scholarly journals Eltrombopag Promotes Megakaryocyte Survival and Signaling in the Presence of Specific Cytotoxic Agents

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3836-3836
Author(s):  
Komal Kumar Javarappa ◽  
Dimitrios Tsallos ◽  
Bulat Zagidullin ◽  
Joseph J Saad ◽  
Jing Tang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Annexin V ◽  
Complete Recovery ◽  
Magnetic Bead ◽  
Signaling Molecules ◽  
Cytotoxic Drugs ◽  
Cytotoxic Agents ◽  
Treatment Regimens ◽  
The Impact

Abstract BACKGROUND: Cytotoxic chemotherapy/agents can cause a range of side effects in cancer patients including anemia, neutropenia and thrombocytopenia, resulting in increased morbidity and mortality. These cytopenias are in part due to massive depletion of bone marrow progenitors. A common target of many chemotherapies is the megakaryocyte (MK), a rare progenitor representing 0.2% of the bone marrow. Loss of MK lineage cells results in thrombocytopenia, therefore, discovering ways to protect MKs from cytotoxic drugs could prevent this life-threatening condition. Here, we show that eltrombopag (EP), a small molecule, non-peptide thrombopoietin receptor (TPO-R) agonist, protects healthy MKs from different cytotoxic agents. We further explored the impact of cytotoxic drugs and EP treatment on signaling molecules active in MKs. METHODS: Peripheral blood and bone marrow samples were collected from healthy donors after informed consent and following protocols in accordance with the Declaration of Helsinki. CD34+ positive cells were isolated by immuno-magnetic bead separation from the samples and expanded for 8-10 days with a cytokine cocktail to induce MK differentiation. After MK expansion, cells were transferred to 96-well plates pretreated with 14 different drugs including cytarabine, gemcitabine, paclitaxel, carboplatin, cisplatin, doxorubicin, vincristine, etoposide, midostaurin, ruxolitinib, panobinostat, azacitidine, venetoclax and navitoclax. The drugs were plated in 5 different doses in a 10,000-fold concentration range, and cells added with or without EP. After 3 days incubation, the cells were analyzed on a high throughput flow cytometer using Annexin V and 7AAD to distinguish live from dead cells. To understand the impact of the drugs and EP on signaling molecules downstream of TPO-R, we analyzed phosphorylation of AKT, ERK, STAT3 and STAT5 in populations defined by MK markers CD41a, CD42b and CD110 (TPO-R). RESULTS: We found that EP overall supports MK survival in the presence of cytotoxic agents. The greatest net effect was observed when EP was combined with BCL2 inhibitors (venetoclax, navitoclax), which resulted in increased MK cell maturation compared to inhibitor alone, while gemcitabine plus EP showed the next best response. EP combined with etoposide, vincristine, cytarabine, paclitaxel, cisplatin and ruxolitinib resulted in an increase of immature MKs without a reduction in mature MK cell numbers. Higher numbers of MKs were observed when EP was combined with midostaurin, carboplatin, panobinostat and doxorubicin, compared to treatment without EP; although these numbers were lower than other tested drugs. Phosphoflow analysis revealed intriguing differences in EP-induced signaling compared to signaling induced by recombinant human thrombopoietin (rhTPO) (Figure 1). Also, EP-induced signaling did not correlate with TPO-R expression. EP activated AKT in all MK subsets, but rhTPO induced AKT only in immature MKs. While rhTPO induced ERK phosphorylation in different MK subsets, EP had no effect on ERK activation. Furthermore, rhTPO activated STAT3 only in mature MKs, but EP induced STAT3 in all MK populations. In contrast, both EP and rhTPO induced phosphorylation of STAT5 in all MK subsets. Overall signaling was inhibited by a selected set of agents (venetoclax, etoposide, midostaurin) compared to basal signaling, but addition of EP prevented the inhibition. Similar to the cell survival analysis, venetoclax combined with EP resulted in complete recovery of signaling activity compared to venetoclax alone. But the response was not as striking for etoposide or midostaurin combination compared to treatment alone. CONCLUSION: Using MKs expanded from CD34+ cells, we found that EP supports MK survival in the presence of several different cytotoxic agents, with the most striking rescue observed when EP was combined with BCL2 inhibitors. Importantly, EP and rhTPO induced distinct signaling patterns, with EP signaling independent of TPO-R expression, suggesting alternate EP targets. The tested drugs inhibited basal signaling in MKs, but this inhibition was prevented by EP. These results suggest addition of EP to cancer treatment regimens may prevent the inhibitory effects of some agents on MKs and support future investigations to demonstrate clinical efficacy of EP in combinations with some of these cytotoxic agents to alleviate thrombocytopenia. Figure 1. Figure 1. Disclosures Javarappa: Novartis Pharma AG: Research Funding. Tsallos:Novartis: Research Funding. Marques Ramos:Novartis: Employment. Pallaud:Novartis: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Orion Pharma: Research Funding.

scholarly journals Bone Marrow Stromal Cells Protect Both FLT3-ITD and FLT3-WT Primary AML Cells from the Anti-Leukemic Activity of the FLT3 Inhibitor AC220

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4377-4377
Author(s):  
Cedric Dos Santos ◽  
Georges Habineza Ndikuyeze ◽  
Michael Nisssan ◽  
Chenghui Zhou ◽  
Xiaochuan Shan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Inhibition ◽  
Stromal Cells ◽  
Research Funding ◽  
Critical Role ◽  
Annexin V ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
The Impact ◽  
Dose Dependent

Abstract FTL3 mutations are found in about 30% of AML patients, conferring a leukemic blast growth advantage, drug therapy resistance in the bone marrow (BM) and poor outcome. Mesenchymal stem/stromal cells (MSCs) are essential components of the bone marrow microenvironment, and growing evidence suggest that MSCs play a critical role in AML chemo-resistance, although the molecular mechanisms involved are poorly understood. The purpose of the study was to (1) establish an novel in vitro co-culture system between primary AML blasts and healthy donor BM-MSCs (HD-MSCs) or AML patient-derived MSCs (AML-MSCs), (2) evaluate the impact of culture with BM-MSCs on the sensitivity of AML cells to AC220 using patients samples with FLT3-ITD (n=4) or FLT3-WT (n=3). We first cultured HD-MSCs (n=5) and AML-MSC (n=3) and observed no phenotypical differences (CD14- CD34- CD45- CD73+ CD90+ CD105+), although HD-MSCs grew faster. We evaluated the effect of co-culturing AML samples (n=6) with HD-MSCs or AML-MSCs for 5 and 12 days on leukemic cell growth and found that both types of MSCs significantly and equally enhanced AML cell proliferation while maintaining blast phenotype. Using clonogenic assays on 4 AML specimens cultured alone or with either HD- or AML-MSCs for 5 and 12 days, we found that co-culture with either source of BM-MSCs drastically increased colony-forming cells number at day 5 and day 12 while CFC number decreased in the absence on BM-MSCs (no colonies at day 12 for the 4 samples), indicating that AML co-culture with HD/AML-MSCs supports the survival and/or proliferation of AML stem/progenitor cells. We next assessed the effect of increasing doses of AC220 (1, 10, 50, 100 and 500nM) on the apoptosis of FLT3-ITD (n=3) and FLT3-WT (n=4) AML cells cultured alone or with HD-MSCs. Exposure to AC220 for 72 hours significantly, and in a dose-dependent manner, increased the apoptosis of AML FLT3-ITD cells in monoculture (n=3, 21±1% of Annexin V positive cells for control, AC220 1nM 29±3.7%, 10nM 31±2.5%, 50nM 32±1.5%, 100nM 34±1.7% and 500nM 38±3.6%). In contrast, AML FLT3-ITD cells co-cultured with HD-MSCs were resistant to the drug (n=3, 21±2.6% of Annexin V positive cells for control, AC220 1nM 23±3%, 10nM 22±3%, 50nM 25±5.7%, 100nM 30±8.3% and 500nM 33±9.5%). Interestingly, we found that AML FLT3-WT are much less sensitive to increasing doses of AC220 compared to ITD samples (n=4, 27±3.9% of Annexin V positive cells for control, AC220 1nM 30±6.5%, 10nM 35±14%, 50nM 37±11%, 100nM 39±13% and 500nM 43±11%), and co-culture with BM-MSCs further decreased the sensitivity of AML FLT3-WT cells to AC220-induced apoptosis (n=4, 19±3.2% of Annexin V positive cells for control, AC220 1nM 17±3.9%, 10nM 20±3.4%, 50nM 19±3.7%, 100nM 21±4.5% and 500nM 26±1%). AC220 treatment for 3 days significantly, and in a dose-dependent manner, inhibited CFCs in AML FLT3-ITD (n=4, with 26±8%, 46±6%, 60±9%, 69±10% and 86±3% inhibition with 1, 10, 50, 100 and 500nM of AC220 respectively) while AML FLT3-ITD co-culture with HD-MSCs were less sensitive (n=4, with 9±10%, 30±6%, 42±9%, 57±11% and 72±7% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Similarly to the AC220-induced apoptosis, we observed that AML FLT3-WT CFCs are less sensitive to AC220-induced growth inhibition compared to ITD samples, although a 3 days exposure to AC220 significantly, and in a dose-dependent manner, inhibited AML FLT3-WT CFCs (n=3, with 38±16%, 44±14%, 58±12%, 70±21% and 81±19% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Interestingly, we observed that co-culture of AML FLT3-WT with stromal cells were significantly more resistant to increasing doses of AC220 (n=3, with 22±7%, 36±5%, 43±8%, 46±8% and 57±6% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Altogether, these results suggest that AML FLT3-ITD cells in monoculture are more sensitive to AC220 treatment compared to AML FLT3-WT primary cells, but more importantly, upon interaction with primary HD-MSCs, both WT and FLT3-ITD primary samples are protected from apoptosis and growth inhibition induced by AC220, indicating a critical role for the BM microenvironment in AC220 resistance. We are currently testing the impact of BM-MSCs co-culture on leukemic stem cell sensitivity to AC220 using transplantation in NSG mice. We will also evaluate if this co-culture model can be predictive of the response to in vivo treatment with AC220 in a patient-derived xenograft model. Disclosures Dos Santos: Janssen R&D: Research Funding. Danet-Desnoyers:Janssen R&D: Research Funding.

scholarly journals Cognitive impairment after cytotoxic chemotherapy

2019 ◽  
Vol 7 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Petra Huehnchen ◽  
Antonia van Kampen ◽  
Wolfgang Boehmerle ◽  
Matthias Endres
Keyword(s):  
Breast Cancer ◽  
Colon Cancer ◽  
Cognitive Impairment ◽  
Cancer Patients ◽  
Cytotoxic Chemotherapy ◽  
Cytotoxic Drugs ◽  
Cytotoxic Agents ◽  
Cancer Type ◽  
Breast Cancer Patients ◽  
The Impact

Abstract Background Neurotoxicity is a frequent side effect of cytotoxic chemotherapy and affects a large number of patients. Despite the high medical need, few research efforts have addressed the impact of cytotoxic agents on cognition (ie, postchemotherapy cognitive impairment; PCCI). One unsolved question is whether individual cytotoxic drugs have differential effects on cognition. We thus examine the current state of research regarding PCCI. Neurological symptoms after targeted therapies and immunotherapies are not part of this review. Methods A literature search was conducted in the PubMed database, and 1215 articles were reviewed for predefined inclusion and exclusion criteria. Thirty articles were included in the systematic review. Results Twenty-five of the included studies report significant cognitive impairment. Of these, 21 studies investigated patients with breast cancer. Patients mainly received combinations of 5-fluorouracil, epirubicin, cyclophosphamide, doxorubicin, and taxanes (FEC/FEC-T). Five studies found no significant cognitive impairment in chemotherapy patients. Of these, 2 studies investigated patients with colon cancer receiving 5-fluorouracil and oxaliplatin (FOLFOX). Independent risk factors for PCCI were patient age, mood alterations, cognitive reserve, and the presence of apolipoprotein E e4 alleles. Conclusions There is evidence that certain chemotherapy regimens cause PCCI more frequently than others as evidenced by 21 out of 23 studies in breast cancer patients (mainly FEC-T), whereas 2 out of 3 studies with colon cancer patients (FOLFOX) did not observe significant changes. Further studies are needed defining patient cohorts by treatment protocol in addition to cancer type to elucidate the effects of individual cytotoxic drugs on cognitive functions.

Mobilization Of Blasts and Leukemia Stem Cells by Anti-CXCR4 Antibody BMS-936564 (MDX 1338) in Patients With Relapsed/Refractory Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3882-3882 ◽  
Author(s):  
Sylvia Chien ◽  
Lauren E. Beyerle ◽  
Brent L. Wood ◽  
Elihu H. Estey ◽  
Frederick R. Appelbaum ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Bone Marrow ◽  
Research Funding ◽  
Annexin V ◽  
Fold Increase ◽  
Cxcr4 Expression ◽  
Leukemia Stem Cells ◽  
Pre Treatment ◽  
Refractory Aml ◽  
Peak Value

Abstract Background Bone marrow homing of AML is dependent on CXCR4, and high levels of CXCR4 expression correlate with worse survival in AML (Rombouts et al 2004, Spoo et al 2007). CXCR4 antagonists overcome environment adhesion mediated drug resistance and enhance chemotherapy induced cytotoxicity (Liesveld et al 2007, Zeng et al 2009, Nervi et al 2009, Beider et al 2010). Plerixafor, a small molecular CXCR4 inhibitor, was studied in a phase I trial of newly diagnosed AML patients (Uy et al ASH 2011), and BMS-936564, a fully-human monoclonal antibody to CXCR4, in combination with MEC (mitoxantrone, etoposide, cytarabine), is currently under study in relapsed/refractory AML. Method The clinical trial completed a phase I dose escalation phase in AML patients, with increasing concentrations of BMS-936564 in 4 dose cohorts (0.3,1,3, and 10 mg/kg) and is currently enrolling a cohort of first salvage AML patients at the maximum dose of 10 mg/kg. The initial cohort of patients at 0.3 mg/kg received three weekly doses of antibody on days 1, 8, 15 [monotherapy period of cycle 1 (21 days)], followed by the same dose of antibody on days 1, 8, 15 of cycle 2 plus MEC chemotherapy [days 1-5 of cycle 2, (28 day cycle)]. After enrollment of the first cohort, the protocol was amended to reduce the monotherapy period to 1 week (1 dose of BMS-936564) in cohorts 1, 3, and 10 mg/kg, followed by the same combination regimen. As a companion study to this trial with the anti-CXCR4 antibody, we are investigating CXCR4 expression, timing of mobilization of leukemic blasts and leukemia stem cells (LSCs), and induction of apoptosis. Mobililzation of LSCs will be critical to eradication of leukemia, as they might serve as a reservoir for drug resistance and future relapse. We analyzed serial blast and LSC populations from blood and bone marrow samples from patients undergoing treatment by flow cytometry for phenotype, CXCR4 and annexin V expression. The putative LSCs were defined as CD34+CD38-CD123+ or by aldehyde dehydrogenase. Results An independent assessment of CXCR4 expression in 56 consecutive AML patients from our institution not related to this clinical trial revealed a mean % expression of 31%, range 1-99%, with mean fluorescence intensity (MFI) of 2092, range 319-7942. A sample of 18 patients showed a correlation in CXCR4 expression between gated blasts derived from blood and bone marrow samples from the same patient (For % expression, r2=0.85, p=5e-8; MFI r2=0.45, p=0.002). Our site has enrolled 24 AML patients thus far on the above noted trial of BMS-936564. Administration of BMS-936564 resulted in brisk mobilization of leukemic blasts in 14/24 patients that initially peaked within at 2-6 hours post start of infusion in most patients, with an average of 2.1-fold increase ± 1.8 fold (range 1.06-8.96 fold), and some blasts continued to be in circulation for days. In most cases, the samples for which mobilization was not observed either did not have circulating blasts at baseline, or were from patients who received lower doses of BMS-936564. In addition, CD34+CD38-CD123+LSCs were also mobilized post-treatment with BMS-936564, and in some cases, continued to rise over the subsequent days, during which the blast population declined. The average rise in %CXCR4 was from 29.3% pre-treatment to 69.8% peak value for blasts, and 23.0% pre-treatment to 75.6% peak value for LSCs. Although a direct correlation between CXCR4 expression by blasts and fold mobilization was not apparent, the highest fold increase in mobilization (∼9-fold) did occur in the patient with a moderately high level of CXCR4 expression, 42.5%. In this patient, there was also a sharp decline in circulating CXCR4 positive cells within 2 days and the patient achieved complete remission. BMS-936564 has demonstrated apoptosis in some preclinical models (Kuhne MR et al Clin Cancer Res v19(2): 357-66 (2013)).) For most patients, there was some increase in annexin V staining observed during the first 96 hours after antibody exposure. One patient sample with initial low baseline level of apoptosis exhibited a rise in annexin V staining (from 6% to 48%) that peaked on day 3 after administration of BMS-936564. Conclusion These data demonstrate that BMS-936564 induces mobilization of both AML blasts and LSCs, which may enhance chemotherapy-induced cytotoxicity in relapsed/refractory AML. Disclosures: Chien: Bristol-Myers Squibb: Research Funding. Cardarelli:BMS: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Becker:Bristol-Myers Squibb: Research Funding.

A Phase I Study of Ruxolitinib Plus Nilotinib in Chronic Phase CML Patients with Molecular Evidence of Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1892-1892 ◽  
Author(s):  
Kendra L. Sweet ◽  
Lori Hazlehurst ◽  
Eva Sahakian ◽  
John J. Powers ◽  
Lisa Nodzon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adverse Events ◽  
Phase I ◽  
Research Funding ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Future Studies ◽  
Fatigue Severity ◽  
Grade 3 ◽  
The Impact

Abstract Background: BCR-ABL tyrosine kinase inhibitors (TKI) are the standard treatment for CP-CML. A subset of patients have profound molecular responses with BCR-ABL transcripts no longer detectable using RT-PCR (MR4.5). The ENESTnd trial compared nilotinib versus imatinib as frontline therapy in CML, and reported an increase in the cumulative incidence of MR4.5 of approximately 11% per year for the first five years in nilotinib treated patients. Discontinuation of TKIs is successful in 40-50% of patients who have a durable MR4.5. The phosphorylation of STAT3-Y705 via the JAK-STAT signaling pathway provides a protective microenvironment for the leukemic stem cells (LSC) and is a well described mechanism of resistance to TKIs. The residual LSCs likely contribute to relapse after TKI discontinuation. Data suggests that by simultaneously blocking JAK2 and TYK2, pSTAT3 is inhibited, thereby eliminating the protective environment in the bone marrow, and sensitizing the LSCs to TKIs. Ruxolitinib is a JAK2 and TYK2 inhibitor. Here we used ruxolitinib in combination with nilotinib in CP-CML patients to establish the maximal tolerated dose (MTD) of ruxolitinib, and obtain preliminary data about the impact of this combination on BCR-ABL transcript levels. Methods: This phase I, dose-escalation study used ruxolitinib plus nilotinib in CP-CML. All subjects were taking nilotinib prior to enrollment. Eligible subjects had a complete cytogenetic response (CCyR), yet had detectable BCR-ABL transcripts by RT-PCR at enrollment. We used a 3+3 design with 3 cohorts. The nilotinib dose remained unchanged, and the three doses of ruxolitinib were 5mg BID, 10mg BID and 15mg BID. Two additional subjects were treated at the MTD. Subjects remained on combination therapy for six months, at which point ruxolitinib was discontinued. RT-PCR was used to measure BCR-ABL transcript levels in the peripheral blood and/or bone marrow at baseline and every 3 months. The primary endpoint was the MTD of ruxolitinib. Secondary endpoints included toxicity assessment, incidence of MR4.5 at six months, change in fatigue severity scores and impact of ruxolitinib on pSTAT3/5 inhibition assessed with a plasma inhibitory assay (PIA) Descriptive statistics were used for baseline demographics, toxicity, MR4.5 and pSTAT3 levels. Subjects completed the fatigue severity index (FSI) questionnaire at baseline and every 3 months. A paired samples t-test was used to measure the difference in fatigue severity over time. Results: A total of 11 patients were enrolled between April 2013 and March 2016. Median age was 41 (25-63). 73% (n=8) were male. 36% (n=4) had received one TKI prior to nilotinib. The nilotinib dose was 300mg (n=8) or 400mg BID (n=3). Median time from diagnosis to enrollment was 11 months (6-135). Each cohort enrolled 3 subjects, and two additional subjects were treated at the MTD. There were no dose limiting toxicities; therefore the MTD/RP2D of ruxolitinib was 15mg PO BID. There were no grade 3/4 adverse events in any cohort, and no clinically significant cytopenias. Grade 1/2 transaminitis occurred in 1 subject in cohorts 1 and 2. No dose reductions were needed. At data cutoff, 9 subjects have completed six months on trial, and 2 remain active. Of those nine, 3 (33%) had ≥1-log reduction in BCR-ABL transcripts from baseline and 4 (44%) achieved MR4.5. One subject in cohort 1 progressed after three months and a kinase domain mutation analysis found a T315I mutation. FSI data available on seven subjects showed a non-significant decline in average fatigue severity from baseline (mean 2.78, SD 1.79) to follow-up (mean 1.86, SD 1.21), p=0.29. Results from the plasma inhibitory assay and updated results of all 11 subjects will be presented at the meeting after all subjects will have completed the trial. Conclusion: Our data suggest that ruxolitinib is safe and tolerable at 15mg PO BID when combined with nilotinib in CP-CML, and with no grade 3/4 adverse events reported, this should be considered the RP2D for future studies. The incidence of MR4.5 after six months was 44% which surpasses that of historical controls, although the sample size is small and a larger study is needed to confirm these results. The combination leads to an improvement in fatigue severity that did not reach statistical significance. This data serves as justification for future studies using ruxolitinib in combination with TKIs to determine the true impact on eradication of MRD in CP-CML. Disclosures Sweet: Karyopharm: Honoraria, Research Funding; Pfizer: Speakers Bureau; Incyte Corporation: Research Funding; Ariad: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Nodzon:Novartis: Speakers Bureau. Pinilla-Ibarz:Janssen: Consultancy, Honoraria; Pharmacyclics: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Novartis: Consultancy; Abbvie: Consultancy, Speakers Bureau.

scholarly journals Targeting the Inflammatory Niche in MDS By Tasquinimod Restores Hematopoietic Support and Suppresses Immune-Checkpoint Expression in Vitro

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2596-2596
Author(s):  
Manja Wobus ◽  
Ekaterina Balaian ◽  
Uta Oelschlaegel ◽  
Russell Towers ◽  
Kristin Möbus ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Immunohistochemical Staining ◽  
Research Funding ◽  
Fold Increase ◽  
Low Risk ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
The Impact

Abstract Introduction Myelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. There is increasing evidence that an aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species. Tasquinimod (TASQ, Active Biotech) is a novel, oral small molecular drug with S100A9 inhibitory activity and it is currently investigated in a phase Ib/IIa trial in relapsed/refractory multiple myeloma (NCT04405167). TASQ has demonstrated anti-angiogenic, antitumor and immunomodulatory properties in a broad range of preclinical solid tumor models; however, little is known about its effects in myeloid malignancies. Aim We investigated the role of S100A9 in cellular models of MDS and the potential of TASQ to target S100A9 within the MDS stroma in vitro. Methods Immunohistochemical staining of S100A9, CD271+ mesenchymal stromal cells (MSCs), CD68+ macrophages and CD66b+ neutrophils in BM tissues from MDS patients and healthy donors was performed with multiplex immunohistochemistry and analyzed with the VECTRA imaging system. MSCs from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) in the presence or absence of TASQ (10µM). Subsequently, TLR4 downstreaming molecules such as IRAK1, gasdermin and NF-kB-p65 were analyzed by Western blot. Moreover, the mRNA expression of further proinflammatory molecules (IL-1b, IL-18, caspase1) and PD-L1 was quantified by real-time PCR. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Additionally, PD-1 mRNA expression was quantified in cocultured HSPCs. Results Immunohistochemical staining of BM tissue demonstrated S100A9 expression mainly by CD66b+ neutrophils and with less extent by CD68+ macrophages. In line with this, we could not detect relevant S100A9 mRNA expression in cultured MDS or healthy MSCs in vitro. Exposure of MDS and healthy MSCs with S100A9 induced TLR4 downstream signalling as demonstrated by increased expression of IRAK1 and NF-kB-p65. We further detected a higher expression of gasdermin, an inductor of pyroptosis, in S100A9 exposed MSCs. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1b and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50%. To evaluate the impact on the hematopoietic support of MSCs, we analysed MSC/HSPC cocultures after treatment with S100A9. We observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs. Interestingly, both the number of CAF-C and CFU could be increased by TASQ pre-treatment. Finally, the PD-1 expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs, nominating this interaction as a potential target of S100A9/TASQ in the MDS BM. Conclusion In summary, we provide evidence that the pathological inflammasome activation in the myelodysplastic bone marrow can be rescued by TASQ at least in part by inhibition of the S100A9 mediated TLR4 downstream signalling including NF-kB-p65 transcription and PD-L1 expression. These effects result in an improved hematopoietic support by MSCs, suggesting a potential efficacy to improve cytopenia in low-risk MDS patients. Disclosures Balaian: Novartis: Honoraria. Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. Platzbecker: AbbVie: Honoraria; Takeda: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Geron: Honoraria. Röllig: Novartis: Honoraria, Research Funding; Jazz: Honoraria; Janssen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Amgen: Honoraria; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

scholarly journals Imetelstat Significantly Reduces Leukemia Stem Cells in Patient-Derived Xenograft Models of Pediatric AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3352-3352
Author(s):  
Sonali P. Barwe ◽  
Fei Huang ◽  
E. Anders Kolb ◽  
Anilkumar Gopalakrishnapillai
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Research Funding ◽  
Annexin V ◽  
Telomerase Activity ◽  
Leukemia Stem Cells ◽  
Patient Derived Xenograft ◽  
Pediatric Aml ◽  
Pdx Models

Abstract Introduction Acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite the use of maximally intensive therapy, 20% of patients experience recurrent disease. These patients are also burdened with significant treatment-related toxicities. To improve survival in pediatric AML, novel targeted therapies that are more effective and less toxic are needed. Telomerase inhibition has been shown to be effective in reducing leukemic burden and eradicating leukemia stem cells (LSCs) in syngeneic mouse models of AML and in patient-derived xenograft (PDX) models of adult AML (Bruedigam et al., 2014). Recent transcriptome analyses demonstrate that the genomic landscape of pediatric AML is distinct from adult AML (Bolouri et al., 2018). In fact, mutations in the telomerase complex components are infrequent in pediatric AML unlike adult AML patients (Aalbers et al., 2013). However, similar to what is seen in adult patients, Aalbers et al. identified that telomere lengths in pediatric AML cells were shortened compared to normal leukocytes, and pediatric AML patients with the shortest telomere length tend to have shorter overall survival. Furthermore, the 5-year survival rate was 88% for pediatric AML patients who had lower telomerase activity, and 43% for those patients with higher telomerase activity, suggesting telomerase activity could be an important prognostic factor in pediatric AML patients (Verstovsek et al., 2003). Imetelstat is an oligonucleotide that specifically binds with high affinity to the RNA template of telomerase and is a potent, competitive inhibitor of telomerase enzymatic activity (Asai et al., 2003; Herbert et al., 2005). In this study, we evaluated if imetelstat has anti-leukemia activity in pediatric AML PDX models. Results The PDX lines tested in this study were derived using samples from pediatric AML patients who were 1-14 years old, representing different FAB subtypes. Mouse passaged pediatric AML PDX lines (n=6) were treated ex vivo with imetelstat or mismatch oligo control and the viability of LSC (CD34+CD38low population) was determined at 48 or 96 h by staining with BV785-human CD45, APC-human CD34, Pacific blue-human CD38, FITC conjugated annexin V and propidium iodide (PI). Imetelstat treatment significantly increased apoptosis/death (PI+/annexin V+) of the LSC population in a dose-dependent manner in all PDX lines evaluated (Fig. 1A, B), while it had limited activity on LSCs in normal pediatric bone marrow samples (n=4). The efficacy of imetelstat either alone or in combination with chemotherapy or azacitidine was evaluated in two distinct PDX models of pediatric AML in vivo. Mice engrafted with both NTPL-377 and DF-2 lived longer when treated with imetelstat than the untreated mice (Fig. 1C, D, n=5 each, P<0.05). Mice receiving standard chemotherapy consisting of cytarabine and daunorubicin or azacitidine showed prolonged survival compared to the untreated mice. Interestingly, sequential administration of imetelstat following chemotherapy treatment provided additional benefit over chemotherapy alone (P<0.01). Concurrent treatment of azacitidine and imetelstat further extended survival of these mice compared to azacitidine alone (P<0.05). At the end of the in vivo studies, the percentage of LSC population was evaluated in the bone marrow of mice post euthanasia. There was a significant reduction of LSC population in mice treated with imetelstat compared to those treated with the mismatch oligo (Fig. 1E, F, P<0.05). Neither chemotherapy nor azacitidine alone affected LSC population compared to untreated mice. However, imetelstat significantly reduced the LSC population when combined with chemotherapy or azacitidine compared to single agent (P<0.05). These results were confirmed by secondary transplantation in mice, which showed delayed engraftment of cells isolated from imetelstat treated mice (Fig. 1G, H). Conclusions Imetelstat treatment of pediatric AML PDX samples showed significant dose- and time-dependent effects on the viability of the LSCs to induce cell apoptosis/death. These results were corroborated in vivo in two distinct PDX models which showed reduced LSC population and increased median survival in mice with imetelstat treatment. Combining imetelstat with chemotherapy or azacitidine further enhanced activity against LSCs, suggesting imetelstat could represent an effective therapeutic strategy for pediatric AML. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Huang: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Gopalakrishnapillai: Geron: Research Funding.

scholarly journals Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 778-778
Author(s):  
Zhihong Zeng ◽  
Yuexi Shi ◽  
Twee Tsao ◽  
Yihua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Signaling Pathways ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Mtor Signaling ◽  
Leukemic Cells ◽  
Survival Signaling

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

scholarly journals Bone Marrow Hematopoietic Dysfunction in Untreated Chronic Lymphocytic Leukemia Is Partially Mediated By Exposure to Constituents of the Leukemic Microenvironment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3132-3132
Author(s):  
Bryce Manso ◽  
Kimberly Gwin ◽  
Charla R Secreto ◽  
Henan Zhang ◽  
Wei Ding ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Healthy Controls ◽  
Advisory Committees ◽  
The Impact

Abstract Peripheral immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) is well-studied and likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the current paradigms of known immune abnormalities are not able to consistently explain these complications and it is not easy to correct CLL patient immune status. Here, we expand on our preliminary reports that demonstrate bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced short-term functional capacity of hematopoietic progenitors in BM using colony forming unit assays (Figure 1A-C) and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations (exemplified by Lin-CD34+ HSPCs, Figure 1D). We further report that protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 are overexpressed in distinct HSPC subsets from CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Interestingly, sustained myelopoiesis, evaluated by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays maintained for five weeks, revealed no difference between healthy controls and CLL patients. These new data indicate that when HSPCs are removed from the leukemic microenvironment for ample in vitro culture time, they recover the ability to sustain myelopoiesis. To further assess the impact of the CLL microenvironment on HSPC biology, isolated HSPCs (CD34+ BM cells) from healthy controls were exposed in vitro to known leukemic microenvironment constituents. Exposure to TNFα, a cytokine constitutively produced by CLL B cells, resulted in rapid increases in PU.1 and GATA-2 proteins (Figure 2A-D). Similarly, addition of TNFα to the LTC-IC assay resulted in a striking ablation of myelopoiesis, even at the highest input cell concentration. Further, overexpression of PU.1 and GATA-2 were observed in HSPCs following co-culture with CLL B cells, a result that was not recapitulated when cells were exposed to IL-10, another cytokine constitutively produced by CLL B cells. These findings indicate specific components of the leukemic microenvironment are involved in HSPC modulation. Together, these findings expand on our previous observations of BM hematopoietic dysfunction in untreated CLL patients and offer new molecular insights into the contribution of the leukemic microenvironment on immunodeficiency in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Differentiation therapy for myeloid malignancies: beyond cytotoxicity

2021 ◽  
Vol 11 (12) ◽  
Author(s):  
Ryan J. Stubbins ◽  
Aly Karsan
Keyword(s):  
Cellular Differentiation ◽  
Cytotoxic Agents ◽  
Novel Agents ◽  
Cellular Mechanisms ◽  
Myeloid Malignancies ◽  
Major Mechanism ◽  
Treatment Regimens ◽  
Reduced Toxicity ◽  
New Treatments ◽  
The Impact

AbstractBlocked cellular differentiation is a central pathologic feature of the myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Treatment regimens promoting differentiation have resulted in incredible cure rates in certain AML subtypes, such as acute promyelocytic leukemia. Over the past several years, we have seen many new therapies for MDS/AML enter clinical practice, including epigenetic therapies (e.g., 5-azacitidine), isocitrate dehydrogenase (IDH) inhibitors, fms-like kinase 3 (FLT3) inhibitors, and lenalidomide for deletion 5q (del5q) MDS. Despite not being developed with the intent of manipulating differentiation, induction of differentiation is a major mechanism by which several of these novel agents function. In this review, we examine the new therapeutic landscape for these diseases, focusing on the role of hematopoietic differentiation and the impact of inflammation and aging. We review how current therapies in MDS/AML promote differentiation as a part of their therapeutic effect, and the cellular mechanisms by which this occurs. We then outline potential novel avenues to achieve differentiation in the myeloid malignancies for therapeutic purposes. This emerging body of knowledge about the importance of relieving differentiation blockade with anti-neoplastic therapies is important to understand how current novel agents function and may open avenues to developing new treatments that explicitly target cellular differentiation. Moving beyond cytotoxic agents has the potential to open new and unexpected avenues in the treatment of myeloid malignancies, hopefully providing more efficacy with reduced toxicity.

scholarly journals Impact of Genomic Alterations on Outcomes in Myelofibrosis Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2301-2301 ◽  
Author(s):  
Raajit K. Rampal ◽  
Roni Tamari ◽  
Nan Zhang ◽  
Caroline Jane McNamara ◽  
Franck Rapaport ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Triple Negative ◽  
Unrelated Donor ◽  
Reduced Intensity Conditioning ◽  
Hematopoietic Stem ◽  
Disease Characteristics ◽  
Donor Type ◽  
The Impact ◽  
Reduced Intensity

Abstract Introduction: The impact of genomic alterations, such as mutations in ASXL1, on the risk of disease progression and leukemic transformation in patients with myelofibrosis (MF) is well established. Further, emerging data suggests that the number and type of mutations may impact response to therapies such as ruxolitinib or imetelstat. Allogeneic hematopoietic stem cell transplant (allo-HSCT) remains the only potentially curative treatment for MF patients. However, the impact of somatic mutations on overall survival (OS) and relapse-free survival (RFS) is poorly understood. Using next-generation sequencing of pre-transplant blood and bone marrow samples from a well clinically-annotated cohort of MF patients who underwent allo-HSCT, we sought to determine the impact of mutational burden on outcomes. Methods: A multicenter retrospective analysis of a cohort of 84 patients was carried out. This included 52 patients treated on the MPD-RC 101 prospective study (NCT00572897), 18 patients treated at Prince Margaret Hospital, and 14 patients treated at Memorial Sloan Kettering Cancer Center. Patient and transplant characteristics are displayed in Table 1. DNA was extracted from pre-transplant bone marrow aspirate samples or peripheral blood samples. High-throughput sequencing of a panel of genes was performed. Average coverage of 829x (standard deviation of ±130) was obtained. Mutect was utilized to call single point variants (comparing our samples to a pool of normal samples) and PINDEL was used to call short insertions and deletions. We excluded all mutations present in at least one database of known non-somatic variants (DBSNP and 1000 genomes) and absent from COSMIC. Univariate Cox regression and Kaplan-Meier graphics were used to investigate the association of patient, transplant, and disease characteristics with OS and RFS. Results: JAK2V617F was the most frequent mutation detected in 41(48.8%) patients (Table 2). Eighteen patients (21.4%) had triple negative disease (negative for JAK2, MPL, and CALR mutations). Univariate analysis included the following: patient characteristics (age, gender), transplant characteristics (related vs. unrelated donor, matched vs. mismatched donor and myeloablative vs. reduced intensity conditioning) and disease characteristics (DIPSS and presence of mutations). Decreased OS was associated with unrelated donor status (HR 2.09, 95% CI: 1.03-4.23, p=0.04), reduced intensity conditioning (HR 4.21, 95% CI: 1.01-17.59, p=0.049), triple negative disease (HR 2.09, 95% CI: 1.02-4.30, p=0.04), and presence of U2AF1 (HR 2.53, 95% CI: 1.10-5.81, p=0.03) or SUZ12 mutations (HR 3.92, 95% CI: 1.19-12.21, p=0.02). Decreased RFS was associated with unrelated donor status (HR 2.27, 95% CI: 1.16-4.45, p=0.02), and the presence of SUZ12 mutation (HR 6.97, 95% CI: 2.37-20.49, p<0.001). A descriptive decrease in RFS in patients with U2AF1 (HR 2.15, 95% CI: 0.94-4.88, p=0.07) was observed but did not reach statistical significance. Importantly, mutations previously reported to be associated with reduced OS and RFS in the non-transplant setting, such as ASXL1, EZH2, IDH1/2, and SRSF2, were not associated with poorer outcomes in this analysis in transplanted patients. In an exploratory multivariate analysis including donor type (related vs. unrelated) and presence of U2AF1 and SUZ12 mutations, there was a significantly reduced OS and RFS in patients who harbor these mutations regardless of donor type (OS: HR 5.30, 95% CI: 2.08-13.47, p<0.001; RFS: HR 5.49, 95% CI: 2.27-13.30, p<0.001). In patients without the above mutations, having an unrelated donor was associated with worse OS (HR 2.55, 95% CI: 1.09-5.96, p=0.03) and RFS (HR 2.61, 95% CI: 1.17-5.83, p=0.02, Figure 1). Conclusions: Our analysis demonstrates that mutations previously associated with poor prognosis in MF, such as ASXL1, do not appear to confer a worsened prognosis in patients undergoing allo-HSCT, suggesting transplant may be able to overcome the impact of these mutations. However, mutations in SUZ12 and U2AF1 are associated with reduced OS in univariate and multivariate analysis (together with donor type). Further studies with larger cohorts of patients are indicated to validate these findings, and to elucidate the impact of these mutations on disease biology. Disclosures Rampal: Incye and CTI: Consultancy. Mascarenhas:Janssen: Research Funding; CTi Biopharma: Research Funding; Promedior: Research Funding; Merk: Research Funding; Incyte: Research Funding. Mesa:Galena: Consultancy; Gilead: Research Funding; Promedior: Research Funding; Incyte: Research Funding; CTI Biopharma: Research Funding; Celgene: Research Funding; Ariad: Consultancy; Novartis: Consultancy. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding.

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