Targeting mTOR and JAK2 in Xenograft Models of CRLF2-Overexpressing Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 249-249
Author(s):  
Shannon L Maude ◽  
Sarah K Tasian ◽  
Tiffaney Vincent ◽  
Junior W Hall ◽  
Racquel Collins-Underwood ◽  
...  
Keyword(s):  
Signal Transduction ◽  
High Risk ◽  
Targeted Therapies ◽  
Peripheral Blood ◽  
Disease Burden ◽  
Mtor Inhibitor ◽  
Jak Inhibitor ◽  
Blast Count ◽  
Xenograft Models

Abstract Abstract 249 CRLF2 genomic rearrangements that lead to overexpression have been identified in a subset of children with clinically high-risk B-precursor ALL and are highly associated with activating JAK2 mutations (Harvey et al., Blood 2010; Mullighan et al., PNAS 2009 and Nat Genet 2009). These children frequently respond poorly to current intensive cytotoxic chemotherapy regimens and suffer high rates of relapse and mortality. New therapies for these patients are urgently needed. These leukemias exhibit gene expression profiles similar to those of BCR-ABL1 positive ALL, suggesting aberrant kinase activation. We previously demonstrated aberrant JAK/STAT and PI3K/mTOR signaling in CRLF2-overexpressing ALL cell lines and primary human samples in vitro, and thus hypothesize that inhibition of these hyperactive signaling networks has therapeutic relevance. To further characterize this high-risk subset of ALL, we have established multiple xenograft models of CRLF2-rearranged and JAK2-mutated ALL, providing a robust platform for preclinical testing of signal transduction inhibitors. In this model, primary human ALL samples are intravenously injected into NOD/SCID/γc null (NSG) mice, and engraftment is determined by flow cytometry of peripheral blood for human CD19+/CD45+ blasts. Eighteen of 21 primary cryopreserved specimens provided by the Children's Oncology Group engrafted successfully. In order to biochemically characterize the xenografts, we measured phosphorylation of relevant signal transduction proteins by phosphoflow cytometry. Spleens of mice xenografted with CRLF2-rearranged ALL had uniformly increased surface staining of human CRLF2, a component of the heterodimeric receptor complex for the cytokine, thymic stromal lymphopoeitin (TSLP). In vitro stimulation of the CRLF2-overexpressing ALL xenograft spleens with TSLP induced phosphorylation of STAT5, Akt, S6, and 4EBP1, but not of ERK 1/2. In additon, in vitro JAK inhibition with INCB018424 abrogated TSLP-induced JAK/STAT and PI3K/mTOR signaling. The mTOR inhibitor sirolimus, mTORC1/2 inhibitor PP242, and PI3K/mTOR inhibitor PI-103 potently inhibited phosphorylation of S6 and 4EBP1 in these xenograft specimens. These data suggest that the JAK/STAT and PI3K/mTOR pathways may interact in these CRLF2-overexpressing leukemias. These data led us to hypothesize that inhibition of the PI3K/mTOR or JAK/STAT pathways may represent potential therapeutic targets; therefore, we utilized these very high-risk ALL xenograft models to study novel, targeted therapies. Once xenografts had engrafted with sufficient disease burden to detect >5% peripheral CD19+/CD45+ blasts, mice were randomized to receive the mTOR inhibitor sirolimus, the JAK inhibitor INCB018424, or vehicle for three to four weeks. Disease burden was assessed weekly by flow cytometric determination of CD19+/CD45+ blast count in peripheral blood, and at sacrifice, by spleen CD19+/CD45+ blast count. To assess potential differential efficacy based on CRLF2 overexpression (CRLF2+) and/or JAK2 activating mutations (JAK2mut), we treated mice with each subtype of ALL. Sirolimus induced a significant decrease in peripheral blast count in 7 of 7 primary ALL xenografts tested (2 JAK2mut/CRLF2+ samples, 1 JAK2mut/CRLF2- sample, 2 JAK2wt/CRLF2+ samples, and 2 JAK2wt/CRLF2- samples) and a significant decrease in spleen blast count in 6 of 7 samples tested. The most profound reduction of disease burden was seen in the JAK2mut/CRLF2+ leukemias. In addition, the JAK inhibitor INCB018424 decreased peripheral blast count and spleen blast count in a JAK2mut/CRLF2+ xenograft. We next determined if sirolimus conferred a survival advantage in xenografts of 2 ALL specimens, a robust responder and an intermediate responder to sirolimus by blast count. Sirolimus treatment significantly prolonged survival of both xenografts (63 days vs. 23 days, p=0.0015; 91 days vs. 58 days, p=0.0027). Additional human ALL xenograft studies of INCB018424 and other kinase inhibitors are ongoing. The preclinical in vivo efficacy of sirolimus and INCB018424 suggests that novel, targeted therapies have therapeutic potential in CRLF2-overexpressing ALL. Based in part on these data, both INCB018424 and temsirolimus (a parenteral ester of sirolimus) are currently being investigated in multi-center early phase clinical trials for children with relapsed or refractory leukemias. Disclosures: No relevant conflicts of interest to declare.

Apoptosis and proliferation ofperipheral blood T-cells as alternative processes in pathogenesis of diabetes mellitus type 1

2019 ◽  
Vol 1 (4) ◽  
pp. 16-20 ◽  
Author(s):  
A. V. Lugovaya ◽  
N. M. Kalinina ◽  
V. Ph. Mitreikin ◽  
Yu. P. Kovaltchuk ◽  
A. V. Artyomova ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
T Cells ◽  
High Risk ◽  
Peripheral Blood ◽  
Cellular Response ◽  
Proliferative Potential ◽  
Autoreactive T Cells ◽  
Alternative Processes

Apoptosis, along with proliferation, is a form of lymphocyte response to activating stimuli. In the early stages of cell differentiation, the apoptotic response prevails and it results to the formation of tolerance to inductor antigen. Mature lymphocytes proliferate in response to stimulation and it means the initial stage in the development of the immune response. Since in this case apoptosis and proliferation acts as alternative processes, their ratio can serve as a measure of the effectiveness of the cellular response to activating signals. The resistance of autoreactive T-cells to apoptosis is the main key point in the development of type 1 diabetes mellitus (T1DM). Autoreactive T-cells migrates from the bloodstream to the islet tissue of the pancreas and take an active part in b cells destruction. The resistance of autoreactive effector T-cells to apoptosis may suggest their high proliferative potential. Therefore, the comparative evaluation of apoptosis and proliferation of peripheral blood lymphocytes can give a more complete picture of their functional state and thus will help to reveal the causes of ineffective peripheral blood T-ceiis apoptosis in patients with T1DM and will help to understand more deeply the pathogenesis of the disease. in this article, the features of proliferative response of peripheral blood T-cells in patients with T1DM and in individuals with high risk of developing T1DM have been studied. Apoptosis of T-cell subpopulations has been investigated. The correlation between the apoptotic markers and the intensity of spontaneous and activation- induced in vitro T-cells proliferation of was revealed. it was determined, that autoreactive peripheral blood T-cells were resistant to apoptosis and demonstrated the increased proliferative potential in patients with T1DM and in individuals with high risk of developing T1DM.

scholarly journals APTO-253 Induces KLF4 to Promote Potent in Vitro Pro-Apoptotic Activity in Hematologic Cancer Cell Lines and Antitumor Efficacy As a Single Agent and in Combination with Azacitidine in Animal Models of Acute Myelogenous Leukemia (AML)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4813-4813 ◽  
Author(s):  
William G Rice ◽  
Avanish Vellanki ◽  
Yoon Lee ◽  
Jeff Lightfoot ◽  
Robert Peralta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Animal Models ◽  
Antitumor Activity ◽  
Single Agent ◽  
Xenograft Model ◽  
Myelogenous Leukemia ◽  
Acute Myelogenous ◽  
Xenograft Models

Abstract APTO-253, a small molecule that mediates anticancer activity through induction of the Krüppel-like factor 4 (KLF4) tumor suppressor, is being developed clinically for the treatment of acute myelogenous leukemia (AML) and high risk myelodysplastic syndromes (MDS). APTO-253 was well tolerated in a Phase I study in patients with solid tumors using a dosing schedule of days 1, 2, 15, 16 of a 28 day cycle (2T-12B-2T-12B), but recent scientific observations guided APTO-253 toward AML and high risk MDS. Indeed, suppression of KLF4 was reported as a key driver in the leukemogenesis of AML and subsets of other hematologic diseases. The vast majority (~90%) of patients with AML aberrantly express the transcription factor CDX2 in human bone marrow stem and progenitor cells (HSPC) (Scholl et al., J Clin Invest. 2007, 117(4):1037-48). The CDX2 protein binds to CDX2 consensus sequences within the KLF4 promoter, thereby suppressing KLF4 expression in HSPC (Faber et al., J Clin Invest. 2013, 123(1):299-314). Based on these observations, the anticancer activity of APTO-253 was examined in AML and other hematological cancers. APTO-253 showed potent antiproliferative activity in vitro against a panel of blood cancer cell lines, with ηM IC50values in AML (6.9 - 305 ηM), acute lymphoblastic leukemia and chronic myeloid leukemia (39 – 250 ηM), non-Hodgkin’s lymphoma (11 – 190 ηM) and multiple myeloma (72 – 180 ηM). To explore in vivo efficacy, dose scheduling studies were initially conducted in the H226 xenograft model in mice. In the H226 model, APTO-253 showed improved antitumor activity when administered for two consecutive days followed by a five day break from dosing (2T-5B) each week, i.e. on days 1,2, 8,9, 15,16, 22,23, compared to the 2T-12B-2T-12B schedule. The 2T-5B schedule was used to evaluate antitumor activity of APTO-253 in several AML xenograft models in mice. In Kasumi-1 AML and KG-1 AML xenograft models, APTO-253 showed significant antitumor activity (p = 0.028 and p=0.0004, respectively) as a single agent when administered using the 2T-5B schedule each week for four weeks compared to control animals. Mice treated with APTO-253 had no overt toxicity based on clinical observations and body weight measurements. Mice bearing HL-60 AML xenograft tumors were treated with APTO-253 for one day or two consecutive days per week for three weeks, either as a single agent or combined with azacitidine, or with azacitidine alone twice per week (on days 1,4, 8, 11, 15 and 18). APTO-253 as a single agent inhibited growth of HL-60 tumors to approximately the same extent as azacitidine. Furthermore, both once weekly and twice weekly dosing of APTO-253 in combination with azacitidine resulted in significantly enhanced antitumor activity relative to either single agent alone (p = 0.0002 and p = 0.0006 for 1X and 2X weekly APTO-253 treatment, respectively, compared to control). Likewise, using a THP-1 AML xenograft model, APTO-253 administered as a single agent using the 2T-5B per week schedule showed significant efficacy, similar to that of azacitidine, while the combination of APTO-253 and azacitidine demonstrated greatly improved antitumor effects relative to either drug alone. APTO-253 was effective and well tolerated as a single agent or in combination with azacitidine in multiple AML xenograft models, plus APTO-253 does not cause bone marrow suppression in animal models or humans. Taken together, our results indicate that APTO-253 may serve as a targeted agent for single agent use and may provide enhanced efficacy to standard of care chemotherapeutics for AML and other hematological malignancies. Disclosures Rice: Lorus Therapeutics Inc.: Employment. Vellanki:Lorus Therapeutics Inc.: Employment. Lee:Lorus Therapeutics Inc.: Employment. Lightfoot:Lorus Therapeutics Inc.: Employment. Peralta:Lorus Therapeutics Inc.: Employment. Jamerlan:Lorus Therapeutics Inc.: Employment. Jin:Lorus Therapeutics Inc.: Employment. Lum:Lorus Therapeutics Inc.: Employment. Cheng:Lorus Therapeutics Inc.: Employment.

The Humanized Anti-CD40 Monoclonal Antibody, SGN-40, Potentiates Chemotherapy Regimens in NHL Xenograft Models Via Pro-Apoptotic Signaling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2342-2342 ◽  
Author(s):  
Timothy S. Lewis ◽  
Renee S. McCormick ◽  
Kim Kissler ◽  
Ivan J. Stone ◽  
Mechthild Jonas ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Monoclonal Antibody ◽  
Antitumor Activity ◽  
Family Member ◽  
Cytotoxic Agents ◽  
P53 Family ◽  
Signaling Mechanism ◽  
Xenograft Models

Abstract SGN-40 is a humanized antibody targeting CD40, a TNF receptor family member expressed on normal B cells, non-Hodgkin’s lymphoma (NHL), multiple myeloma, and a variety of carcinomas. Previous studies have shown that SGN-40 triggers proapoptotic signal transduction, mediates effector function (ADCC), and has in vivo antitumor activity in CD40+ lymphoma xenograft models. We now report in vivo efficacy data for SGN-40 in combination with the anti-CD20 monoclonal antibody, rituximab, and approved chemotherapy regimens for the treatment of NHL. The growth of subcutaneous Ramos tumors in SCID mice was delayed following SGN-40 or rituximab treatment. However, the combination of SGN-40 + rituximab (S-R) significantly improved efficacy over either antibody alone. SGN-40 was then tested with ICE (ifosfamide, carboplatin, etoposide) chemotherapy with or without rituximab (S-R-ICE and S-ICE). These studies demonstrated that both S-R-ICE and S-ICE treated mice had lower tumor burden than R-ICE or SGN-40 treated animals. Additionally, the effect of SGN-40 in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy with or without rituximab (S-R-CHOP and S-CHOP) was examined. S-R-CHOP and S-CHOP therapies showed a significant delay in tumor growth compared with R-CHOP or SGN-40 alone. Furthermore, the efficacy observed in S-R-ICE and S-R-CHOP treatments exceeded the S-R combination, suggesting that SGN-40 chemosensitizes lymphoma cells by a signaling mechanism in addition to augmenting ADCC when combined with rituximab. To better understand the chemosensitization effect of SGN-40 in xenograft models, signal transduction events triggered by SGN-40 were examined in vitro. SGN-40 treatment caused the sustained degradation of the BCL-6 protooncogene in several lymphoma cell lines, following prolonged MAP Kinase pathway activation. BCL-6 is implicated in lymphomagenesis of germinal center derived lymphomas, and is proteasomally degraded after phosphorylation by ERK1/2 MAPK. Immunohistochemical analyses of Ramos tumors harvested from mice following treatment with SGN-40 or S-CHOP revealed elevated numbers of apoptotic cells versus untreated tumors. A distinct downregulation of BCL-6 staining in Ramos tumor cells was also observed in SGN-40 and S-CHOP treated animals, correlating with increased cell death. Finally, in some NHL lines SGN-40 upregulated the p53 family member TAp63alpha, a chemo-sensitizing transcription factor capable of inducing apoptosis when overexpressed. When combined with cytotoxic agents, SGN-40 caused a greater induction of TAp63alpha compared with chemotherapy alone, a potential mechanism underlying the improved antitumor activity seen in combination studies. Collectively, these data suggest that SGN-40 signaling occurs at the tumor site, likely contributing directly to tumor cell killing and chemosensitization. These preclinical studies support our earlier work suggesting that addition of SGN-40 to standard therapeutic regimens may improve the outcome for patients with NHL.

Thymic Stromal Lymphopoietin Stimulation of Pediatric Acute Lymphoblastic Leukemias with CRLF2 Alterations Induces JAK/STAT and PI3K Phosphosignaling

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 410-410 ◽  
Author(s):  
Sarah K Tasian ◽  
Michelle Y Doral ◽  
Brent L Wood ◽  
Michael J Borowitz ◽  
J. Racquel Collins-Underwood ◽  
...  
Keyword(s):  
High Risk ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Thymic Stromal Lymphopoietin ◽  
Jak Inhibitor ◽  
Oncology Group ◽  
Term Survival ◽  
Phosphoflow Cytometry ◽  
Stimulation Of

Abstract Abstract 410 Collaborative genomic profiling efforts though the National Cancer Institute's TARGET Initiative and the Children's Oncology Group have identified CRLF2 and JAK mutations in a subset of children with high-risk acute lymphoblastic leukemia (ALL), but few biochemical studies have assessed the functional sequelae of these genetic alterations. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor chain, which heterodimerizes with the IL-7 receptor alpha chain (IL-7Rα). Children with high CRLF2-expressing ALL detected by gene expression profiling have high rates of minimal residual disease at end-induction (Day 29), and approximately 70% of these patients ultimately relapse (Harvey et al., Blood 2010). We hypothesize that characterization of aberrant signaling networks in these leukemias will facilitate identification of potential targets for small molecule inhibitor therapies. Using phosphoflow cytometry, we analyzed the phosphorylation status of key signaling molecules after stimulation with TSLP, IL-7, or pervanadate (an irreversible proximal membrane phosphatase inhibitor used as a positive control) in 2 human ALL cell lines with CRLF2 and JAK2 mutations and in 43 fresh or cryopreserved diagnostic primary patient samples, 27 of which overexpressed CRLF2 through P2RY8-CRLF2 fusion or CRLF2-IgH translocation and 16 of which did not have CRLF2 or JAK mutations (controls). Cells were rested in serum-free media for 60 minutes at 37°C, then stimulated with TSLP, IL-7, or pervanadate for 30 minutes to induce signaling. Cells were also exposed to the JAK inhibitor XL019 (Exelixis) for 60 minutes and/or subsequently stimulated with the aforementioned cytokines or pervanadate to determine the effects of JAK inhibition on signaling. Cells were then processed for phosphoflow cytometry according to our previously published methodologies (Kotecha et al., Cancer Cell 2008). High CRLF2-expressing leukemias (n = 27) with or without concomitant JAK mutations demonstrated strong surface staining of the TSLP receptor, as well as CD10, CD19, and CD127 (IL-7Rα). In vitro stimulation of leukemic blasts with TSLP elicited phosphorylation of STAT5 and S6, but not ERK 1/2, in leukemias with JAK and/or CRLF2 alterations. Control leukemias without CRLF2 and JAK mutations (n=16) did not stain for the TSLP receptor, and TSLP stimulation did not elicit phosphosignaling through the JAK/STAT, PI3K, or MAPK pathways. STAT5 and S6 phosphorylation in the high CRLF2-expressing leukemias was further abrogated by in vitro JAK inhibition with XL019. Surprisingly, despite flow cytometric staining for CD127, stimulation with IL-7 did not elicit phosphosignaling through these epitopes in high CRLF2-expressing or control leukemic blasts, although it did predictably phosphorylate STAT5 in control T and non-blast B cells contained within the primary patient leukemia samples. These results suggest that the JAK/STAT and PI3K pathways, but not the MAPK pathway, are involved in TSLP receptor signaling in high CRLF2-expressing ALL +/− JAK mutations and may represent druggable targets. Phosphoflow cytometry is an efficient method of interrogating intracellular signaling at a single-cell level in primary human samples and, furthermore, can be used for rapid identification of patients at time of leukemia diagnosis with high CRLF2-expressing ALL who exhibit the TSLP phosphosignature. Therapy for this subset of high-risk patients might be modified to include a targeted therapeutic (such as a JAK inhibitor) to improve initial treatment responses and, ultimately, to enhance long-term survival. To this end, we have developed a Children's Oncology Group Phase I clinical trial of JAK inhibition for patients with relapsed or refractory leukemias (including those with CRLF2 and JAK mutations) and will validate the use of phosphoflow cytometry and other biologic assays to assess in vivo target inhibition during therapy. We ultimately envision incorporation of JAK inhibitor therapy into a systemic chemotherapy backbone for patients with high CRLF2-expressing ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PLEKHA4 Promotes Wnt/β-catenin Signaling-Mediated G1/S Transition and Proliferation in Melanoma

2020 ◽  
Author(s):  
Adnan Shami Shah ◽  
Xiaofu Cao ◽  
Andrew C. White ◽  
Jeremy M. Baskin
Keyword(s):  
Small Molecules ◽  
Ubiquitin Ligase ◽  
Targeted Therapies ◽  
Drug Target ◽  
Binding Partners ◽  
Xenograft Models ◽  
Melanoma Patients ◽  
Cell Cycle Transition ◽  
Braf Mutant

ABSTRACTMelanoma patients incur substantial mortality, despite promising recent advances in targeted therapies and immunotherapies. In particular, inhibitors targeting BRAF-mutant melanoma can lead to resistance, and no targeted therapies exist for NRAS-mutant melanoma, motivating the search for additional therapeutic targets and vulnerable pathways. Here, we identify a regulator of Wnt/β-catenin signaling, PLEKHA4, as a factor required for melanoma proliferation and survival. PLEKHA4 knockdown in vitro leads to lower Dishevelled levels, attenuated Wnt/β-catenin signaling, and a block of progression through the G1/S cell cycle transition. In mouse xenograft models, inducible PLEKHA4 knockdown attenuated tumor growth in BRAF- and NRAS-mutant melanomas and synergized with the clinically used inhibitor encorafenib in a BRAF-mutant model. As an E3 ubiquitin ligase regulator with both lipid and protein binding partners, PLEKHA4 presents several opportunities for targeting with small molecules. Our work identifies PLEKHA4 as a promising drug target for melanoma and clarifies a controversial role for Wnt/β-catenin signaling in the control of melanoma proliferation.

scholarly journals Quantitative Modeling of Azacitidine Resistance in Patients with Myelodysplastic Syndrome Identifies Distinct Phenotypes of Disease Progression: Evidence for the Presence of a Disease Versus Native Clone Effect

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1723-1723
Author(s):  
Roman M Shapiro ◽  
Adam R Stinchcombe
Keyword(s):  
Peripheral Blood ◽  
Maximum Rate ◽  
Disease Burden ◽  
Laboratory Data ◽  
Model Parameters ◽  
Bone Marrow Blast ◽  
Self Renewal ◽  
Renewal Time ◽  
Blast Count ◽  
Linear Manner

Introduction Myelodysplastic syndrome (MDS) patients who are treated with azacitidine (AZA) may develop drug resistance in a number of different ways. Distinguishing the mechanisms underlying disease progression to leukemia while on AZA as opposed to clonal evolution without an increasing blast count is challenging due to the difficulty of studying MDS clones and their effect on non-diseased hematopoietic stem cells (HSCs). The application of a robust mathematical model of hematopoiesis to MDS patients allows for the calculation of kinetic parameters reflective of the function of the dominant hematopoietic clones, and infers the behaviour of both HSCs and MDS clones. We demonstrate the application of such a model applied to IPSS int-2/hi risk MDS patients treated with AZA, and show how different natural histories of disease can be explained by changes in model parameters over time. We also demonstrate how interactions between modeled HSCs and MDS clones can be inferred from the model. Methods A database of 97 IPSS int-2/hi risk MDS patients treated with AZA was previously constructed containing longitudinal peripheral blood count and laboratory data during the period 2008-2016, and was used for model fitting. Of these patients, 79 patients had sufficient data for modeling. A mathematical model of hematopoiesis was developed based on a formulation by Colijn and Mackey and modified to include a bone marrow blast compartment. Hematopoietic kinetic parameters were fit to de-identified patient laboratory data using a Kalman filter. The model data input was adjusted for red blood cell and platelet transfusion frequency and weighted so that parameter fits were made insensitive to periods of acute illness as identified from chart review and ancillary laboratory values. The resulting fit parameters represented a weighted average of disease and native HSCs contributions. Model parameters were evaluated with respect to time and sensitivity analysis was done identifying optimal correlation with the development of AZA resistance. A novel analysis was developed to determine if the contributions of the native and disease HSCs to peripheral blood counts are in proportion to their clonal burdens, or if the AZA-resistant phenotype reflected an additional effect of the MDS clones on the native HSCs. Results A schematic of the improved model of hematopoiesis adapted to MDS is shown along with the distribution of data collected over time (Fig 1A-B). The model fits for three representative patients are shown with different disease courses: AZA resistance with a rapidly rising blast count (patient 90), AZA resistance with a minimal rise in the blast count (patient 86), and AZA resistance with cytogenetic evolution without an increase in the blast count (patient 74). The model was fit to the longitudinal peripheral blood counts and bone marrow blast count data (Fig 2A-D). Development of AZA resistance in patient 74 was best correlated with a reduction in the average HSC self-renewal time (τS in Fig 2E), and this reduction was related to disease burden in a linear manner (Fig 2F, leftmost panel). Similarly, the development of AZA resistance in patient 86 was best correlated with an increase in the intrinsic threshold rate at which a stem cell differentiates (k0 in Fig 2E), and this increase was related to disease burden in a non-linear manner (Fig 2F, middle panel). For patient 90, the development of AZA resistance was best correlated with a decrease in the maximum rate of HSC differentiation (f0 in Fig 2E), and this decrease was related to disease burden in a non-linear manner (Fig 2F, rightmost panel). Discussion MDS patients in our cohort developed AZA resistance in distinct ways, and this correlated with changes in either HSC self-renewal time, differentiation threshold, or maximum differentiation rate. In the former case, the linearity in modeled HSC self-renewal time with respect to disease burden suggested this parameter change is accounted for by an expanding MDS clone. In the latter two cases, the mechanism of AZA resistance is hypothesized to be associated with an increased MDS clone threshold for differentiation as well as decreased maximum rate of HSC differentiation. The non-linearity of the association between these rate changes and disease burden suggests they were driven in large part by an external influence from the MDS clone on the HSCs. Further validation of these findings in a larger cohort of MDS patients is anticipated. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Phase 1 Study of TP-3654, an Orally-Delivered PIM Kinase Inhibitor, in Patients with Intermediate-2 or High-Risk Primary or Secondary Myelofibrosis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Claudia Lebedinsky ◽  
Stephen P. Anthony ◽  
Golam Mohi ◽  
Huyuan Yang ◽  
Jian Mei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Adverse Events ◽  
Dose Escalation ◽  
Peripheral Blood ◽  
Phase 1 ◽  
Jak Inhibitor ◽  
Jak Inhibitors ◽  
Phase 1 Study ◽  
To Receive

Background: Myelofibrosis (MF) is characterized by bone marrow fibrosis (BMF) and ineffective extramedullary hematopoiesis resulting in splenomegaly and debilitating symptoms. An activating Janus kinase 2 (JAK2) mutation (V617F) has been frequently observed in MF. Ruxolitinib (a JAK1/JAK2 inhibitor) reduces splenomegaly and improves constitutional symptoms, but appears to offer a modest reduction of BMF. Patients with MF who are intolerant to JAK inhibitors or their components, or for whom JAK inhibitors have failed, have limited treatment options. The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine kinases are overexpressed in hematological malignancies. Because PIM kinase expression is regulated by JAK2 signaling, PIM kinase inhibition is a potential therapeutic target for JAK2 mutant-driven malignancies, such as MF. TP-3654, an investigational agent, has been shown to reduce proliferation and increase apoptosis in murine and human hematopoietic cells expressing the JAK2V617F mutation. TP-3654 alone reduced leukocytosis and spleen size in an in vivo murine model of JAK2V617F-induced MF, as well as an apparent reduction of BMF. A phase 1 study is being conducted to evaluate TP-3654 monotherapy in patients with MF. Study Design and Methods: This phase 1, multicenter, dose-escalation, open-label study is evaluating TP-3654 monotherapy in patients with MF who previously failed a JAK inhibitor or who are ineligible to receive ruxolitinib or fedratinib (NCT04176198). Primary objectives are to determine the incidence of dose-limiting toxicities (DLT) at escalated doses of TP-3654 and treatment emergent adverse events. Secondary objectives are to determine QT interval changes, establish the pharmacokinetic profile, and assess preliminary disease activity. Exploratory objectives are pharmacodynamic markers in peripheral blood and bone marrow biopsy samples. Eligible patients have a primary or secondary MF (post-polycythemia vera-MF/post-essential thrombocythemia-MF) based on the World Health Organization diagnostic criteria and intermediate-2 or high-risk primary or secondary MF based on the Dynamic International Prognostic Scoring System; previously failed, or are ineligible to receive, treatment with a JAK inhibitor; grade ≥2 MF, as confirmed by BM biopsy ≤12 weeks prior to screening; have a platelet count >50x109/L, absolute neutrophil count ≥1x109/L, hemoglobin level ≥8 g/L, peripheral blood blast counts <10%, Eastern Cooperative Oncology Group performance status ≤2, life expectancy ≥3 months, and adequate renal and hepatic function; have spleen length ≥5 cm by palpation or spleen volume ≥450 cm3 by computerized tomography/magnetic resonance imaging, and show ≥2 measurable symptoms per the MF Symptom Assessment Form, version 4.0. Patients must not have received prior systemic antineoplastic therapy or any experimental therapy within 14 days or 5 half-lives before TP-3654; had major surgery ≤2 weeks before first study dose; have had splenic irradiation ≤6 months prior to screening; have acute myeloid leukemia or myelodysplastic syndrome; or had prior stem cell transplantation (SCT) or be eligible for allogeneic bone marrow or SCT. Enrollment of approximately 50 patients is planned. Patients will receive oral TP-3654. Dose-escalation will be performed using a Bayesian logistic regression model with escalation with overdose control. Adverse events occurring during the first cycle will be considered in the determination of the maximum tolerated dose (MTD). Dose escalation will continue until identification of the MTD or a suitable recommended phase 2 dose. This study is currently recruiting patients. Disclosures Lebedinsky: Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Anthony:Exact Sciences: Consultancy; Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Mohi:Tolero Pharmaceuticals Inc.: Research Funding. Yang:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Mei:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Braendle:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment.

A Pilot Trial of Rapamycin with Glucocorticoids In Children and Adults with Relapsed ALL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3244-3244
Author(s):  
Krysta D Schlis ◽  
Matthew Stubbs ◽  
Daniel J. DeAngelo ◽  
Donna Neuberg ◽  
Suzanne E. Dahlberg ◽  
...  
Keyword(s):  
Western Blot ◽  
Ribosomal Protein ◽  
Mtor Inhibitor ◽  
Mtor Inhibition ◽  
Blood Samples ◽  
Protein Levels ◽  
Blast Count ◽  
B Lineage

Abstract Abstract 3244 Background: The mammalian target of rapamycin (mTOR) has been identified as a potential therapeutic target in acute lymphoblastic leukemia (ALL). Of particular interest is the potential for mTOR inhibitors to reverse lymphoblast resistance to glucocorticoids. Multiple studies have demonstrated that resistance of lymphoblasts to glucocorticoids, both in vitro and in vivo, predicts a poor clinical outcome in ALL. We have previously demonstrated that rapamycin can reverse glucocorticoid resistance in vitro via suppression of the anti-apoptotic protein MCL-1. Based on this preclinical data, a pilot study was conducted to assess the impact of rapamycin on markers of glucocorticoid resistance in patients with relapsed ALL. Patients and Methods: Protocol therapy consisted of a 5-day investigational window of either glucocorticoids alone (methylprednisolone 32 mg/m2/day IV or prednisone 40 mg/m2/day by mouth) or in combination with rapamycin (12 mg/m2 loading dose followed by 9 mg/m2 divided bid). Peripheral blood samples were obtained and lymphoblasts extracted by Ficoll gradient prior to the first dose of study drug(s) and at 3, 6, 24 and 120 hours (5 days) following the initiation of therapy. Levels of the antiapoptotic protein MCL-1 and phospho-S6 ribosomal protein (a downstream marker of mTOR inhibition) were assessed in the lymphoblasts by Western blot analysis. After completion of the 5-day therapy, patients were treated with multiagent chemotherapy at the discretion of their treating physician. Results: Six patients with a first or subsequent bone marrow relapse of B-lineage ALL were enrolled. Five patients received rapamycin with glucocorticoids and 1 patient received glucocorticoids alone. The median age was 9 years (range: 1 year to 47 years) and 50 % were male. Sufficient protein to perform Western blot analysis was obtained from 5 patients (4 treated with rapamycin/ glucocorticoids and 1 with glucocorticoids alone). Of the 4 assessable rapamycin-treated patients, 3 demonstrated a marked decrease in MCL-1 protein levels after initiation of study drugs which was evident by 6 hours. This was accompanied by a concomitant decrease in phospho-S6 ribosomal protein levels, suggesting successful mTOR inhibition. The fourth patient treated with rapamycin and glucocorticoids had no change in MCL-1 expression and no phospho-S6 ribosomal protein detected at any time point. As controls, we analyzed samples from the one patient from this trial treated with glucocorticoids alone, as well as peripheral blood samples obtained from three patients with newly diagnosed B-lineage ALL who were treated with glucocorticoids (methylprednisolone 32 mg/m2/day) alone for 3 days. Three of these 4 patients demonstrated no change in MCL-1 or phospho-S6 ribosomal protein levels. The fourth patient treated with glucocorticoids alone demonstrated variable MCL-1 protein levels with no change in phospho-S6 ribosomal protein levels. Data from these patients indicates that, in the absence of rapamycin, glucocorticoid therapy alone does not appear to alter MCL-1 or phospho-S6 ribosomal protein levels. Daily circulating absolute blast counts were also monitored for patients enrolled on the study. Notably, one patient treated with rapamycin/ glucocorticoids demonstrated a steady decrease in the circulating absolute blast count during the 5 days of therapy, but had a rebound in the absolute blast count when rapamycin was discontinued despite continued treatment with glucocorticoids. Conculsions: Similar to the findings in preclinical studies, rapamycin suppressed MCL-1 expression in vivo in patients with relapsed ALL. This finding suggests that combining glucocorticoids with an mTOR inhibitor in therapeutic regiments for high-risk and relapsed ALL patients may improve the likelihood of glucocorticoid-induced apoptosis. We are currently preparing to conduct a clinical trial of an mTOR inhibitor combined with multiagent reinduction chemotherapy (including glucocorticoids) for relapsed ALL. Disclosures: Silverman: Enzon: Consultancy, Honoraria; EUSA: Consultancy, Honoraria.

5-Azacytidine Specifically Depletes Regulatory T Cells (Tregs) in Myelodysplastic Syndrome (MDS) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 787-787 ◽  
Author(s):  
Benedetta Costantini ◽  
Shahram Y Kordasti ◽  
Austin G Kulasekararaj ◽  
Jie Jiang ◽  
Thomas Seidl ◽  
...  
Keyword(s):  
T Cells ◽  
High Risk ◽  
Regulatory T Cells ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
In Vitro Study ◽  
Tnf Α ◽  
T Helpers

Abstract Abstract 787 Introduction: The hypomethylating agent 5-azacytidine (5-azaC) leads to improved survival compared to conventional care regimens in patients with intermediate-2 and high-risk MDS and Acute Myeloid Leukaemia (AML) with less than 30% blasts. The precise mode of action of 5-azaC is uncertain, however a combination of cytotoxicity and demethylation is partly responsible for its anti-leukemic activity. In addition, 5-azaC has a profound effect on immune function and inhibits T cell proliferation and activation, blocking cell cycle in the G0 to G1 phase and decreases the production of pro-inflammatory cytokines, suggesting a possible in vivo and in vitro immunomodulating role that may contribute to its anti-leukemic activity. The aim of this study was to investigate the effects of 5-azaC on different subsets of CD4+ T cells, including regulatory T cells (Tregs) and T helpers (Th1, Th2, and Th17). Patients and methods: Seventy intermediate-2/high risk MDS patients and 10 healthy age matched donors (HDs) were studied. CD4+ and CD8+ T cells subsets (percentages and absolute numbers) were investigated by flow cytometry. All patients have received 5-azaC and peripheral blood samples were collected at diagnosis and after 1, 3, 6, 9 and 12 month from initial treatment. On average 3 samples were collected per patient. In vitro study: 5-azaC was added to pre-stimulated PBMCs from 4 HDs and 3 high-risk MDS patients to facilitate the drug incorporation. After 48 hours of initial stimulation, 5-azaC was added every 24 h up to 96 h on two different concentrations (1 μM and 2 μM). For each timepoint (t0, t+24, t+48, t+72, t+96) cells were stained with CD3, CD4, CD25, CD127 and Foxp3 for Tregs and with CD3, CD4, IFN-γ, TNF-α, IL-4, IL-17 for T helpers after an additional 4 hours stimulation with PMA/Ionomycin. Results: In vivo results: Numbers and percentages of Tregs were significantly higher in patients' peripheral blood prior to treatment compared to HDs (0.7% v 0.08%, p<0.001 and 1.1 × 107/L v 4.6 × 106/L, p=0.01). However, after 12 months of treatment with 5-azaC the number of Tregs decreased to the normal level. Absolute numbers and percentages of Tregs were also higher in non-responder patients compared to responders after treatment (1.2 × 107/L v 7.3 × 106/L, p=0.01). Although the number of Th1 and Th17 cells did not change significantly following treatment with 5-azaC, the Th1/Tregs and Th17/Tregs ratios were significantly decreased in non-responders (p=0.02), whereas these ratios remained stable in responder patients. In vitro results: There were no changes in the number or frequency of Th1, Th2 or Th17 when 5-azaC (1 μM and 2 μM) was added to patients' T cells. However, numbers and frequencies of Tregs dropped significantly compared to HDs' T cells (p=0.034). The ratio of Th1/Tregs and Th17/Tregs were also higher in patients' treated PBMCs after in vitro 5-azaC. There was a significant decrease in the percentages and numbers of Th1 cells (15.4% v 2.7%, p=0.043 and 1.42 × 103 v 6.44 × 104, p=0.021), Th17 cells (1.01% v 0.07%, p = 0.021 and 4.22 × 103 v 7.2 × 102, p=0.021) Th1/Tregs ratio (79.8 v 1.5, p=0.043), and Th17/Tregs ratio (5.2 v 0.1, p=0.021) in HDs' PBMCs treated with 2 μM compared to untreated cells. 5-azaC also reduced the absolute numbers of CD4+TNF-α+ T cells (1.07 × 105 v 5.37 × 103, p=0.021) and Th2 (9.24 × 103 v 1.25 × 102, p=0.021) in HDs' T cells. There was no preferential apoptosis in any subsets of T cells confirmed by Annexin V staining. However, it is interesting to note that the telomere length of Tregs treated with 5-azaC was longer than untreated Tregs, suggesting a decrease of their proliferation. Conclusion: Our study suggests that 5-azaC can induce a significant decrease in the number of Tregs in patients (in vivo and in vitro) and HDs, and therefore creates a pro-inflammatory state, despite a small decrease in the number of Th1 and Th17 cells. These changes are more significant in patients who responded to 5-azaC rather than in non-responders. Surprisingly, our in vitro study suggests that 5-azaC leads to a marked reduction in Tregs. As there is not a Tregs' specific apoptosis following 5-azaC treatment, we speculate that the reduction in Tregs' number is mainly due to de-methylation of transcription factors which leads to conversion of Tregs into other T cell subsets (ie Th1 or Th17). Disclosures: Mufti: Celgene: Research Funding.

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