasts, 4 developed a refractory anemia with excess of blasts or AML within a follow up period of 12 months. There was a trend for a lower CD62L/CD54 ratio for MDS patients who developed an AML compared with patients who did not. In conclusion, the CD62L/CD54 ratio is significantly decreased in MDS compared with healthy volunteers and even more decreased in AML with prior MDS. Both CD62L and CD54 are involved in regulation of proliferation and apoptosis of the HPC. A decreased adhesion ratio in low risk MDS patients might reflect HPC damage at an early stage of the disease with an increased proliferative capacity and a decreased apoptotic profile. Interestingly, a low CD62L/CD54 ratio showed a significant inverse correlation with the IPSS cytogenetic risk category. Due to an absence of metaphases in a proportion of MDS patients, cytogenetics is not always available. The CD62L/CD54 ratio might serve as a surrogate marker for poor prognosis cytogenetics in case no karyotype is available. Low risk MDS patients who developed an AML within 12 months tended to have a lower CD62L/CD54 ratio. Although these results are promising, sample size and follow up period needs to be extended. The CD62L/CD54 ratio might add to prognostication of MDS patients and might identify MDS patients with <5% blasts who are at risk for development of an AML. Disclosures: Ossenkoppele: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 23-23
Author(s):  
Alexander Schaeffer ◽  
Ewelina Czlonka ◽  
Irene Tirado-González ◽  
Thomas Böse ◽  
Jennifer Beauvarlet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
Standard Of Care ◽  
Bone Marrow Niche ◽  
Advisory Committees ◽  
Bone Marrow Biopsies ◽  
Primary Bone ◽  
Primary Bone Marrow

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

The Pro-Inflammatory Cytokine Interleukin-1 Is Essential for Clonal Expansion and Disease Progression in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 866-866
Author(s):  
Alyssa Carey ◽  
Megan M Cleary ◽  
David K Edwards ◽  
Christopher A. Eide ◽  
Elie Traer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Disease Progression ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Clonal Expansion ◽  
Advisory Committees ◽  
Clonogenic Potential ◽  
Equity Ownership

Abstract Background: Recent 'omics'-based approaches have revealed that acute myeloid leukemia (AML) has significant genetic heterogeneity, and that clonal evolution in AML occurs through stepwise acquisition of somatic mutations. However, extrinsic factors may also influence this process. Here we provide strong evidence that inflammatory cytokines secreted in the AML microenvironment play a critical role in clonal expansion and disease progression. Specifically, we show that the proinflammatory cytokine IL-1 promotes in vitro clonal expansion in a large subset of primary AML samples and in vivo disease progression in a murine AML model. Methods: We used several functional assays to determine which extrinsic cytokine-activated pathways are essential for AML cell survival. We analyzed 90 primary AML samples ex vivo in the absence and presence of dose gradients of 98 distinct cytokines. We used stromal cells conditioned media and a recombinant cytokine cocktail (IL-3, IL-6, SCF, GCSF, FLT3L) for positive controls. The distributions for the positive and negative control allowed us to derive rigorous empirical thresholds to define cytokine dependence for individual samples. We also performed siRNA screens targeting 188 cytokine and growth factor receptors, and measured 30 different cytokines secreted in the plasma of peripheral blood and bone marrow samples using a multiplex Luminex assay. Functionally relevant pathways were validated using shRNA and genetic mouse models. Results: Several of the 98 cytokines promoted the growth of primary AML cells, including IL-1, GM-CSF, G-CSF, TPO, CXCL-1, IL-3, M-CSF, MCPs, TNF-α, and BMP-4. Many of these are known to induce an inflammatory response and cluster with the growth response to IL-1, a master mediator of innate immunity and inflammation. IL-1α and IL-1β had the most profound effect on the clonal expansion of myeloid progenitors, leading to 3- to 20-fold growth increase in 70% (63/90) of primary AML samples. Consistent with our findings from the cytokine screen, treatment of CD34+ AML cells with IL-1 led to increases in cell growth, survival, and clonogenic potential. Paradoxically, IL-1 suppressed both growth and colony formation in normal CD34+ cells. In support of these ex vivo findings, IL-1β was overexpressed in IL-1-sensitive AML bone marrow and peripheral blood samples compared to non-sensitive AML samples and normal samples. Intracellular FACS showed that 80% of the total IL-1β is secreted by monocytes, and to some extent by myeloid progenitors and stromal cells, but not by B or T cells in the AML bone marrow microenvironment. Consistent with this, most of the IL-1-sensitive AML samples exhibited monocytic and myelomonocytic features. These results suggest that IL-1 secreted in the bone marrow microenvironment regulates AML cell growth in a paracrine manner. Silencing of the IL-1 receptor, IL1R1, reduced the viability of AML primary samples by 60-80% and led to a significant ablation of clonogenic potential (80% reduction) of oncogene-induced leukemic cells (AML1-ETO9a, NRASG12D and MLL-ENL) in mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer (39 days; range: 28-118) than did recipients of wild-type marrow (30 days; range: 27-61; p=0.012). Mechanistically, IL-1β regulates phosphorylation of p38MAPK, a downstream component of the IL-1 pathway, in AML progenitors and IL-1-sensitive AML samples exhibit more p38 phosphorylation as compared to normal cells. Conversely, knocking down IL1R1 or treating AML cells with p38MAPK inhibitors such as doramapimod reduced the growth of AML cells by decreasing p38MAPK phosphorylation. Conclusion: These results demonstrate a novel role for IL-1 and its receptor in promoting clonal growth and disease progression in a large subset of AML patients. Our findings suggest that AML patients may benefit from drugs targeting IL-1/IL1R1/p38MAPK signaling because of their potential to enhance normal hematopoiesis while inhibiting AML -- a significant clinical advantage over traditional chemotherapy, which kills both normal and leukemic cells.Since IL-1 and its receptors are not mutated in these patients, our data also highlight the importance of ex vivo functional screening for microenvironmental stimuli for the identification of novel therapeutic targets. Disclosures Tyner: Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Research Funding. Druker:Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Millipore: Patents & Royalties; McGraw Hill: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sage Bionetworks: Research Funding; ARIAD: Research Funding; AstraZeneca: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Oncotide Pharmaceuticals: Research Funding. Agarwal:CTI BioPharma: Research Funding.

scholarly journals In Human Visualization of Ibrutinib-Induced CLL Compartment Shift

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1750-1750
Author(s):  
Marius E Mayerhoefer ◽  
Alexander Haug ◽  
Ulrich Jaeger ◽  
Lukas Kazianka ◽  
Verena Pichler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Lymph Nodes ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Relative Increase ◽  
Whole Body ◽  
Advisory Committees ◽  
Treatment Naïve

BACKGROUND. B-cell receptor (BCR) signaling is a central driver for chronic lymphocytic leukemia (CLL), and its targeting through irreversible inhibition of Bruton's tyrosine kinase (BTK) by ibrutinib has significantly improved the prognosis of CLL patients. Ibrutinib treatment has become standard of care, and, recently, has advanced to the first-line setting. A treatment-induced, sometimes dramatic increase of peripheral lymphocytosis, has emerged as a class effect of BTK inhibitors in CLL. Mechanistically, BTK blocking by ibrutinib might affect adhesion molecules and chemokine receptors, such as CXCR4 and CXCR5, thus interfering with the protective tissue microenvironment and mobilizing tissue-resident CLL cells from the lymph nodes and bone marrow into the peripheral blood. This hypostasized mechanism of a "compartment shift," however, has not yet been demonstrated experimentally or visually. Positron emission tomography (PET) with [68Ga]Pentixafor, a radiotracer that specifically targets the CXCR4 receptor, was recently established as a sensitive approach with which to detect CLL in vivo. As a proof-of-concept, we here present three CLL patients to demonstrate the potential of [68Ga]Pentixafor-PET/MR imaging to functionally track CLL cells along the redistribution induced by ibrutinib. METHODS. Three CLL patients were included: patient 1 was a 74-year-old treatment-naïve male, with unmutated IGHV status and high-risk cytogenetics, including del17p13 and TP-53 mutation; patient 2 was a 53-year-old female with late relapse (12 years after allogeneic stem cell transplantation), IGHV was unmutated, and cytogenetic abnormalities included del11q22 and del13q14; and patient 3 was a 59-year-old treatment-naïve male with unmutated IGHV and del11q22 and del13q14. Whole-body PET/MRI with injection of 150 MBq of [68Ga]Pentixafor was performed pre and on ibrutinib treatment (patient 1: one week; patient 2: two weeks; and patient 3: three weeks after start of the first therapy cycle, respectively). Treatment consisted of continuous oral administration of 420 mg of ibrutinib. Mean standardized [68Ga]Pentixafor uptake values (SUVmean) of involved lymph nodes, the bone marrow, and the spleen were measured. Isolated peripheral blood mononuclear cells (PBMCs) were stained with fluorescence-labeled antibodies, and measurements were performed on flow cytometer. RESULTS. In all three cases - at one week, two weeks, and three weeks on ibrutinib treatment - CXCR4 density, as measured on [68Ga]Pentixafor-PET, shifted from the bone marrow and lymph nodes toward the spleen (Figs. 1-3). In patient 1, the SUVmean decreased in the bone marrow (-9.8%) and lymph nodes (-12.0%), whereas it increased markedly in the spleen (+29.8%). At flow cytometry, this patient exhibited an increase of CXCR4-high (tissue-resident) CLL cells upon ibrutinib treatment (+56.7% relative to baseline). In patient 2, the SUVmean decreased in the bone marrow (-14.7%) and lymph nodes (-27.3%), whereas it more than doubled in the spleen (+133.0%). At flow cytometry, individual CLL cells demonstrated a relative increase (+39.1%) of CXCR4 positivity. In patients 3, the SUVmean decreased in the bone marrow (-27.6% %) and lymph nodes (-41.9%), whereas it increased markedly in the spleen (+26.1%). At flow cytometry, the relative increase of CXCR4-high CLL cells was +58.3%. CONCLUSIONS. We here provide the first pictures of the early functional treatment effects of ibrutinib. While our analyses confirmed a shift of CXCR4 positive CLL cells from lymph nodes to peripheral blood, they also revealed that ibrutinib rapidly released CLL cells from the bone marrow. Also, unexpectedly, CLL cells redistributed to the orthotopic splenic cavernous system. Visualization of CLL on ibrutinib supports the pre-existing clinical hypothesis of a "compartment shift", however it also modified and refined the mechanistic model by describing early clearing of the bone marrow and re-distribution to the peripheral blood and the spleen. Disclosures Jaeger: Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria. Wester:Scintomics: Other: Spouse CEO of Company; CXCR4-targeted radiopharmaceuticals: Other: Inventor; Scintomics GmbH, Germany: Other: Shareholder. Staber:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Speakers Bureau; Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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